**Measuring of DNA Damage by Quantitative PCR**

## Ayse Gul Mutlu

*Mehmet Akif Ersoy University, Arts and Sciences Faculty, Department of Biology, Burdur Turkey* 

## **1. Introduction**

282 Polymerase Chain Reaction

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#### **1.1 QPCR; principles and development**

PCR is an in vitro method of nucleic acid synthesis by which a particular segment of DNA can be specifically replicated. It involves two oligonucleotide primer that flank the DNA fragment to be amplified and repeated cycles of heat denaturation of the DNA, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase. Successive cycles of amplification essentially double the amount of the target DNA synthesized in the previous cycle (1).

Recent advances of the fluorometric dyes allow the very sensitive and quick quantitation of DNA. Before the invention of fluorometric quantitative PCR (QPCR) method the researchers who measured a gene's amount, have used the different methods like competetive PCR, solid phase assays, HPLC, dot blot or immunoassay (2). Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers (3).

The detection of gene-spesific damage and repair has been studied in nuclear and mitochondrial DNA by the use of southern analysis. But this method requires knowledge of the restriction sites flanking the damaged site, the use of large quantities of DNA, and incision of DNA lesions with a spesific endonuclease (4). Govan and collegues has reported a new approach to measuring of DNA damage in 1990 (5). This PCR based quantitative technique has been improved by Kalinowski and collegues (6). Principle of this analysis is that lesions present in the DNA, block the progression of any thermostable polymerase on the template. So the DNA amplification decreases in the damaged template when compared to the undamaged DNA (4). QPCR is a suitable method for the measuring damage and repair in the subgene level functional units like promotor regions, exons and introns (7). Method also useful to determining DNA damage and repair that originated by the genotoxic agents and oxidative stress (8,9,10). The method capable of detect 1 lesion/105 nucleotides from as little as 5 ng of total genomic DNA (4).

DNA extraction, pre quantitation of DNA template, PCR amplification and quantitation of PCR products are crucial for success of the application (Figure 1). Quantity and quality of the DNA sample is important. We use mini column based kits for DNA extraction in our laboratory. These extraction kits and carefully pipetting, minimize the artificial DNA

Measuring of DNA Damage by Quantitative PCR 285

Hot start PCR improve specifity of PCR reaction. Hot start PCR is reported to minimize

Optimization might involve changes in nucleic acids preparation, in primer usage, in buffer usage and in cycling parameters. One of the recent developments in PCR optimization is to recognize the importance of eliminating some undesired hybridization events that often happen in the first cycle and can carry potentially devastating effects. Theoretically, if the amplification precedes with an efficiency of 100%, the amount of amplicons is doubling at each cycle. However, in most PCR procedures, the overall efficiency is less than 100% and a typical amplification runs with a constant efficiency of about 70-80% from the 15th cycle to the 30th cycle, depending on the amount of starting material. The increase in the amount of amplicons stays exponential only for a limited number of cycles, after which the amplification rate reaches a plateau. The factors that contribute to this plateau phenomenon include substrate saturation of enzyme, product strand reannealing, and incomplete product strand separation. In this latter phase, the quantitated amount of amplified product is no longer proportional to the starting amount of target molecules. Therefore, to make PCR suitable in quantitative settings, it is imperative that a balance be found between a constant efficiency and an exponential phase in the amplification process. This will ultimately depend on the number of cycles, on the amount of targets in the starting material, and on

We run a 50% template control and a nontemplate control in PCR in our laboratory. 50% template control should given a 50% reduction of the amplification signal (values between 40%-60% reduction are acceptable). The nontemplate control would detect contamination

Fig. 2. PCR band of 10 kb mtDNA fragment of Mus musculus (Balb C). (Band 1: λ *Hind III*

We study mtDNA damage by QPCR method in different organisms like fruit flies, mice and snails in our laboratory. In our research that we used mice, oxidative mtDNA damage that

nontarget amplification and the formation of primer-dimer (14) .

the system of detection and quantitation of the amplified product (15).

with spurious DNA or PCR products (4).

**3. Measuring of mtDNA damage on mice** 

digest marker DNA)

damages. Pico Green dsDNA quantitation kit is used for both template DNA quantitation and the analysis of PCR products as fluorometrically 485 nm excitation, 530 nm emission. Pico Green and SYBR green are substantially more sensitive for quantifying DNA concentrations than ethidium bromide and some other fluorimetric dyes (11). Initial DNA template quantity in the all PCR tubes must be the same. mtDNA damage is quantified by comparing the relative efficiency of amplification of long fragments of DNA and normalizing this to gene copy numbers by the amplification of smaller fragments, which have a statistically negligible likelihood of containing damaged bases. To calculate relative amplification, the long QPCR values are divided by the corresponding short QPCR results to account for potential copy number differences between samples. Decreased relative amplification is an indicator of the damaged DNA (4, 12).

Calculation of the relative amplification

Fig. 1. Flowchart of the QPCR assay for measuring of DNA damage

## **2. Optimization of the assay; the crucial steps**

Crucial step of the QPCR is PCR optimization. Thermal conditions, especially annealing temperature must be optimized. Extention temperature may be lower for long PCR amplifications. mtDNA amplification may needs some adjuvants. We use in our laboratory DMSO (%4) for improve the efficiency of the PCR reaction. Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5%–10% (vol/vol). However, in the multiplex reactions, these adjuvants give conflicting results. For example, 5% DMSO improve the amplification of some products and decrease the amount of others. There are similar results with 5% glycerol. Therefore, the usefulness of these adjuvants needs to be tested in each case. Also BSA may increase the efficiency of the PCR (13).

damages. Pico Green dsDNA quantitation kit is used for both template DNA quantitation and the analysis of PCR products as fluorometrically 485 nm excitation, 530 nm emission. Pico Green and SYBR green are substantially more sensitive for quantifying DNA concentrations than ethidium bromide and some other fluorimetric dyes (11). Initial DNA template quantity in the all PCR tubes must be the same. mtDNA damage is quantified by comparing the relative efficiency of amplification of long fragments of DNA and normalizing this to gene copy numbers by the amplification of smaller fragments, which have a statistically negligible likelihood of containing damaged bases. To calculate relative amplification, the long QPCR values are divided by the corresponding short QPCR results to account for potential copy number differences between samples. Decreased relative

DNA isolation

↓

Quantitation of the isolated DNA (template quantitation)

↓

PCR (Starting amounts of the template DNA must be same in all of the samples)

↓

PCR products quantitation

↓

Calculation of the relative amplification

Crucial step of the QPCR is PCR optimization. Thermal conditions, especially annealing temperature must be optimized. Extention temperature may be lower for long PCR amplifications. mtDNA amplification may needs some adjuvants. We use in our laboratory DMSO (%4) for improve the efficiency of the PCR reaction. Various authors recommend DMSO and glycerol to improve amplification efficiency (higher amount of product) and specificity (no unspecific products) of PCR, when used in concentrations varying between 5%–10% (vol/vol). However, in the multiplex reactions, these adjuvants give conflicting results. For example, 5% DMSO improve the amplification of some products and decrease the amount of others. There are similar results with 5% glycerol. Therefore, the usefulness of these adjuvants needs to be tested in each case. Also BSA may increase the efficiency of the PCR (13).

Fig. 1. Flowchart of the QPCR assay for measuring of DNA damage

**2. Optimization of the assay; the crucial steps** 

amplification is an indicator of the damaged DNA (4, 12).

Hot start PCR improve specifity of PCR reaction. Hot start PCR is reported to minimize nontarget amplification and the formation of primer-dimer (14) .

Optimization might involve changes in nucleic acids preparation, in primer usage, in buffer usage and in cycling parameters. One of the recent developments in PCR optimization is to recognize the importance of eliminating some undesired hybridization events that often happen in the first cycle and can carry potentially devastating effects. Theoretically, if the amplification precedes with an efficiency of 100%, the amount of amplicons is doubling at each cycle. However, in most PCR procedures, the overall efficiency is less than 100% and a typical amplification runs with a constant efficiency of about 70-80% from the 15th cycle to the 30th cycle, depending on the amount of starting material. The increase in the amount of amplicons stays exponential only for a limited number of cycles, after which the amplification rate reaches a plateau. The factors that contribute to this plateau phenomenon include substrate saturation of enzyme, product strand reannealing, and incomplete product strand separation. In this latter phase, the quantitated amount of amplified product is no longer proportional to the starting amount of target molecules. Therefore, to make PCR suitable in quantitative settings, it is imperative that a balance be found between a constant efficiency and an exponential phase in the amplification process. This will ultimately depend on the number of cycles, on the amount of targets in the starting material, and on the system of detection and quantitation of the amplified product (15).

We run a 50% template control and a nontemplate control in PCR in our laboratory. 50% template control should given a 50% reduction of the amplification signal (values between 40%-60% reduction are acceptable). The nontemplate control would detect contamination with spurious DNA or PCR products (4).

## **3. Measuring of mtDNA damage on mice**

We study mtDNA damage by QPCR method in different organisms like fruit flies, mice and snails in our laboratory. In our research that we used mice, oxidative mtDNA damage that

Measuring of DNA Damage by Quantitative PCR 287

Many carcinogens in the cigarette smoke like PAHs, nitrosamine and cisplatin bind mitochondrial DNA (mtDNA) preferentially (22). The antioxidants are used frequently as food supplements may be effective to preventing cigarette smoke damage on mtDNA. Damages that are created by CS may be prevented by vitamin E (Vit E) and selenium (Se)

Genomic DNA mini column kit (SIGMA) was used for total DNA isolation according to the technical bulletin. We used Pico Green dsDNA quantitation kit for both template DNA quantitation and the analysis of PCR products as fluorometrically 485 nm excitation, 530 nm emission (23). A crucial step of quantitative PCR is the concentration of the DNA sample. In fact, the accuracy of the assay relies on initial template quantity because all of the samples must have exactly the same amount of DNA. The Pico Green dye has not only proved efficient in regarding to template quantitation but also to PCR product analysis (10). Hot Start ready mix Taq (SIGMA) were used for PCR. In this mix, taq polymerase combines the performance enhancements of Taq antibody for hot start. When the temperature is raised above 70ºC in the first denaturation step of the cycling process, the complex dissociates and the polymerase becomes fully active. DMSO as 4% of total volume and 20 ng of template total DNA were added into the each PCR tube. Mouse 117 bp Mouse 117 bp mtDNA

13597 5'- CCC AGC TAC TAC CAT CAT TCA AGT- 3'

Table 1. QPCR primers for measuring of mtDNA damage in *Mus musculus* and *Drosophila* 

13688 5'- GAT GGT TTG GGA GAT TGG TTG ATG T- 3' (Table 1)

which are powerful antioxidants.

fragment (small fragment) primers were:

*Mus musculus* primers for long fragment (10085 bp):

*Mus musculus* primers for small fragment (117 bp):

*Drosophila* primers for long fragment (10629 bp): 1880 5'- ATGGTGGAGCTTCAGTTGATTT - 3' 12487 5'- CAACCTTTTTGTGATGCGATTA - 3'

*Drosophila* primers for small fragment (100 bp):

*melanogaster*

11426 5'- TAAGAAAATTCCGAGGGATTCA - 3' 11504 5'- GGTCGAGCTCCAATTCAAGTTA - 3'

13597 5'- CCC AGC TAC TAC CAT CAT TCA AGT- 3'

13688 5'- GAT GGT TTG GGA GAT TGG TTG ATG T- 3' (4)

3278 5'- GCC AGC CTG ACC CAT AGC CAT AAT AT- 3' 13337 5'- GAG AGA TTT TAT GGG TGT AAT GCG G- 3' (4)

created by cigarette smoke and protective effects of VitE and selenium was investigated (Figure 3).

Fig. 3. Vitamin supplementation

DNA damage that is originated by cigarette smoke in various organs is declared by some research (16,17). Tobacco smoking contains many thousands of chemicals including a plethora of mutagens. Many carcinogens undergo metabolic activation in mammalian tissues to reactive intermediates that interact with and modify informational macromolecules, such as DNA with potentially mutagenic consequences (18). PAHs (Polycyclic aromatic hydrocarbons) cause irreversible DNA damage via covalent binding or oxidation (19). However genetic damage reflecting individual exposure and susceptibility to PAH may play a role in disease development (20). Tobacco smoke contains major classes of carcinogens that include PAHs, aromatic amines and tobaccospecific nitrosamines. In addition, toxic compounds such as formaldehyde, acetaldehyde, acrolein, short-lived radicals and reactive oxygen intermediates generated by redox cycling from catechol and hydroquinone and nitric oxide (NO) may also contribute to the toxic and carcinogenic effects of tobacco smoke. Direct DNAdamaging compounds that are present in cigarette smoke (CS) have previously been reported to include reactive oxygen intermediates, peroxynitrite, ethylating agents and unidentified compounds (21).

created by cigarette smoke and protective effects of VitE and selenium was investigated

DNA damage that is originated by cigarette smoke in various organs is declared by some research (16,17). Tobacco smoking contains many thousands of chemicals including a plethora of mutagens. Many carcinogens undergo metabolic activation in mammalian tissues to reactive intermediates that interact with and modify informational macromolecules, such as DNA with potentially mutagenic consequences (18). PAHs (Polycyclic aromatic hydrocarbons) cause irreversible DNA damage via covalent binding or oxidation (19). However genetic damage reflecting individual exposure and susceptibility to PAH may play a role in disease development (20). Tobacco smoke contains major classes of carcinogens that include PAHs, aromatic amines and tobaccospecific nitrosamines. In addition, toxic compounds such as formaldehyde, acetaldehyde, acrolein, short-lived radicals and reactive oxygen intermediates generated by redox cycling from catechol and hydroquinone and nitric oxide (NO) may also contribute to the toxic and carcinogenic effects of tobacco smoke. Direct DNAdamaging compounds that are present in cigarette smoke (CS) have previously been reported to include reactive oxygen intermediates,

peroxynitrite, ethylating agents and unidentified compounds (21).

(Figure 3).

Fig. 3. Vitamin supplementation

Many carcinogens in the cigarette smoke like PAHs, nitrosamine and cisplatin bind mitochondrial DNA (mtDNA) preferentially (22). The antioxidants are used frequently as food supplements may be effective to preventing cigarette smoke damage on mtDNA. Damages that are created by CS may be prevented by vitamin E (Vit E) and selenium (Se) which are powerful antioxidants.

Genomic DNA mini column kit (SIGMA) was used for total DNA isolation according to the technical bulletin. We used Pico Green dsDNA quantitation kit for both template DNA quantitation and the analysis of PCR products as fluorometrically 485 nm excitation, 530 nm emission (23). A crucial step of quantitative PCR is the concentration of the DNA sample. In fact, the accuracy of the assay relies on initial template quantity because all of the samples must have exactly the same amount of DNA. The Pico Green dye has not only proved efficient in regarding to template quantitation but also to PCR product analysis (10). Hot Start ready mix Taq (SIGMA) were used for PCR. In this mix, taq polymerase combines the performance enhancements of Taq antibody for hot start. When the temperature is raised above 70ºC in the first denaturation step of the cycling process, the complex dissociates and the polymerase becomes fully active. DMSO as 4% of total volume and 20 ng of template total DNA were added into the each PCR tube. Mouse 117 bp Mouse 117 bp mtDNA fragment (small fragment) primers were:

#### 13597 5'- CCC AGC TAC TAC CAT CAT TCA AGT- 3'

#### 13688 5'- GAT GGT TTG GGA GAT TGG TTG ATG T- 3' (Table 1)

*Mus musculus* primers for long fragment (10085 bp):

3278 5'- GCC AGC CTG ACC CAT AGC CAT AAT AT- 3'

13337 5'- GAG AGA TTT TAT GGG TGT AAT GCG G- 3' (4)

*Mus musculus* primers for small fragment (117 bp):

13597 5'- CCC AGC TAC TAC CAT CAT TCA AGT- 3'

13688 5'- GAT GGT TTG GGA GAT TGG TTG ATG T- 3' (4)

*Drosophila* primers for long fragment (10629 bp):

1880 5'- ATGGTGGAGCTTCAGTTGATTT - 3'

12487 5'- CAACCTTTTTGTGATGCGATTA - 3'

*Drosophila* primers for small fragment (100 bp):

11426 5'- TAAGAAAATTCCGAGGGATTCA - 3'

11504 5'- GGTCGAGCTCCAATTCAAGTTA - 3'

Table 1. QPCR primers for measuring of mtDNA damage in *Mus musculus* and *Drosophila melanogaster*

Measuring of DNA Damage by Quantitative PCR 289

3278 5'- GCC AGC CTG ACC CAT AGC CAT AAT AT- 3'

13337 5'- GAG AGA TTT TAT GGG TGT AAT GCG G- 3'

For long fragment PCR amplification, DNA was denatured initially at 75ºC for 2 min and 95ºC for 1 min, and then the reaction underwent 21 PCR cycles of 94ºC for 15 sec, 59ºC for 30 sec, and 65 ºC for 11 min. Final extension was allowed to proceed at 72ºC for 10 min (Table 2). For small fragment PCR amplification, DNA was denatured initially at 75 ºC for 2 min and 95 ºC for 15 sec, and then the reaction underwent 19 PCR cycles of 94ºC for 30 sec, 50ºC for 45 sec, and 72 ºC for 45 sec. Final extension was allowed to proceed at 72ºC for 10 min (23). We were always run a 50% template control and a nontemplate control in PCR. To calculate relative amplification, the long QPCR values were divided by the corresponding short QPCR results to account for potential copy number differences between samples (mtDNA/total DNA value may be different in 20 ng template total DNA of each PCR tube)

We detected mtDNA damage in the mouse heart succesfully. According to these relative amplification results "cigarette smoke" application group was significantly different from all other groups. mtDNA damage was significantly higher in the cigarette smoke group than the other groups. However "Cigarette Smoke+Vitamin E+Selenium" group had lowest

**4. Measuring of oxidative mtDNA damage and copy number on Drosophila**  The free radical theory of aging postulates that aging changes are caused by free radical reactions. Aging is the progressive accumulation of changes with time that are responsible for the ever-increasing likelihood of disease and death. These irreversible changes are attributed to the aging process. This process is now the major cause of death in the developed countries. The aging process may be due to free radical reactions (25). The free radical theory of aging posits that the accumulation of macromolecular damage induced by toxic reactive oxygen species (ROS) plays a central role in the aging process. The mitochondria are the principal generator of ROS during the conversion of molecular oxygen to energy production where approximately 0.4% to 4% of the molecular oxygen metabolized by the mitochondrial electron transport chain is converted to ROS (26). Cellular damage caused by radicals may induce cancer, neurodegeneration and autoimmun disease (27). Toxic materials may produce ROS and generate oxidative damage on mitochondrial DNA (mtDNA) (23). mtDNA damages may trigger mitochondrial dysfunction (28). Damage to mtDNA could be potentially more important than deletions in nDNA, because the entire mitochondrial genome codes for genes that are expressed while nDNA contains a large amount of non-transcribed sequences. Also, mtDNA, unlike nDNA, is continuously replicated, even in terminally differentiated cells, such as neurons and cardiomyocytes; hence, somatic mtDNA damage potentially causes more adverse effects on cellular functions

Mouse 10 kb mtDNA fragment (Figure 2) primers were:

(3,4,10,23). The copy number results not indicate the damage.

(4).

mean damage (23,24).

than does somatic nDNA damage (29).

Thermal conditions for *Mus musculus* long fragment (10085 bp):

75ºC for 2 min

95ºC for 1 min

**94ºC for 15 sec** 

**59ºC for 30 sec 21 cycles** 

**65 ºC for 11 min** 

72ºC for 10 min

Thermal conditions for *Mus musculus* small fragment (117 bp):

75 ºC for 2 min

95 ºC for 15 sec

**94ºC for 30 sec** 

**50ºC for 45 sec 19 cycles** 

**72 ºC for 45 sec.** 

72ºC for 10 min

Thermal conditions for *Drosophila* long fragment (10629 bp):

75ºC for 1 min

95ºC for 1 min

**94ºC for 15 sec** 

**52ºC for 45 sec 21 cycles** 

**65 ºC for 11 min** 

68ºC for 10 min.

Thermal conditions for *Drosophila* small fragment (100 bp):

75 ºC for 2 min

95 ºC for 15 sec

**94ºC for 30 sec** 

**55ºC for 45 sec 21 cycles** 

**72 ºC for 45 sec** 

72ºC for 10 min

Table 2. Thermal conditions for QPCR in *Mus musculus* and *Drosophila melanogaster* 

Mouse 10 kb mtDNA fragment (Figure 2) primers were:

## 3278 5'- GCC AGC CTG ACC CAT AGC CAT AAT AT- 3' 13337 5'- GAG AGA TTT TAT GGG TGT AAT GCG G- 3'

(4).

288 Polymerase Chain Reaction

Thermal conditions for *Mus musculus* long fragment (10085 bp):

Thermal conditions for *Mus musculus* small fragment (117 bp):

Thermal conditions for *Drosophila* long fragment (10629 bp):

Thermal conditions for *Drosophila* small fragment (100 bp):

Table 2. Thermal conditions for QPCR in *Mus musculus* and *Drosophila melanogaster* 

75ºC for 2 min 95ºC for 1 min **94ºC for 15 sec** 

**65 ºC for 11 min**  72ºC for 10 min

75 ºC for 2 min 95 ºC for 15 sec **94ºC for 30 sec** 

**72 ºC for 45 sec.**  72ºC for 10 min

75ºC for 1 min 95ºC for 1 min **94ºC for 15 sec** 

**65 ºC for 11 min**  68ºC for 10 min.

75 ºC for 2 min 95 ºC for 15 sec **94ºC for 30 sec** 

**72 ºC for 45 sec**  72ºC for 10 min

**59ºC for 30 sec 21 cycles** 

**50ºC for 45 sec 19 cycles** 

**52ºC for 45 sec 21 cycles** 

**55ºC for 45 sec 21 cycles** 

For long fragment PCR amplification, DNA was denatured initially at 75ºC for 2 min and 95ºC for 1 min, and then the reaction underwent 21 PCR cycles of 94ºC for 15 sec, 59ºC for 30 sec, and 65 ºC for 11 min. Final extension was allowed to proceed at 72ºC for 10 min (Table 2). For small fragment PCR amplification, DNA was denatured initially at 75 ºC for 2 min and 95 ºC for 15 sec, and then the reaction underwent 19 PCR cycles of 94ºC for 30 sec, 50ºC for 45 sec, and 72 ºC for 45 sec. Final extension was allowed to proceed at 72ºC for 10 min (23).

We were always run a 50% template control and a nontemplate control in PCR. To calculate relative amplification, the long QPCR values were divided by the corresponding short QPCR results to account for potential copy number differences between samples (mtDNA/total DNA value may be different in 20 ng template total DNA of each PCR tube) (3,4,10,23). The copy number results not indicate the damage.

We detected mtDNA damage in the mouse heart succesfully. According to these relative amplification results "cigarette smoke" application group was significantly different from all other groups. mtDNA damage was significantly higher in the cigarette smoke group than the other groups. However "Cigarette Smoke+Vitamin E+Selenium" group had lowest mean damage (23,24).

## **4. Measuring of oxidative mtDNA damage and copy number on Drosophila**

The free radical theory of aging postulates that aging changes are caused by free radical reactions. Aging is the progressive accumulation of changes with time that are responsible for the ever-increasing likelihood of disease and death. These irreversible changes are attributed to the aging process. This process is now the major cause of death in the developed countries. The aging process may be due to free radical reactions (25). The free radical theory of aging posits that the accumulation of macromolecular damage induced by toxic reactive oxygen species (ROS) plays a central role in the aging process. The mitochondria are the principal generator of ROS during the conversion of molecular oxygen to energy production where approximately 0.4% to 4% of the molecular oxygen metabolized by the mitochondrial electron transport chain is converted to ROS (26). Cellular damage caused by radicals may induce cancer, neurodegeneration and autoimmun disease (27). Toxic materials may produce ROS and generate oxidative damage on mitochondrial DNA (mtDNA) (23). mtDNA damages may trigger mitochondrial dysfunction (28). Damage to mtDNA could be potentially more important than deletions in nDNA, because the entire mitochondrial genome codes for genes that are expressed while nDNA contains a large amount of non-transcribed sequences. Also, mtDNA, unlike nDNA, is continuously replicated, even in terminally differentiated cells, such as neurons and cardiomyocytes; hence, somatic mtDNA damage potentially causes more adverse effects on cellular functions than does somatic nDNA damage (29).

Measuring of DNA Damage by Quantitative PCR 291

Fig. 4. QUBIT 2.0 fluorometer were used for both template DNA quantitation and the

QPCR is a suitable method for the measuring damage and repair in the subgene level functional units like promotor regions, exons and introns (7). Recent advances of the fluorometric dyes allow the very sensitive and quick quantitation of DNA. Before the invention of fluorometric quantitative PCR (QPCR) method, the researchers who measured a gene's amount, have used the different methods like competetive PCR, solid phase assays, HPLC, dot blot or immunoassay (2). Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers (3). Method also useful to determining DNA damage and repair that originated by the genotoxic agents and oxidative stress (8,9,10). Crucial step of the QPCR is PCR optimization. Thermal conditions, especially annealing temperature must be optimized. Important points of the optimization:

2. Optimization of he extention temperature (Extention temperature may be lower for

4. Hot start PCR (minimize nontarget amplification and the formation of primer-dimer)

analysis of PCR products as fluorometrically

1. Determination of annealing temperature

long PCR amplifications) 3. Adjuvants (if necessary)

5. Determination of cycling number

**5. Conclusions** 

Cereals naturally contain a wide variety of polyphenols such as the hydroxycinnamic acids, ferulic, vanillic, and *p*-coumaric acids which show a strong antioxidant power and may help to protect from oxidative stress and, therefore, can decrease the risk of contracting many diseases. Flavonoids are present in small quantities, even though their numerous biological effects and their implications for inflammation and chronic diseases have been widely described. The mechanisms of action of polyphenols go beyond the modulation of oxidative stress-related pathways (30).

Wheat is an important component of the human diet. But the distribution of phytochemicals (total phenolics, flavonoids, ferulic acid, and carotenoids) and hydrophilic and lipophilic antioxidant activity in milled fractions (endosperm and bran/germ) are different each other. Different milled fractions of wheat have different profiles of both hydrophilic and lipophilic phytochemicals. Total phenolic content of bran/germ fractions is 15−18-fold higher than that of endosperm fractions. Hydrophilic antioxidant activity of bran/germ samples is 13−27-fold higher than that of the respective endosperm samples. Similarly, lipophilic antioxidant activity is 28−89-fold higher in the bran/germ fractions. In whole-wheat flour, the bran/germ fraction contribute 83% of the total phenolic content, 79% of the total flavonoid content, 51% of the total lutein, 78% of the total zeaxanthin, 42% of the total βcryptoxanthin, 85% of the total hydrophilic antioxidant activity, and 94% of the total lipophilic antioxidant activity (31).

Aim of our study was investigate the effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in the aging process of *Drosophila* (32).

Genomic DNA kits (invitrogen) were used for total DNA isolation according to the technical bulletin. İnvitrogen (Molecular Probes) Pico Green dsDNA quantitation dye and QUBIT 2.0 fluorometer were used for both template DNA quantitation and the analysis of PCR products as fluorometrically (Figure 4). DMSO as 4% of total volume and 5 ng of template total DNA were added into the each PCR tube.

Primers for Drosophila mtDNA 100bp fragment were designed as;

11426 5'- TAAGAAAATTCCGAGGGATTCA - 3'

11525 5'- GGTCGAGCTCCAATTCAAGTTA - 3'

Primers for Drosophila mtDNA 10629 bp fragment were designed as;

1880 5'- ATGGTGGAGCTTCAGTTGATTT - 3'

12508 5'- CAACCTTTTTGTGATGCGATTA - 3' (Table 1)

For long fragment PCR amplification, DNA was denatured initially at 75ºC for 1 min and 95ºC for 1 min, and then the reaction underwent 21 PCR cycles of 94ºC for 15 sec, 52ºC for 45 sec, and 65 ºC for 11 min. Final extension was allowed to proceed at 68ºC for 10 min (Table 2).

For small fragment PCR amplification, DNA was denatured initially at 75 ºC for 2 min and 95 ºC for 15 sec, and then the reaction underwent 21 PCR cycles of 94ºC for 30 sec, 55ºC for 45 sec, and 72 ºC for 45 sec. Final extension was allowed to proceed at 72ºC for 10 min.

Cereals naturally contain a wide variety of polyphenols such as the hydroxycinnamic acids, ferulic, vanillic, and *p*-coumaric acids which show a strong antioxidant power and may help to protect from oxidative stress and, therefore, can decrease the risk of contracting many diseases. Flavonoids are present in small quantities, even though their numerous biological effects and their implications for inflammation and chronic diseases have been widely described. The mechanisms of action of polyphenols go beyond the modulation of oxidative

Wheat is an important component of the human diet. But the distribution of phytochemicals (total phenolics, flavonoids, ferulic acid, and carotenoids) and hydrophilic and lipophilic antioxidant activity in milled fractions (endosperm and bran/germ) are different each other. Different milled fractions of wheat have different profiles of both hydrophilic and lipophilic phytochemicals. Total phenolic content of bran/germ fractions is 15−18-fold higher than that of endosperm fractions. Hydrophilic antioxidant activity of bran/germ samples is 13−27-fold higher than that of the respective endosperm samples. Similarly, lipophilic antioxidant activity is 28−89-fold higher in the bran/germ fractions. In whole-wheat flour, the bran/germ fraction contribute 83% of the total phenolic content, 79% of the total flavonoid content, 51% of the total lutein, 78% of the total zeaxanthin, 42% of the total βcryptoxanthin, 85% of the total hydrophilic antioxidant activity, and 94% of the total

Aim of our study was investigate the effects of a wheat germ rich diet on oxidative mtDNA damage, mtDNA copy number and antioxidant enzyme activities in the aging process of

Genomic DNA kits (invitrogen) were used for total DNA isolation according to the technical bulletin. İnvitrogen (Molecular Probes) Pico Green dsDNA quantitation dye and QUBIT 2.0 fluorometer were used for both template DNA quantitation and the analysis of PCR products as fluorometrically (Figure 4). DMSO as 4% of total volume and 5 ng of template

11426 5'- TAAGAAAATTCCGAGGGATTCA - 3'

11525 5'- GGTCGAGCTCCAATTCAAGTTA - 3'

1880 5'- ATGGTGGAGCTTCAGTTGATTT - 3'

 12508 5'- CAACCTTTTTGTGATGCGATTA - 3' (Table 1) For long fragment PCR amplification, DNA was denatured initially at 75ºC for 1 min and 95ºC for 1 min, and then the reaction underwent 21 PCR cycles of 94ºC for 15 sec, 52ºC for 45 sec, and 65 ºC for 11 min. Final extension was allowed to proceed at 68ºC for 10 min (Table 2). For small fragment PCR amplification, DNA was denatured initially at 75 ºC for 2 min and 95 ºC for 15 sec, and then the reaction underwent 21 PCR cycles of 94ºC for 30 sec, 55ºC for 45 sec, and 72 ºC for 45 sec. Final extension was allowed to proceed at 72ºC for 10 min.

stress-related pathways (30).

lipophilic antioxidant activity (31).

total DNA were added into the each PCR tube.

Primers for Drosophila mtDNA 100bp fragment were designed as;

Primers for Drosophila mtDNA 10629 bp fragment were designed as;

*Drosophila* (32).

Fig. 4. QUBIT 2.0 fluorometer were used for both template DNA quantitation and the analysis of PCR products as fluorometrically

## **5. Conclusions**

QPCR is a suitable method for the measuring damage and repair in the subgene level functional units like promotor regions, exons and introns (7). Recent advances of the fluorometric dyes allow the very sensitive and quick quantitation of DNA. Before the invention of fluorometric quantitative PCR (QPCR) method, the researchers who measured a gene's amount, have used the different methods like competetive PCR, solid phase assays, HPLC, dot blot or immunoassay (2). Many of the applications of real-time Q-PCR include measuring mRNA expression levels, DNA copy number, transgene copy number and expression analysis, allelic discrimination, and measuring viral titers (3). Method also useful to determining DNA damage and repair that originated by the genotoxic agents and oxidative stress (8,9,10). Crucial step of the QPCR is PCR optimization. Thermal conditions, especially annealing temperature must be optimized. Important points of the optimization:


Measuring of DNA Damage by Quantitative PCR 293

[13] O. Henegariu, N.A. Heerema, S.R. Dlouhy, G.H. Vance and P.H. Vogt, 1997. Multiplex PCR: Critical Parameters and Step-by-Step Protocol. BioTechniques, 23: 504-511. [14] Erlich HA, Gelfand D, Sninsky JJ, 1991. Recent Advances in the Polymerase Chain

[15] Ferre F, 1992. Quantitative or semi-quantitative PCR: reality versus myth. Genome

[16] Izzotti A, Balanksy RM, Blagoeva PM, Mircheva Z, Tulimiero L, Cartiglia C, De Flora S,

[17] Izzotti A, Bagnasco M, D'Agostini F, Cartiglia C, Lubet RA, Kelloff G, De Flora S, 1999.

[18] Phillips DH, 2002. Smoking related DNA and protein adducts in human tissues.

[19] Gelboin HV, 1980. Benzo α pyrene metabolism, activation and carcinogenesis: role and

[20] Rundle A, Tang D, Hibshoosh H, Estabrook A, Schnabel F, Cao W, Grumet S, Perera

[22] Sawyer DE, Van Houten B. (1999) Repair of DNA damage in mitochondria. Mutat Res,

[23] Mutlu AG, Fiskin K, 2009. Can Vitamin E and Selenium Prevent Cigarette Smoke-

[24] Mutlu AG., Fiskin K, 2009. Oxidative mtDNA damage in the heart tissue of cigarette

[26] Lim H, Bodmer R and Perrin L, 2006. Drosophila aging 2005-2006. Exp Gerontol, 41:

[27] Rodriguez C, Mayo JC, Sainz RM, Antolin I, Herrera F, Martin V and Reiter RJ, 2004.

[28] Lesnefsky EJ, Moghaddas S, Tandler B, Kerner J and Hoppel CL, 2001. Mitochondrial

[29] Liang F-Q and Godley BF, 2003. Oxidative stres induced mtDNA damage in human

macular degeneration. Experimental Eye Research, 76: 397-403.

[25] Harman D, 2006. Free Radicals in Aging. Moll Cell Biochem, 84: 155-61.

environmental cigarette smoke. Carcinogenesis, 20: 1499-1506.

1998. DNA alterations in rat organs after chronic exposure to cigarette smoke

Formation and persistence of nucleotide alterations in rats exposed whole-body to

regulation of mixed-function oxidases and related enzymes. Physiol Rev, 60:1107–

FP, 2000. The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer. Carcinogenesis, 21: 1281-1289. [21] Yang Q, Hergenhahn M, Weninger A, Bartsch H, 1999. Cigarette smoke induces direct

DNA damage in the human B-lymphoid cell line Raji. Carcinogenesis, 20: 1769-

Derived Oxidative mtDNA Damage? Turkish Journal of Biochemistry, 34 (3); 167-

smoke exposed mice and protective effects of Vitamn E and Selenium. IUBMB Life. 61: 328-329. (III. International Congress of Molecular Medicine, İstanbul, 5-8 May

Regulation of antioxidant enzymes: a significant role for melatonin. J Pineal Res, 36:

dysfunction in cardiac disease: ischemia –reperfusion, aging and heart failure. J

retinal pigment epithelial cells: a possible mechanism for RPE aging and ge-related

Reaction. Science, 252: 1643-1651.

Carcinogenesis, 23: 1979- 2004.

and/or ethanol digestion. FASEB J, 12: 753-758.

Research, 2:1-9.

66.

1775.

172.

2009).

1-9.

1213-1216.

Mol Cell Cardiol, 33: 1065-1089.

434: 161- 176.

6. Running of %50 template and nontemplate controls in PCR (50% template control should given a 50% reduction of the amplification signal -values between 40%-60% reduction are acceptable-. The nontemplate control would detect contamination with spurious DNA or PCR products)

We detected mtDNA damage that originated by the genotoxic agents, oxidative stress and age, above mentioned conditions in our various studies (23,24,32). Also, QPCR method is suitable for the nutritional studies and some cancer researches.

#### **6. References**


6. Running of %50 template and nontemplate controls in PCR (50% template control should given a 50% reduction of the amplification signal -values between 40%-60% reduction are acceptable-. The nontemplate control would detect contamination with

We detected mtDNA damage that originated by the genotoxic agents, oxidative stress and age, above mentioned conditions in our various studies (23,24,32). Also, QPCR method is

[1] Saiki RK, Amplification of genomic DNA. PCR Protocols: a guide to methods and

[2] Wong A, Cortopassi G, Reproducible QPCR of mitochondrial and nuclear DNA copy

[3] Ginzinger DG, 2002. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Experimental Hematology, 30(6): 503-512. [4] Santos JH, Mandavilli BS, Van Houten B, Measuring oxidative mtDNA damage and

[5] Govan HL, Valles-Ayoub Y, Braun J, 1990. Fine-mapping of DNA damage and repair in specific genomic segments. Nucleic Acids Research, 18 (13):3823-3830. [6] Kalinowski DP, Illenye S, Van Houten B, 1992. Analysis of DNA damage and repair in

[7] Grimaldi KA, Bingham JP, Souhami RL, Hartley JA, 1994. DNA damage by anticancer

[8] Van Houten B, Cheng S, Chen Y, 2000. Measuring gene spesific nucleotide excision

[9] Ayala-Torres S, Chen Y, Suoblada T, Rosenblatt J, Van Houten B, 2000. Analysis of gene spesific DNA damage and repair using QPCR. Methods, 22: 135-147. [10] Yakes FM, Van Houten B, 1997. Mitochondrial DNA damage is more extensive and

[11] Rengarajan K, Cristol SM, Mehta M, Nickerson JM, 2002. Quantifying DNA

[12] Venkatraman A, Landar A, Davis AJ, Chamlee L, Sandersoni T, Kim H, Page G,

nanogram quantities of DNA. Mutat Res, 25; 460(2):81-94.

(eds.Copeland). pp.129-149, Humana Press Inc, Totawa, NJ. 2002.

applications (eds. Innis, Gelfand, Sninsky, White). Academic Press, California.

number using the LightCycler. Mitochondrial DNA methods and Protocols

repair using QPCR. Mitochondrial DNA methods and Protocols (eds.Copeland).

murine leukemia L1210 cells using a QPCR assay. Nucleic Acids Research,

reagents and its repair: mapping in cells in the subgene level with QPCR reaction.

repair in human cells using quantitative amplification of long targets from

persists longer than nuclear DNA damage in human cells following oxidative stres.

concentrations using fluorimetry: A comparison of fluorophores. Molecular Vision,

Pompilius M, Ballinger S, Darley-UsmarV, Bailey SM, 2004. Modification of the mitochondrial proteome in response to the stres of ethanol-dependent hepatoxicity.

spurious DNA or PCR products)

**6. References** 

1990.

20(13):3485-3494.

8: 416-421.

Anal Biochem, 222(1):236-242.

Proc Natl Acad Sci USA, 94: 514-519.

J Biol Chem, 279: 22092-22101.

suitable for the nutritional studies and some cancer researches.

pp.159-176, Humana Press Inc, Totawa, NJ. 2002.


**15** 

**Detection of** *Apple Chlorotic Leaf Spot Virus*

*Department of Horticultural, Agricultural College of Shihezi University, Shihezi* 

*Apple chlorotic leaf spot virus* (ACLSV) is the type member of the Trichovirus genus, the family *Flexiviridae* (Martelli et al., 1994; Adams et al., 2004) and is known to infect most pome and stone fruit tree species, including apple, peach, pear, plum, almond, cherry and apricot (Lister, 1970; Németh, 1986). ACLSV has a worldwide distribution and induces a large variety of symptoms in sensitive fruit trees (Németh, 1986; Dunez & Delbos, 1988; Desvignes & Boyé, 1989). However, In Japan, this virus is one of the causative agents of topworking disease and induces lethal decline in apple trees grown on Maruba kaido (Malus prunifolia var. ringo) rootstocks (Yanase, 1974). Other severe symptoms of stone fruit trees in Europe caused by ACLSV including bark split and pseudopox in plum, bark split in cherry, pseudopox and graft incompatibility in apricot and ring pattern mosaic in pear (Dunez et al., 1972; Desvignes & Boyé, 1989; Cieślińska et al., 1995; Jelkmann & Kunze, 1995). ACLSV has very flexuous filamentous particles, approximately 640 to 760 nm in length and consisting of a single-stranded positive-sense RNA with Mr of 2·48 x 106 and

*In situ* detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, *in situ* detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. In certain cases, they also can provide increased specificity and more rapid analyses. *In situ* reverse transcription polymerase chain reaction (RT-PCR) is a molecular biological-cytological method. *In situ* RT-PCR combined the sensitiveness of PCR amplification with spatial localization of products to monitor the appearance of specific transcripts in the tissue sections. Therefore, *in situ* RT-PCR defined a powerful tool for the low abundance transcript detection (Pesquet et al., 2004). Hasse et al. (1990) first reported the *in situ* PCR technology, which combined the strong points of PCR and *in situ* hybridization. It was widely used for all kinds of disease and genetic studies in human and animal (Gressens & Martin, 1994; Staskus et al., 1991; Nuovo et al., 1991; Bagasra et al., 1992; Cohen, 1996; Chen & Fuggle, 1993; Höfler et al., 1995). The first application of *in situ* RT-PCR for the plant tissue was reported by Woo et al. (1995). Most recently, this

multiple copies of a 22 kDa coat protein (CP) (Yoshikawa & Takahashi, 1988).

**1. Introduction** 

 \*

Corresponding Author

**in Tissues of Pear Using** *In Situ* **RT-PCR** 

**and Primed** *In Situ* **Labeling** 

Na Liu, Jianxin Niu\* and Ying Zhao

*People's Republic of China* 


## **Detection of** *Apple Chlorotic Leaf Spot Virus* **in Tissues of Pear Using** *In Situ* **RT-PCR and Primed** *In Situ* **Labeling**

Na Liu, Jianxin Niu\* and Ying Zhao *Department of Horticultural, Agricultural College of Shihezi University, Shihezi People's Republic of China* 

## **1. Introduction**

294 Polymerase Chain Reaction

[30] Alvarez P, Alvarado C, Puerto M, Schlumberger A, Jimenez L and De la Fuente M,

[31] Adom KK, Sorrells ME and Liu RH, 2005. Phytochemicals and antioxidant activity of milled fractions of different wheat varieties. J Agric Food Chem, 53: 2297-2306. [32] Mutlu AG, 2011. Bugday embriyosunca zengin bir diyetin, Drosophila'nn yaslanma

921.

2006. Improvement of leucocyte functions in prematurely aging mice after five weeks of diet supplementation with polyphenol-rich cereals. Nutrition, 22: 913-

surecinde, oksidatif mtDNA hasar, mtDNA kopya says, ve antioksidan enzim aktiviteleri üzerine etkileri. Türkiye Klinikleri Tp Bilimleri Dergisi, 31(6): 132.

> *Apple chlorotic leaf spot virus* (ACLSV) is the type member of the Trichovirus genus, the family *Flexiviridae* (Martelli et al., 1994; Adams et al., 2004) and is known to infect most pome and stone fruit tree species, including apple, peach, pear, plum, almond, cherry and apricot (Lister, 1970; Németh, 1986). ACLSV has a worldwide distribution and induces a large variety of symptoms in sensitive fruit trees (Németh, 1986; Dunez & Delbos, 1988; Desvignes & Boyé, 1989). However, In Japan, this virus is one of the causative agents of topworking disease and induces lethal decline in apple trees grown on Maruba kaido (Malus prunifolia var. ringo) rootstocks (Yanase, 1974). Other severe symptoms of stone fruit trees in Europe caused by ACLSV including bark split and pseudopox in plum, bark split in cherry, pseudopox and graft incompatibility in apricot and ring pattern mosaic in pear (Dunez et al., 1972; Desvignes & Boyé, 1989; Cieślińska et al., 1995; Jelkmann & Kunze, 1995). ACLSV has very flexuous filamentous particles, approximately 640 to 760 nm in length and consisting of a single-stranded positive-sense RNA with Mr of 2·48 x 106 and multiple copies of a 22 kDa coat protein (CP) (Yoshikawa & Takahashi, 1988).

> *In situ* detection techniques allow specific nucleic acid sequences to be exposed in morphologically preserved tissue sections. In combination with immunocytochemistry, *in situ* detection can relate microscopic topological information to gene activity at the transcript or protein levels in specific tissues. In certain cases, they also can provide increased specificity and more rapid analyses. *In situ* reverse transcription polymerase chain reaction (RT-PCR) is a molecular biological-cytological method. *In situ* RT-PCR combined the sensitiveness of PCR amplification with spatial localization of products to monitor the appearance of specific transcripts in the tissue sections. Therefore, *in situ* RT-PCR defined a powerful tool for the low abundance transcript detection (Pesquet et al., 2004). Hasse et al. (1990) first reported the *in situ* PCR technology, which combined the strong points of PCR and *in situ* hybridization. It was widely used for all kinds of disease and genetic studies in human and animal (Gressens & Martin, 1994; Staskus et al., 1991; Nuovo et al., 1991; Bagasra et al., 1992; Cohen, 1996; Chen & Fuggle, 1993; Höfler et al., 1995). The first application of *in situ* RT-PCR for the plant tissue was reported by Woo et al. (1995). Most recently, this

<sup>\*</sup> Corresponding Author

Detection of *Apple Chlorotic Leaf Spot Virus*

Table 1. Oligonucleotide primers used to PRINS

it immediately by electrophoresis or stored at – 20℃.

incubated at 42℃ for 1 h.

**3.1 Total RNA extraction and RT-PCR** 

targets.

**3. Methods** 

in Tissues of Pear Using *In Situ* RT-PCR and Primed *In Situ* Labeling 297

Blast search of the primer sequences showed that they were specific for their intended

Total RNAs were extracted from phloem infected by ACLSV. The 200 mg fresh Pear phloem tissue were grinded in liquide nitrogen for a fine powder and transferred to a 1.5 mL eppendorf tube which has added 800 μL extraction buffer (50 mmol·L-1 Tris-Cl pH 8.0, 140 mmol·L-1 NaCl, 10 mmol·L-1 EDTA, 4% SDS, 3% PVP, 15% ethanol, 5% β-mercaptoethanol), well mixed by invertion of the tube. Added 500 μL Tris-saturated phenol (pH 8.0): chloroform: isoamyl alcohol (25: 24: 1) to the tube, sepaeated by centrifugation at 12 000 rpm for 15 min at 4℃. Transferred the supernatant by hand-suction to a fresh tube and mixed with an equal volume of Tris-saturated phenol (pH 8.0): chloroform: isoamyl alcohol (25: 24: 1), followed by centrifugation at 12 000 rpm at 4 ℃ for 15 min. The supernatant was transferred to a fresh tube and mixed with an equal volume of chloroform: isoamyl alcohol (24: 1) and then centrifugation at 12 000 rpm at 4℃ for 10 min. Transferred the supernatant to a fresh tube and added 2.0 volumes of LiCl. Precipitated at –20℃ for 2-3 h. RNA was separated by centrifugation at 12 000 rpm for 15 min at 4°C. Removed the supernatant by hand-suction, washed the pellet two times by 70% ethanol, air-dry at room temperature. Suspended the pellet in 20-30 μL of TE solution or DEPC-treated sterile water and analysed

The reverse transcription mixture contained 1.0 μL specific reverse primer and 5.0 μL of total RNA and 9.5 μL of ddH2O. The mixture was kept at 70℃ for 5 min, and then immediately transferred to ice for 5 min. Then 2.5 μL of dNTPs (10 mM each), 5.0 μL of 5×M-MLV buffer, 1.0 μL of RNasin ribonuclease inhibitor (40 U·μL-1), 1.0 μL of M-MLV reverse transcriptase (200 U·μL-1) and made the total volume of 25.0 μL. The mixture was

Primer Primer Sequence (5'-3') Annealing Temp (℃)

acls Pa 1 CTTTACGAGCCCATTTCTTGCC 61.5 acls Ps 1 GAACATAGCGATACAGGGGACC 60.3 acls Pa 2 TGCCTCACACACTTGGCGGAG 60.6 acls Ps 2 CGATACAGGGGACCTCGGAAC 61.5 acls Pa 3 GCCTTTACGAGCCCATTTCTTG 59.5 acls Ps 3 AGGGGACCTCGGAACAAACAG 60.5 acls Pa 4 GTACAAAAGAGGTTTGTGAAG 54.2 acls Ps 4 GTGCTGGTGGAGGTGAAATC 57.4 acls Pa 5 CAATCTGAAGGAGGTAGTCGGT 56.4 acls Ps 5 TTCAGGCGTAGTAGAAAAGAGG 57.7

method had not been used to a large extent in plants (Greer et al., 1991; Johansen, 1997; Matsuda et al., 1997).

The primed *in situ* labeling (PRINS) procedure is a fast and efficient alternative to conventional fluorescence *in situ* hybridization for nucleic acid detection. According to the PRINS method, laboratory-synthesized oligonucleotide probes are used instead of cloned DNA for the *in situ* localization of individual genes. The PRINS primers are annealed to complementary target sequences on tissues and are extended in the presence of labeled nucleotides (Koch et al., 1995) utilizing *Taq* DNA polymerase. Since its introduction, the PRINS protocol has been continuously optimized, and numerous applications have been developed (Thomas et al., 2001; Yan et al., 2001; Xu et al., 2002; Tharapel & Wachtel, 2006a, 2006b; Wachtel & Tharapel, 2006; Kaczmarek et al., 2007). The technique has thus proved to be a useful tool for *in situ* screening, and has become a simple and efficient complement to conventional and molecular cytogenetic methods.

In this paper, we optimized the *in situ* RT-PCR and PRINS method for increased sensitivity to localize the virus in plant tissues with ACLSV. Based on this research, through observing distribution of amplified cDNA in tissues, we can analysis the virus infection. In this way, it can provide a new approach to detection virus in fruit trees, as well as investigate the formation, distribution and transformation of virus and produce innocuity fruit trees.

## **2. Materials**

#### **2.1 Virus sources**

Leaves were collected from Korla pear in Shayidong commercial orchard of Korla, Xinjiang, China. Virus-free healthy leaves were used as negative controls.

#### **2.2 Reagents and enzymes**

*Taq* DNA Polymerase, dNTPs, dATP, dGTP, dCTP, dTTP, PMD19-T were all purchased from TakaRa (China); M-MLV Reverse Transcriptase, T4 DNA ligase were from Fermentas (USA); TIANprep Mini Plasmid Kit and TIANgel Midi purification Kit were from TIANGEN (China); SuperScript II RNase H-Reverse Transcriptase were from Invitrogen (EU); Proteinase K were from Merk (Germany); Digoxigenin-11- dUTP, alkaline phosphatase labeled antidigoxin, anti-digoxin- fluorescence, Ribonuclease inhibitor, DNaseI were purchased from ROCH (USA); Nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro- 3-indolylphosphate (BCIP) were purchased from Shanghai Sangon (China); others were all analysis purity made in China. *E. coli* DH5α as preserved strains were stored at Biotechnology Laboratory of Horticultural Department, Agriculture College, Shihezi University, China.

### **2.3 Primer design**

The sequences were amplified by *in situ* RT-PCR reaction with specific primers, which were designed according to the cDNA sequence of ACLSV (Sato et al., 1993). Primer sequences are as follows: forward primer (P3) 5′-GGCAACCCTGGAACAGA-3′ and the reverse primer (P4) 5′-CAGACCCTTATTGAAG TCGAA-3′.

The sequences were amplified by PRINS reaction with specific primers, which were designed according to the cDNA sequence of ACLSV from GenBank D14996 (Table 1). A


Blast search of the primer sequences showed that they were specific for their intended targets.

Table 1. Oligonucleotide primers used to PRINS

## **3. Methods**

296 Polymerase Chain Reaction

method had not been used to a large extent in plants (Greer et al., 1991; Johansen, 1997;

The primed *in situ* labeling (PRINS) procedure is a fast and efficient alternative to conventional fluorescence *in situ* hybridization for nucleic acid detection. According to the PRINS method, laboratory-synthesized oligonucleotide probes are used instead of cloned DNA for the *in situ* localization of individual genes. The PRINS primers are annealed to complementary target sequences on tissues and are extended in the presence of labeled nucleotides (Koch et al., 1995) utilizing *Taq* DNA polymerase. Since its introduction, the PRINS protocol has been continuously optimized, and numerous applications have been developed (Thomas et al., 2001; Yan et al., 2001; Xu et al., 2002; Tharapel & Wachtel, 2006a, 2006b; Wachtel & Tharapel, 2006; Kaczmarek et al., 2007). The technique has thus proved to be a useful tool for *in situ* screening, and has become a simple and efficient complement to

In this paper, we optimized the *in situ* RT-PCR and PRINS method for increased sensitivity to localize the virus in plant tissues with ACLSV. Based on this research, through observing distribution of amplified cDNA in tissues, we can analysis the virus infection. In this way, it can provide a new approach to detection virus in fruit trees, as well as investigate the formation, distribution and transformation of virus and produce innocuity fruit trees.

Leaves were collected from Korla pear in Shayidong commercial orchard of Korla, Xinjiang,

*Taq* DNA Polymerase, dNTPs, dATP, dGTP, dCTP, dTTP, PMD19-T were all purchased from TakaRa (China); M-MLV Reverse Transcriptase, T4 DNA ligase were from Fermentas (USA); TIANprep Mini Plasmid Kit and TIANgel Midi purification Kit were from TIANGEN (China); SuperScript II RNase H-Reverse Transcriptase were from Invitrogen (EU); Proteinase K were from Merk (Germany); Digoxigenin-11- dUTP, alkaline phosphatase labeled antidigoxin, anti-digoxin- fluorescence, Ribonuclease inhibitor, DNaseI were purchased from ROCH (USA); Nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro- 3-indolylphosphate (BCIP) were purchased from Shanghai Sangon (China); others were all analysis purity made in China. *E. coli* DH5α as preserved strains were stored at Biotechnology Laboratory of

The sequences were amplified by *in situ* RT-PCR reaction with specific primers, which were designed according to the cDNA sequence of ACLSV (Sato et al., 1993). Primer sequences are as follows: forward primer (P3) 5′-GGCAACCCTGGAACAGA-3′ and the reverse primer

The sequences were amplified by PRINS reaction with specific primers, which were designed according to the cDNA sequence of ACLSV from GenBank D14996 (Table 1). A

Matsuda et al., 1997).

**2. Materials** 

**2.1 Virus sources** 

**2.3 Primer design** 

(P4) 5′-CAGACCCTTATTGAAG TCGAA-3′.

**2.2 Reagents and enzymes** 

conventional and molecular cytogenetic methods.

China. Virus-free healthy leaves were used as negative controls.

Horticultural Department, Agriculture College, Shihezi University, China.

### **3.1 Total RNA extraction and RT-PCR**

Total RNAs were extracted from phloem infected by ACLSV. The 200 mg fresh Pear phloem tissue were grinded in liquide nitrogen for a fine powder and transferred to a 1.5 mL eppendorf tube which has added 800 μL extraction buffer (50 mmol·L-1 Tris-Cl pH 8.0, 140 mmol·L-1 NaCl, 10 mmol·L-1 EDTA, 4% SDS, 3% PVP, 15% ethanol, 5% β-mercaptoethanol), well mixed by invertion of the tube. Added 500 μL Tris-saturated phenol (pH 8.0): chloroform: isoamyl alcohol (25: 24: 1) to the tube, sepaeated by centrifugation at 12 000 rpm for 15 min at 4℃. Transferred the supernatant by hand-suction to a fresh tube and mixed with an equal volume of Tris-saturated phenol (pH 8.0): chloroform: isoamyl alcohol (25: 24: 1), followed by centrifugation at 12 000 rpm at 4 ℃ for 15 min. The supernatant was transferred to a fresh tube and mixed with an equal volume of chloroform: isoamyl alcohol (24: 1) and then centrifugation at 12 000 rpm at 4℃ for 10 min. Transferred the supernatant to a fresh tube and added 2.0 volumes of LiCl. Precipitated at –20℃ for 2-3 h. RNA was separated by centrifugation at 12 000 rpm for 15 min at 4°C. Removed the supernatant by hand-suction, washed the pellet two times by 70% ethanol, air-dry at room temperature. Suspended the pellet in 20-30 μL of TE solution or DEPC-treated sterile water and analysed it immediately by electrophoresis or stored at – 20℃.

The reverse transcription mixture contained 1.0 μL specific reverse primer and 5.0 μL of total RNA and 9.5 μL of ddH2O. The mixture was kept at 70℃ for 5 min, and then immediately transferred to ice for 5 min. Then 2.5 μL of dNTPs (10 mM each), 5.0 μL of 5×M-MLV buffer, 1.0 μL of RNasin ribonuclease inhibitor (40 U·μL-1), 1.0 μL of M-MLV reverse transcriptase (200 U·μL-1) and made the total volume of 25.0 μL. The mixture was incubated at 42℃ for 1 h.

Detection of *Apple Chlorotic Leaf Spot Virus*

in Tissues of Pear Using *In Situ* RT-PCR and Primed *In Situ* Labeling 299

2. Proteinase K treatment: Added 1 µg·mL-1 Proteinase K digested 10-45 min at 37℃, stopped reaction by washings for 5 min in PBS buffer and transferred to DEPC-treated

3. DNaseI treatment: For each slide, 4.0 µL 10×DNase I buffer, 4.0 µL DNase I (10 U·µL-1), 1.0 µL Ribonuclease inhibitor (40 U·µL-1) and DEPC water added to 20.0 µL in a 0.5 mL microtube. Applied the reaction solution onto the slide and put it into humidified

4. Wash the slide two times in DEPC-treated sterile water for 5 min each and in alcohol for

For each slide, 4.0 µL 5×Frist-Strand Buffer (MgCl2+ 15 mM), 2.0 µL dNTPs (10 mM each), 1.0 µL RNasin (40 U·µL-1), 1.0 µL Antisense primer (20 µM), 2.0 µL DDT (0.1 M), 1.0 µL SuperScript II RT (200 U·µL-1), and DEPC water added to 20.0 µL in a 0.5 mL microtube. Applied the reaction solution onto the slide and put it into a humidified chamber and incubated at 42℃ for 1 h, then inactived at 92℃ for 1 min. Washed the slide two times for 5

The reaction was consisted of 2.5 µL 10 × PCR buffer (Mg2+ free), 0.5 µL dNTP (10 mmol·µL-1), 1.0 µL each primer (20 pmol·µL-1), 2.5 µL Dig-11-dUTP (1 nmol·µL-1), 1.0 µL *Taq* DNA polymerase (2.5 U·µL-1) and distilled water to 25.0 µL. Mounted the slide with genic frame, added the reaction solution, and covered the slide with a cover slip ,then put the slide on the flate bloke of the thermocycler. Cycling parameters consisted of 94℃ for 3 min, 94℃ for 2 min and 35 cycles of a two-step PCR with an annealing temperature of 56℃ for 1 min. Removed the cover slip and inactivated at 94℃ for 2 min. Washed the slid two times for 10 min each in washing buffer with gentle shaking. Several slides were used as negative controls for each *in situ* RT-PCR experiment. One slide was healthy plant, the other slides

1. Mounted the slide with 100 µL blocking buffer (100 mmol·L-1 Tris-HCl, pH 7.5, 150 mmol·L-1 NaCl, and 3% BSA). Incubated the slide in a humidified chamber at 37℃ for

2. Added anti-Dig-alkaline phosphatase (1: 100 in blocking buffer), and incubated the

3. Stopped the reaction by rinsing the slide with washing buffer (100 mmol·L-1 Tris-HCl, pH 7.5, 150 mmol·L-1 NaCl) two times for 10 min each at room temperature with gentle

4. Developed the color reaction by adding 100 µL of NBT/BCIP solution to the slide and incubated the slide in a humidified chamber for 60 min in the dark at room

completely removed, then left the slide at room temperature for air-dry.

sterile water for 5 min at room temperature, then air-dry.

were amplified without primers, *Taq* DNA polymerase, or RT step.

slide in a humidified chamber for 30 min at room temperature.

temperature. Then rinsed the slide with water to stop the reaction.

30min. Drained the blocking buffer from the slide.

chamber and incubated at 37℃ overnight.

5 min at room temperature.

**3.6** *In situ* **RT-PCR detection 3.6.1** *In situ* **RT-PCR reaction** 

**3.6.2 Immunoenzymatic detection** 

shaking.

**3.5** *In situ* **reverse transcription reaction** 

min each in distilled water at room temperature.

and transferred to ethanol for 5 min, repeated more times until the paraffin was

PCR reaction volumes were 20.0 μL, and contained 2.0 μL of 10×PCR buffer, 0.5 μL of dNTPs (each 10 mM), 2.0 μL of primers, 2.0 μL of cDNA, 0.2 μL (5U·μL-1) *Tap* DNA polymerase and 13.3 μL of ddH2O. PCR was carried out with an initial denaturation of 4 min at 94℃, followed by 35 cycles of 30s, 94℃; 30s, 55℃; 1 min, 72℃; and then by a final elongation step of 7 min at 72℃.

## **3.2 Cloning and sequencing**

The amplified PCR products were gel purified and extracted using TIANgel Midi Purification Kit (TIANGEN, China). The purified DNA fragments were ligated into the PMD19-T vector (TaKaRa Biotechnology, China) following the manufacturer's instruction, and used to transform *E. coli* DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion before sequencing. Two clones from independent PCR reactions were sequenced from both directions.

## **3.3 Tissue embedding and preparation of slide**


## **3.4 Pretreatment of slides**

1. De-waxed: Removed the slides from the refrigerator, put the slide into the oven incubated for 1-3 h, at 60℃ in order to melt paraffin. Rinsed the slide in xylene for 5 min and transferred to ethanol for 5 min, repeated more times until the paraffin was completely removed, then left the slide at room temperature for air-dry.


## **3.5** *In situ* **reverse transcription reaction**

For each slide, 4.0 µL 5×Frist-Strand Buffer (MgCl2+ 15 mM), 2.0 µL dNTPs (10 mM each), 1.0 µL RNasin (40 U·µL-1), 1.0 µL Antisense primer (20 µM), 2.0 µL DDT (0.1 M), 1.0 µL SuperScript II RT (200 U·µL-1), and DEPC water added to 20.0 µL in a 0.5 mL microtube. Applied the reaction solution onto the slide and put it into a humidified chamber and incubated at 42℃ for 1 h, then inactived at 92℃ for 1 min. Washed the slide two times for 5 min each in distilled water at room temperature.

## **3.6** *In situ* **RT-PCR detection**

298 Polymerase Chain Reaction

PCR reaction volumes were 20.0 μL, and contained 2.0 μL of 10×PCR buffer, 0.5 μL of dNTPs (each 10 mM), 2.0 μL of primers, 2.0 μL of cDNA, 0.2 μL (5U·μL-1) *Tap* DNA polymerase and 13.3 μL of ddH2O. PCR was carried out with an initial denaturation of 4 min at 94℃, followed by 35 cycles of 30s, 94℃; 30s, 55℃; 1 min, 72℃; and then by a final

The amplified PCR products were gel purified and extracted using TIANgel Midi Purification Kit (TIANGEN, China). The purified DNA fragments were ligated into the PMD19-T vector (TaKaRa Biotechnology, China) following the manufacturer's instruction, and used to transform *E. coli* DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion before sequencing. Two clones from independent PCR

1. Slide disposal: After rinsed, ultrasonic cleaned and high temperature baked, the slide must be pre-prepared with poly-L-lysine for 5 min, and then incubated it at 26℃

2. Tissues fixation: Leaves were cut into small pieces (3×2 mm) and rinsed the tissues in 4% paraformaldehyde immediately for 1h at room temperature with gentle shaking. 3. Dehydration: Washed the tissues in PBS buffer two times (5 min each), immersed the tissues in series of concentration of ethanol (50%, 70%, 85%, 95% and 100%) for 1h,

4. Transparences: Put the tissues into pure alcohol: xylene (1: 1) and pure xylene for 1 h,

5. Low-temperature wax infiltration: Put the tissues into the container which contained transparence and paraffin, covered the container with lid, and incubated at 38℃

6. High-temperature wax infiltration: Removed the lid, and put the container into incubator at 58℃, and then changed the pure paraffin three times for 2 h each. 7. Paraffin-embedding: Pour melted paraffin wax to pre-folded carton for embedding. 8. Sectioning: Tissue sections (2-16 μm) were obtained by a conventional rotary microtome. If very thin sections were required, a retracting rotary microtome should be used to avoid the compression of the tissue block by the up-stroke of the knife and

9. Stretched section: Wax sections needed to be stretched before adhesion to the glass slide. Sections were lifted onto a layer of de-gassed water on a slide held on a warmed flat plate (45℃). Once the sections was stretched, drained away the excess water and left the slide into incubator at 40℃, overnight, the section has dried onto the slide, stored at

1. De-waxed: Removed the slides from the refrigerator, put the slide into the oven incubated for 1-3 h, at 60℃ in order to melt paraffin. Rinsed the slide in xylene for 5 min

sections should be mounted onto poly-L-lysine-coated pre-prepared slides.

overnight, sealed and stored at room temperature for use within 10 d.

elongation step of 7 min at 72℃.

**3.2 Cloning and sequencing** 

reactions were sequenced from both directions.

respectively, at room temperature.

respectively, at room temperature.

overnight.


**3.4 Pretreatment of slides** 

**3.3 Tissue embedding and preparation of slide** 

## **3.6.1** *In situ* **RT-PCR reaction**

The reaction was consisted of 2.5 µL 10 × PCR buffer (Mg2+ free), 0.5 µL dNTP (10 mmol·µL-1), 1.0 µL each primer (20 pmol·µL-1), 2.5 µL Dig-11-dUTP (1 nmol·µL-1), 1.0 µL *Taq* DNA polymerase (2.5 U·µL-1) and distilled water to 25.0 µL. Mounted the slide with genic frame, added the reaction solution, and covered the slide with a cover slip ,then put the slide on the flate bloke of the thermocycler. Cycling parameters consisted of 94℃ for 3 min, 94℃ for 2 min and 35 cycles of a two-step PCR with an annealing temperature of 56℃ for 1 min. Removed the cover slip and inactivated at 94℃ for 2 min. Washed the slid two times for 10 min each in washing buffer with gentle shaking. Several slides were used as negative controls for each *in situ* RT-PCR experiment. One slide was healthy plant, the other slides were amplified without primers, *Taq* DNA polymerase, or RT step.

### **3.6.2 Immunoenzymatic detection**


Detection of *Apple Chlorotic Leaf Spot Virus*

**4.1 Detection ACLSV by RT-PCR** 

Fig. 1. The productions of RT-PCR of ACLSV M: Marker; 1-4: productions; 5: negative control

showed that sections were not stained.

treatment 20 min was more moderate.

**4.3 The effect of treatment with proteinase K** 

**4.4 The effect of RT-component concentration** 

analysis system.

**4. Results** 

in Tissues of Pear Using *In Situ* RT-PCR and Primed *In Situ* Labeling 301

DAPI/FITC/Rhodamine, AxioCam Camera module and Video Test-FISH 4.0 image

Total RNA were extract from the phloem of pear which were infected with ACLSV, first strand cDNA synthesis was obtained by reverse transcription using specific primer and 358 bp fragment was amplified by P3/P4 primers as shown in Figure. 1. The purified DNA fragments were ligated into the PMD19-T vector and transformed into *E. coli* DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion before sequencing.

**4.2 Detection the reliability of alkaline phosphatase chromogenic system** 

The slide were digested by 1µg·mL-1 Proteinase K for 20 min at 37℃, and incubated at 37℃ overnight with DNase I. Washed the slide two times for 10 min each in PBS buffer. Mounted the slide with blocking buffer and incubated at 37℃ for 30min. Added anti-Dig-alkaline phosphatase (1:100 in blocking buffer) and incubated the slide in a moist chamber for 60 min at room temperature, then washed the slide two times for 10 min each in PBS buffer at room temperature with gentle shaking. Added NBT/BCIP solution to the slide and incubated the slide in a humidified chamber for 60 min in the dark at room temperature. The result

After treated with Proteinase K treatment for 10 min or 15 min, the organization performed a piece of blue, which indicated that Proteinase K digested inadequately. Morphology was fuzzy when digested for 30 min or 40 min, illustrating excessive digestion. Proteinase K

The results showed there was no signal when RNasin was less than 0.2 U·µL-1, and it was enhanced with the increased RNasin. The concentration of dNTPs was above 0.4 mmol·L-1, the signal was appeared; the concentration of SuperScript II ranged from 0.1U·µL-1 to 1.3


## **3.7 PRINS detection**

## **3.7.1 PRINS reaction**


## **3.7.2 Visualization of PRINS products**


## **3.7.3 Signal detection and image analysis**

Olympus BX51 fluorescence microscope system was adopted for this process. This system contained Olympus UPlanFI 100×/1.30 Oil ∞/0.17 C1field lens, pass band filter with DAPI/FITC/Rhodamine, AxioCam Camera module and Video Test-FISH 4.0 image analysis system.

## **4. Results**

300 Polymerase Chain Reaction

5. Rinsed the slide in series of concentration of ethanol, 50%, 70%, 85%, 95%, and 100% for

7. Covered the section with the cover slip using mounting solution, air-dry. Then the sections were ready for data recording, which could view under bright field microscopy

2. Denature the samples by immersing them in 70% formamide/2×SSC, at 72℃ for 2 min. 3. Dehydrate the slides in a series (70%, 90%, and 100%) of ice-cold ethanol washes (4℃)

4. Prepare reaction mixture in a final volume of 25.0 µL consisted of specific primers (20 µM) 10.0 µL, 0.1% BSA 2.5 μL, 0.2 mM dNTPs 2.5 μL (each), 0.02 mM dTTP 1.0 μL, 0.02 mM Dig-11-dUTP 3.0 μL, *Taq* buffer 2.5 μL, *Taq* DNA polymerase (2.5 U·μL-1) 1.0 μL and distilled water to 25.0 µL. Kept the mix on ice during preparation and until

5. Reaction mixture incubated at annealing temperature and incubated the denatured the slide for 7 min at annealing temperature. Applied the reaction mixture and covered the working area of the slide completely with a 22 × 22 cover slip on the denatured the

6. Set up the PRINS program and start the reaction. The program was carried out on a programmable thermal cycler equipped with a flat plate for slides. The program consisted of one cycle of 9 min at annealing temperature with an additional 30 min at

7. After extension, the slide was removed from cycler, the cover slip was removed, and the slide washed in NE solution (500 mM NaCl, 50 mM EDTA, pH 8.0) at 72℃ for 5 min, and transferred the slide to 4×SSC/0.2% Tween-20 at 50℃ for 5 min to stop the reaction.

1. For each slide, added 10 µg·mL-1 avidin-Rhodamine and 20 µg·mL-1 anti-digoxigenin-FITC. 2. Placed slides in a humidified chamber for 30 min at room temperature, worked in the

3. The slide was rinsed in preheated solutions (1×PBS/0.2%Tween-20, 37℃; 0.5×PBS/0.2% Tween-20, 37℃; 0.2×PBS/0.2%Tween-20, 37℃) for 5min, respectively, air-dried. 4. Mounted the slide with 3µg·mL-1 of DAPI/antifade solution under a 22×22 coverslip

5. Let the excess mounting medium dry. Approximately 1 h, permanently seal the slide

Olympus BX51 fluorescence microscope system was adopted for this process. This system contained Olympus UPlanFI 100×/1.30 Oil ∞/0.17 C1field lens, pass band filter with

slide, and then transferred to the heating block of the thermal.

dark as much as possible to avoid fluorescence bleaching.

with nail polish. Slide can be maintained at 4℃ until scored.

2 min, respectively, at room temperature for dehydration. 6. Put the slide into pure xylene for 3 min for transparent.

through stained with Alcian Blue.

1. Immersed slides in 0.02 N HCl for 20 min.

before allowing them to air-dry.

application to the slide.

72℃ for extension.

**3.7.2 Visualization of PRINS products** 

counterstained for 10min, in dark.

**3.7.3 Signal detection and image analysis** 

**3.7 PRINS detection 3.7.1 PRINS reaction** 

## **4.1 Detection ACLSV by RT-PCR**

Total RNA were extract from the phloem of pear which were infected with ACLSV, first strand cDNA synthesis was obtained by reverse transcription using specific primer and 358 bp fragment was amplified by P3/P4 primers as shown in Figure. 1. The purified DNA fragments were ligated into the PMD19-T vector and transformed into *E. coli* DH5α. The positive clones were confirmed by PCR and restriction enzyme digestion before sequencing.


Fig. 1. The productions of RT-PCR of ACLSV M: Marker; 1-4: productions; 5: negative control

## **4.2 Detection the reliability of alkaline phosphatase chromogenic system**

The slide were digested by 1µg·mL-1 Proteinase K for 20 min at 37℃, and incubated at 37℃ overnight with DNase I. Washed the slide two times for 10 min each in PBS buffer. Mounted the slide with blocking buffer and incubated at 37℃ for 30min. Added anti-Dig-alkaline phosphatase (1:100 in blocking buffer) and incubated the slide in a moist chamber for 60 min at room temperature, then washed the slide two times for 10 min each in PBS buffer at room temperature with gentle shaking. Added NBT/BCIP solution to the slide and incubated the slide in a humidified chamber for 60 min in the dark at room temperature. The result showed that sections were not stained.

## **4.3 The effect of treatment with proteinase K**

After treated with Proteinase K treatment for 10 min or 15 min, the organization performed a piece of blue, which indicated that Proteinase K digested inadequately. Morphology was fuzzy when digested for 30 min or 40 min, illustrating excessive digestion. Proteinase K treatment 20 min was more moderate.

## **4.4 The effect of RT-component concentration**

The results showed there was no signal when RNasin was less than 0.2 U·µL-1, and it was enhanced with the increased RNasin. The concentration of dNTPs was above 0.4 mmol·L-1, the signal was appeared; the concentration of SuperScript II ranged from 0.1U·µL-1 to 1.3

Detection of *Apple Chlorotic Leaf Spot Virus*

**4.6 PRINS-Rhodamine staining** 

enzyme).

**4.7 PRINS-FITC staining** 

in Tissues of Pear Using *In Situ* RT-PCR and Primed *In Situ* Labeling 303

synthesis (Figure. 2). The concentration of *Taq* DNA polymerase with 2 U·100µL-1-10 U·100µL-1 could satisfy amplification and showed stronger signals, which indicated that the

Applied PRINS-Rhodamine staining detected ACLSV showed that the infected leaves of pear tissues were presented red fluorescence positive signals (Fig. 4, A~D, arrows showing the locations), which were consistent with the results of *In situ* RT-PCR detection (Niu et al., 2007). Healthy leaves and infected leaves without SuperScript II RT, fluorescent antibody

**A B C D** 

**E F G H** 

suitable concentration of *Taq* DNA polymerase was 2 U·100µL-1 (Figure. 3).

and *Taq* DNA polymerase, did not present red fluorescence signals (Fig. 5, E~H).

Fig. 4. PRINS-Rhodamine staining results of ACLSV in pear tissues

polymerase, did not present red fluorescence signals (Fig. 2, E~H).

A-D: Labeled results of virus infected pear leaves from the same positions of different trees; E: Labeled results of healthy pear leave (control); F-H: PRINS-Rhodamine staining results of ACLSV in pear tissues (control: Left out of SuperScript II RT, fluorescence antibody, *Taq*

FITC fluorochrome was more sensitive to the temperature and pH, and the efficiency was lower than Rhodamine staining, and the results showed inconspicuous signals. Applied PRINS-FITC staining detected ACLSV showed that the infected leaves of pear tissues were presented green fluorescence positive signals (Fig. 5, A~D, arrows showing the locations), which were consistent with the results of *In situ* RT-PCR detection (Niu et al., 2007). Healthy leaves and infected leaves without SuperScript II RT, fluorescent antibody and *Taq* DNA

U·µL-1 and the signal was enhanced with the increase concentration of SuperScript II; the concentration of primers above 0.9 µmol·L-1 were effective, less than 0.8 µmol·L-1 could not synthesized sufficient quantities of cDNA and above 1.2 µmol·L-1 could produce non-specific product.

## **4.5 The effect of other factors**

The result showed that positive signals were appeared on the slide only when the annealing temperature at 56℃, which indicated that the suitable temperature was 56℃. Amplification with 10-20 cycles, the signals were not appeared, 25 cycles appeared weaker blue signal, 30- 35 cycles showed stronger signals, which demonstrated that fewer cycles led to lower

Fig. 2. The effect of cycle number on *In situ* RT-PCR A: 10 cycles; B: 15 cycles; C: 20 cycles; D: 25 cycles; E: 30 cycles

Fig. 3. The effect of the different *Taq* DNA polymerase concentration on the detection of *In situ* RT-PCR A: 2 U·100 µL-1; B: 4 U·100 µL-1; C: 6 U·100 µL-1; D: 8 U·100 µL-1; E: 10 U ·100 µL-1

synthesis (Figure. 2). The concentration of *Taq* DNA polymerase with 2 U·100µL-1-10 U·100µL-1 could satisfy amplification and showed stronger signals, which indicated that the suitable concentration of *Taq* DNA polymerase was 2 U·100µL-1 (Figure. 3).

## **4.6 PRINS-Rhodamine staining**

302 Polymerase Chain Reaction

U·µL-1 and the signal was enhanced with the increase concentration of SuperScript II; the concentration of primers above 0.9 µmol·L-1 were effective, less than 0.8 µmol·L-1 could not synthesized sufficient quantities of cDNA and above 1.2 µmol·L-1 could produce non-specific

The result showed that positive signals were appeared on the slide only when the annealing temperature at 56℃, which indicated that the suitable temperature was 56℃. Amplification with 10-20 cycles, the signals were not appeared, 25 cycles appeared weaker blue signal, 30- 35 cycles showed stronger signals, which demonstrated that fewer cycles led to lower

Fig. 3. The effect of the different *Taq* DNA polymerase concentration on the detection of *In* 

A: 2 U·100 µL-1; B: 4 U·100 µL-1; C: 6 U·100 µL-1; D: 8 U·100 µL-1; E: 10 U ·100 µL-1

product.

*situ* RT-PCR

**4.5 The effect of other factors** 

Fig. 2. The effect of cycle number on *In situ* RT-PCR

A: 10 cycles; B: 15 cycles; C: 20 cycles; D: 25 cycles; E: 30 cycles

Applied PRINS-Rhodamine staining detected ACLSV showed that the infected leaves of pear tissues were presented red fluorescence positive signals (Fig. 4, A~D, arrows showing the locations), which were consistent with the results of *In situ* RT-PCR detection (Niu et al., 2007). Healthy leaves and infected leaves without SuperScript II RT, fluorescent antibody and *Taq* DNA polymerase, did not present red fluorescence signals (Fig. 5, E~H).

Fig. 4. PRINS-Rhodamine staining results of ACLSV in pear tissues A-D: Labeled results of virus infected pear leaves from the same positions of different trees; E: Labeled results of healthy pear leave (control); F-H: PRINS-Rhodamine staining results of ACLSV in pear tissues (control: Left out of SuperScript II RT, fluorescence antibody, *Taq* enzyme).

### **4.7 PRINS-FITC staining**

FITC fluorochrome was more sensitive to the temperature and pH, and the efficiency was lower than Rhodamine staining, and the results showed inconspicuous signals. Applied PRINS-FITC staining detected ACLSV showed that the infected leaves of pear tissues were presented green fluorescence positive signals (Fig. 5, A~D, arrows showing the locations), which were consistent with the results of *In situ* RT-PCR detection (Niu et al., 2007). Healthy leaves and infected leaves without SuperScript II RT, fluorescent antibody and *Taq* DNA polymerase, did not present red fluorescence signals (Fig. 2, E~H).

Detection of *Apple Chlorotic Leaf Spot Virus*

ratio to 1: 3 generated strong signals.

**6. Conclusions** 

to reduce washing processing steps were necessary.

more extensive application prospects in plant virus detection.

primers for PRINS, and achieved clearly fluorescence signals.

in Tissues of Pear Using *In Situ* RT-PCR and Primed *In Situ* Labeling 305

However, too many primers would likely lead to primer-dimers or non-specific hybridization. In PRINS reaction system, primer extensions strictly followed the principle of complementary base pairing, and ensure the specificity labeling. Synthesis of labeled DNA will remain in the amplified position and not diffusion. In this study, we used five specific

Terkelsen et al., (1993) using repeated primed *in situ* labeling (repeated-PRINS). This change of strategy results in a localized accumulation of sequence-specific labeled DNA, resulting in up to a 15-fold amplification of the signal as compared to the standard PRINS method. Ni et al., (1998) results showed that the repeated-PRINS technology could to enhance the signal; however, repeated heat denaturation and extension process for long time which induced the cell loss normal forms. In our study, we pretreatment species with appropriate concentration of protease K, and the optimal time of proteinase K digestion was necessary. The tissues slices were treated with proteinase K for 10, 20, 30, and 45 min. The best results were achieved after 20 min of the proteinase K digestion. The morphology of the tissue was well retained, and interpretation of results was unambiguous. The signal was recognized as fluorescence-signals the site of the label. The 10 min durations turned out to be too short and led to lack of signal. The extension of the reaction time up to 45 min produced morphological distortions to the point that interpretation of results became impossible. In addition, our research showed that increasing the ratio of dTTP and labeled-dUTP could improve the signal intensity. In general, the ratio of dTTP and labeled-dUTP was 1: 1 could generate enough strong signals. We increased the dTTP and labeled-dUTP concentration

In this study, two fluorescence labeling were used, FITC and Rhodamine, respectively. Fluorescent-FITC was used *in situ* labeling showed sensitive on PH and easy to decay. In the conditions of susceptible pH or strong UV irradiation, the fluorescence excitation rapid decay. In addition, increase the times of washing, the tissues were more easily damaged and higher backgrounds were obtained. Therefore, on the basis of complete elution, appropriate

Primed *in situ* labeling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence *in situ* hybridization (FISH). PRINS is based on sequencespecific annealing of unlabelled oligonucleotide primer under stringent conditions to the DNA of denaturated. Compared to FISH, PRINS is faster and does not require preparation of labeled probes, the process costs much less in terms of reagents (Velagelati et al., 1998; Tharapel & Kadandale, 2002; Pellestor et al., 2002), and hybridization signal is stronger, more specific and easy to control. In addition, we believe that this modified PRINS technique can have very meaningful applications in molecular cytogenetics. It can be used for the visualization and mapping of genetic loci on chromosomes, and for detection of the presence or absence of small DNA segments involved in genetic diseases. PRINS will have a

ACLSV of leave sections of Korla Pear were detected by *in situ* RT-PCR and PRINS, and the positive materials were found obviously alcian blue and fluorescence signals in mesophyll cells. The results showed that *in situ* RT-PCR and PRINS, which had two staining methods

Fig. 5. PRINS-FITC staining results of ACLSV in pear tissues A-D: Labeled results of virus infected pear leaves from the same positions of different trees; E: Labeled results of healthy pear leave (control); F-H: PRINS- Rhodamine staining results of ACLSV in pear tissues (control: Left out of SuperScript II RT, fluorescence antibody, *Taq* enzyme).

## **5. Discussion**

The study is based on virus RNA as a template to reverse transcription cDNA and *in situ* amplification. Before amplification, the slides treatment with DNA exonuclease without RNA enzyme overnight digest the original genomic DNA in tissues which can eliminating DNA fragment decorated by polymerase which could form false-positive amplification (Long et al., 1993). In our studies, the known virus-free material of pear tree used as the negative control did not appear specificity of fluorescence signals. Negative control without SuperScript II RT, fluorescence antibody, *Taq* enzyme showed the same result of virus-free material. Signals did not display without RT steps indicated that the products were amplified by cDNA, which excluded the possible of experimental reagents cross produced fluorescent complex and attached to the tissue surface induced fluorescence signals. In our studies, ACLSV of leave sections of Korla Pear were detected by *in situ* RT-PCR and PRINS, the results showed that the positive materials were found obviously alcian blue and fluorescence signals in mesophyll cells, while the negative control tissue did not appear. It was indicated that ACLSV mainly distributed in the palisade tissue of the mesophyll cells, and the same results as *in situ* RT-PCR detection (Niu et al., 2007). In addition, the results showed that the thickness of section had a great influence on detection. Thin slices can easy to cause the tissues were not complete, and the cell of thick slices were multiple and overlapping, which unfavorable for observing, and seriously affect the detection results. So, in order to obtain desire results of detection, the 4-6 µm of sections were used.

Because of the *in situ* amplified cDNA in tissues, we must consider the number of primers to use. A single primer would not allow a strong enough signal for fluorescent detection.

**A B C D** 

**E F G H** 

A-D: Labeled results of virus infected pear leaves from the same positions of different trees; E: Labeled results of healthy pear leave (control); F-H: PRINS- Rhodamine staining results of ACLSV in pear tissues (control: Left out of SuperScript II RT, fluorescence antibody, *Taq*

The study is based on virus RNA as a template to reverse transcription cDNA and *in situ* amplification. Before amplification, the slides treatment with DNA exonuclease without RNA enzyme overnight digest the original genomic DNA in tissues which can eliminating DNA fragment decorated by polymerase which could form false-positive amplification (Long et al., 1993). In our studies, the known virus-free material of pear tree used as the negative control did not appear specificity of fluorescence signals. Negative control without SuperScript II RT, fluorescence antibody, *Taq* enzyme showed the same result of virus-free material. Signals did not display without RT steps indicated that the products were amplified by cDNA, which excluded the possible of experimental reagents cross produced fluorescent complex and attached to the tissue surface induced fluorescence signals. In our studies, ACLSV of leave sections of Korla Pear were detected by *in situ* RT-PCR and PRINS, the results showed that the positive materials were found obviously alcian blue and fluorescence signals in mesophyll cells, while the negative control tissue did not appear. It was indicated that ACLSV mainly distributed in the palisade tissue of the mesophyll cells, and the same results as *in situ* RT-PCR detection (Niu et al., 2007). In addition, the results showed that the thickness of section had a great influence on detection. Thin slices can easy to cause the tissues were not complete, and the cell of thick slices were multiple and overlapping, which unfavorable for observing, and seriously affect the detection results. So,

in order to obtain desire results of detection, the 4-6 µm of sections were used.

Because of the *in situ* amplified cDNA in tissues, we must consider the number of primers to use. A single primer would not allow a strong enough signal for fluorescent detection.

Fig. 5. PRINS-FITC staining results of ACLSV in pear tissues

enzyme).

**5. Discussion** 

However, too many primers would likely lead to primer-dimers or non-specific hybridization. In PRINS reaction system, primer extensions strictly followed the principle of complementary base pairing, and ensure the specificity labeling. Synthesis of labeled DNA will remain in the amplified position and not diffusion. In this study, we used five specific primers for PRINS, and achieved clearly fluorescence signals.

Terkelsen et al., (1993) using repeated primed *in situ* labeling (repeated-PRINS). This change of strategy results in a localized accumulation of sequence-specific labeled DNA, resulting in up to a 15-fold amplification of the signal as compared to the standard PRINS method. Ni et al., (1998) results showed that the repeated-PRINS technology could to enhance the signal; however, repeated heat denaturation and extension process for long time which induced the cell loss normal forms. In our study, we pretreatment species with appropriate concentration of protease K, and the optimal time of proteinase K digestion was necessary. The tissues slices were treated with proteinase K for 10, 20, 30, and 45 min. The best results were achieved after 20 min of the proteinase K digestion. The morphology of the tissue was well retained, and interpretation of results was unambiguous. The signal was recognized as fluorescence-signals the site of the label. The 10 min durations turned out to be too short and led to lack of signal. The extension of the reaction time up to 45 min produced morphological distortions to the point that interpretation of results became impossible. In addition, our research showed that increasing the ratio of dTTP and labeled-dUTP could improve the signal intensity. In general, the ratio of dTTP and labeled-dUTP was 1: 1 could generate enough strong signals. We increased the dTTP and labeled-dUTP concentration ratio to 1: 3 generated strong signals.

In this study, two fluorescence labeling were used, FITC and Rhodamine, respectively. Fluorescent-FITC was used *in situ* labeling showed sensitive on PH and easy to decay. In the conditions of susceptible pH or strong UV irradiation, the fluorescence excitation rapid decay. In addition, increase the times of washing, the tissues were more easily damaged and higher backgrounds were obtained. Therefore, on the basis of complete elution, appropriate to reduce washing processing steps were necessary.

Primed *in situ* labeling (PRINS) of nucleic acids was developed as an alternative to traditionally used fluorescence *in situ* hybridization (FISH). PRINS is based on sequencespecific annealing of unlabelled oligonucleotide primer under stringent conditions to the DNA of denaturated. Compared to FISH, PRINS is faster and does not require preparation of labeled probes, the process costs much less in terms of reagents (Velagelati et al., 1998; Tharapel & Kadandale, 2002; Pellestor et al., 2002), and hybridization signal is stronger, more specific and easy to control. In addition, we believe that this modified PRINS technique can have very meaningful applications in molecular cytogenetics. It can be used for the visualization and mapping of genetic loci on chromosomes, and for detection of the presence or absence of small DNA segments involved in genetic diseases. PRINS will have a more extensive application prospects in plant virus detection.

## **6. Conclusions**

ACLSV of leave sections of Korla Pear were detected by *in situ* RT-PCR and PRINS, and the positive materials were found obviously alcian blue and fluorescence signals in mesophyll cells. The results showed that *in situ* RT-PCR and PRINS, which had two staining methods

Detection of *Apple Chlorotic Leaf Spot Virus*

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## **7. Acknowledgements**

This stsudy was supported by National Natural Science Foundation of China (30360066), the National Key Technologies R&D Program of China (2003BA546C), the Foundation Science and Technology Commission Xinjiang Production and Construction Crops, China (NKB02SDXNK01 SW) and Natural Science and Technology Innovation of Shihezi University, China (ZRKX200707).

## **8. References**


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**7. Acknowledgements** 

**8. References** 

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**16** 

*1Cameroon 2,3France 4South Africa* 

**Application of PCR Technologies** 

Born Céline3, Aubouy Agnès4 and Nkenfou Céline1

 *and Management of HIV/AIDS (CIRCB), Yaoundé, 2Institut de Recherche pour le Developpement (IRD), UMR 216 (IRD/UPD) Faculté de Pharmacie, Paris, 3Institut de Recherche pour le Developpement (IRD), UMR 152, Université Paul Sabatier, Toulouse,* 

**from Central Africa** 

**to Humans, Animals, Plants and Pathogens** 

Ouwe Missi Oukem-Boyer Odile1, Migot-Nabias Florence2,

*1Chantal Biya International Reference Centre for Research on Prevention* 

*4University of Stellenbosch, Department of Botany and Zoology, Stellenbosch,* 

The Central African region, also called Atlantic Equatorial Africa, harbors one of the biggest worldwide biodiversity. It is true for human, with a great diversity of ethnic groups, but also for animals, plants, and microorganisms including pathogen species. Although this region is lagging behind in various domains, few research centers and laboratories have been able to develop sophisticated research work for diagnostics, fundamental research, and operational research, using polymerase chain reaction (PCR) techniques. This present paper intends to give an overview of the use of PCR technology in Central Africa and its various applications in the field of genetics, phylogeography, ecology, botany, and infectious diseases, which may have a broad impact on interspecies relationships, diagnostics of

We will successively describe the main research findings in humans, animals, plants and pathogens from Central Africa, and show how the PCR has allowed scientists from this region to contribute significantly to generalized knowledge in these fields. Then, we'll discuss opportunities and challenges in conducting such kind of research in these particular

Since the nineties, the extensive use of molecular techniques has contributed to deepen the knowledge on human genetics. In most studies related to Central Africa, such

**1. Introduction** 

**2. Humans** 

diseases, environment and biodiversity.

limited-resources settings before concluding this chapter.


## **Application of PCR Technologies to Humans, Animals, Plants and Pathogens from Central Africa**

Ouwe Missi Oukem-Boyer Odile1, Migot-Nabias Florence2, Born Céline3, Aubouy Agnès4 and Nkenfou Céline1 *1Chantal Biya International Reference Centre for Research on Prevention and Management of HIV/AIDS (CIRCB), Yaoundé, 2Institut de Recherche pour le Developpement (IRD), UMR 216 (IRD/UPD) Faculté de Pharmacie, Paris, 3Institut de Recherche pour le Developpement (IRD), UMR 152, Université Paul Sabatier, Toulouse, 4University of Stellenbosch, Department of Botany and Zoology, Stellenbosch, 1Cameroon 2,3France 4South Africa* 

## **1. Introduction**

308 Polymerase Chain Reaction

Pellestor, F.; Imbert, I. & Andréo, B. (2002a). Rapid chromosome detection by PRINS in

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Velagelati, G. V.; Shulman, L. P.; Phillips, O. P.; Tharapel, S. A. & Tharapel, A. T. (1998).

*Gynecology*, Vol. 178, No.6, (June 2001), pp. 1311-1320, ISSN ISSN: 0002-9378 Wachtel, S. S. & Tharapel, A. T. (2006). PRINS for the detection of unique sequences, In:

Woo, H. H., Brigham, L. A. & Hawes, M. C. (1995). In-cell RT-PCR in a single, detached

Xu, J.; Wang, A. & Chen, J M. (2002). New developments in primed in situ labeling (PRINS)

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*Station*, *Japan* Series C1, pp. 47-109 ISSN 0916-5851 0385-2326

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human sperm. *American journal of Medical Genetics*, Vol.107, No.2, (January 2002),

detection by in situ RT-PCR in isolated plant cells and tissues. *The Plant Journal*,

genome of an apple isolate of apple chlorotic leaf spot virus. *Journal of General* 

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gene deletions in cancer. *American journal of Medical Genetics*, Vol.107, No.2,

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plant cell. *Plant Molecular Biology Reporter*, Vol.13, No.4, (December 1995), pp. 355–

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grooving and apple chlorotic leaf spot viruses. *Journal of General Virology*, Vol.69,

The Central African region, also called Atlantic Equatorial Africa, harbors one of the biggest worldwide biodiversity. It is true for human, with a great diversity of ethnic groups, but also for animals, plants, and microorganisms including pathogen species. Although this region is lagging behind in various domains, few research centers and laboratories have been able to develop sophisticated research work for diagnostics, fundamental research, and operational research, using polymerase chain reaction (PCR) techniques. This present paper intends to give an overview of the use of PCR technology in Central Africa and its various applications in the field of genetics, phylogeography, ecology, botany, and infectious diseases, which may have a broad impact on interspecies relationships, diagnostics of diseases, environment and biodiversity.

We will successively describe the main research findings in humans, animals, plants and pathogens from Central Africa, and show how the PCR has allowed scientists from this region to contribute significantly to generalized knowledge in these fields. Then, we'll discuss opportunities and challenges in conducting such kind of research in these particular limited-resources settings before concluding this chapter.

#### **2. Humans**

Since the nineties, the extensive use of molecular techniques has contributed to deepen the knowledge on human genetics. In most studies related to Central Africa, such

Application of PCR Technologies

**2.2 Red blood cell polymorphisms** 

and Simonin techniques) are both simple and robust.

to Humans, Animals, Plants and Pathogens from Central Africa 311

Few studies investigated the extensive allelic diversity in the class I loci (to date, more than 250 HLA-A, 500 HLA-B, and 120 HLA-C alleles) by means of molecular methods among populations of Central Africa [5]. In populations as geographically close as Cameroonians (Yaoundé) [8] and Gabonese (Dienga, South-East of Gabon) [9], the two most frequently detected HLA-A and HLA-B allele families diverged, illustrating the patchwork representation of the different genetic backgrounds (Cameroon: HLA-A\*23, A\*29, HLA-B\*53 and B\*58; Gabon: HLA-A\*19, A\*10, HLA-B\*17 and B\*70). In Cameroon, where populations are very heterogeneous in their origin, culture and language, the most frequently encountered HLA-A, HLA-B and HLA-C alleles differed in four ethnic groups distributed from the north to the south of the country, reflecting the complex migrations and admixtures that occurred in this area located in the borders of Central and west Africa, before that populations settled [10].

Red blood cell polymorphisms are frequently found in areas where malaria is currently or was historically endemic. This observation led to the idea that some of these polymorphisms might provide a relative advantage for survival [11]. The best-characterized polymorphism in this context is the sickle cell trait (HbAS), comprising heterozygous carriage of hemoglobin (Hb) S, which results from a valine substitution for glutamic acid at position 6 of the hemoglobin β chain. HbAS provides carriers with a high degree of protection against severe *Plasmodium falciparum* malaria during early life, which explains the relatively high penetrance of this mutation— in some areas reaching 30%—in sub-Saharan African communities exposed to high rates of infection with *P. falciparum* [12]. The mutation in the homozygous state (HbSS) leads to the disease referred to as "sickle cell anemia," a lifethreatening condition that usually results in early death [13, 14]. HbAS in such populations thus exemplifies a balanced polymorphism that confers a selective advantage to the heterozygote [15]. Molecular determination of the HbS carriage is assessed by PCR-RFLP, where a 369-bp segment of the codon 6 in the beta-globine gene, encompassing the A>T substitution, is amplified, before being digested with the restriction endonuclease *DdeI*.

In sub-Saharan populations, the ABO blood group distribution is in large part dominated by the O blood group, with prevalence rates of at least 50%. Strong hypotheses favor a selection pressure exerted by the plasmodial parasite on its host cell, and include i) the worldwide distribution of the ABO blood groups with a type O predominance in malarious regions of the world [16], ii) the fact that *Plasmodium falciparum* has substantially affected the human genome and was present when the ABO polymorphisms arose [17], iii) the associations of ABO blood groups and clinical outcome of malaria with the observation of a degree of protection conferred by blood group O against severe courses of the disease [18] and iv) the potential role that erythrocyte surface antigens may play in cytoadhesion of infected erythrocytes to micro vessel endothelia and in parasite invasion [19]. No molecular method is used for the determination of ABO blood groups, as hematological methods (Beth-Vincent

G6PD is a cytoplasmic enzyme allowing cells to withstand oxidant stress. It is encoded by one of the most polymorphic genes in humans, located on the X chromosome. In Africa, G6PD is represented by three major variants, G6PD B (normal), G6PD A (90% enzyme activity) and G6PD A- (12% enzyme activity) [20]. The location of the G6PD gene on the X chromosome and the subsequent variable X-chromosome inactivation implies that the expression of G6PD

methodologies have often been used in the context of immunogenetics or genetic epidemiology of infectious diseases. The host genetic background is as important as immunity in the individual fight against infections. These studies were a fabulous opportunity to investigate the richness and extreme diversity of the genetic polymorphisms that characterize populations from Central Africa.

## **2.1 HLA characterization**

The major histocompatibility complex (MHC) is one of the most polymorphic genetic systems of many species, including human leukocyte antigen (HLA) in humans. The class I and class II MHC genes encode cell-surface heterodimers that play an important role in antigen presentation, tolerance, and self/non-self recognition. The HLA molecules bind intracellularly processed antigenic peptides, forming complexes that are the ligands of the antigen receptors of T lymphocytes. In addition, the class I and class II histocompatibility antigens play an important role in allogeneic transplantation. Matching for the alleles at the class I and class II MHC loci impacts the outcome of both solid-organ and hematopoietic stem cell allogeneic transplants.

The HLA class II typing of 167 unrelated Gabonese individuals living in the village of Dienga, located in the South-East of Gabon (province of the Haut-Ogooué) was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [2]. All individuals belonged to the Banzabi ethnic group, which represents the second most important population grouping in Gabon after the Fang, with 55,000 to 60,000 individuals living in an area of 32,000 km2. At the date of realization, in 1996, restriction endonuclease mapping of the PCR products provided profiles that allowed identification of 135 major alleles or groups of alleles among the 184 known DRB1 alleles [3]. Similarly, 9, 24 and 53 major alleles or groups of alleles were recognizable out of a total of 19, 35 and 83 DQA1, DQB1 and DPB1 alleles respectively, so far reported in the literature. For each locus, the PCR-RFLP identified alleles include all major alleles, while unidentifiable alleles were corresponding to rare and newly described alleles. The most frequent alleles at each locus were DRB1\*1501–3 (0.31), DQA1\*0102 (0.50), DQB1\*0602 (0.42) and DPB1\*0402 (0.29). The estimation of the haplotype frequencies as well as the observation of the segregation of several haplotypes using additional HLA typing of relatives, revealed that the three-locus haplotype DRB1\*1501–3-DQA1\*0102- DQB1\*0602 was found at the highest frequency (0.31) among these individuals. This haplotype is not typically African and has already been described in Caucasians, but its presence at high frequency is exclusive to populations originating from Central Africa, and can thus be designated as a particular genetic marker of these populations. On the other hand, the absence in the Gabonese Banzabi group of DRB1\*04 and the concomitant predominance at equal prevalence rates of DRB1\*02 and DRB1\*05, conforms to the other sub-Saharan population groups which have already been typed for their DR1-DR10 allospecificities [4]. Similarly, the predominant alleles observed at the DQA1, DQB1 and DPB1 loci studied have already been described in other sub-Saharan populations [5]. As an example, the determination of DRB1- DQA1-DQB1 haplotype frequencies for 230 Gabonese individuals belonging to tribes as different as Fang, Kele, Myene, Punu, Sira and Tsogo, revealed, as for the Banzabi group, the highest frequency (0.24) for the DRB1\*15/16-DQA1\*0102-DQB1\*0602 haplotype [6]. The same predominant haplotype was observed with a high frequency of 0.27 among 126 healthy individuals in Cameroon, by means of a determination by high-resolution PCR using sequence-specific oligonucleotide probes (PCR-SSOP) and/or DNA sequencing [7].

Few studies investigated the extensive allelic diversity in the class I loci (to date, more than 250 HLA-A, 500 HLA-B, and 120 HLA-C alleles) by means of molecular methods among populations of Central Africa [5]. In populations as geographically close as Cameroonians (Yaoundé) [8] and Gabonese (Dienga, South-East of Gabon) [9], the two most frequently detected HLA-A and HLA-B allele families diverged, illustrating the patchwork representation of the different genetic backgrounds (Cameroon: HLA-A\*23, A\*29, HLA-B\*53 and B\*58; Gabon: HLA-A\*19, A\*10, HLA-B\*17 and B\*70). In Cameroon, where populations are very heterogeneous in their origin, culture and language, the most frequently encountered HLA-A, HLA-B and HLA-C alleles differed in four ethnic groups distributed from the north to the south of the country, reflecting the complex migrations and admixtures that occurred in this area located in the borders of Central and west Africa, before that populations settled [10].

## **2.2 Red blood cell polymorphisms**

310 Polymerase Chain Reaction

methodologies have often been used in the context of immunogenetics or genetic epidemiology of infectious diseases. The host genetic background is as important as immunity in the individual fight against infections. These studies were a fabulous opportunity to investigate the richness and extreme diversity of the genetic polymorphisms

The major histocompatibility complex (MHC) is one of the most polymorphic genetic systems of many species, including human leukocyte antigen (HLA) in humans. The class I and class II MHC genes encode cell-surface heterodimers that play an important role in antigen presentation, tolerance, and self/non-self recognition. The HLA molecules bind intracellularly processed antigenic peptides, forming complexes that are the ligands of the antigen receptors of T lymphocytes. In addition, the class I and class II histocompatibility antigens play an important role in allogeneic transplantation. Matching for the alleles at the class I and class II MHC loci impacts the outcome of both solid-organ and hematopoietic

The HLA class II typing of 167 unrelated Gabonese individuals living in the village of Dienga, located in the South-East of Gabon (province of the Haut-Ogooué) was assessed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [2]. All individuals belonged to the Banzabi ethnic group, which represents the second most important population grouping in Gabon after the Fang, with 55,000 to 60,000 individuals living in an area of 32,000 km2. At the date of realization, in 1996, restriction endonuclease mapping of the PCR products provided profiles that allowed identification of 135 major alleles or groups of alleles among the 184 known DRB1 alleles [3]. Similarly, 9, 24 and 53 major alleles or groups of alleles were recognizable out of a total of 19, 35 and 83 DQA1, DQB1 and DPB1 alleles respectively, so far reported in the literature. For each locus, the PCR-RFLP identified alleles include all major alleles, while unidentifiable alleles were corresponding to rare and newly described alleles. The most frequent alleles at each locus were DRB1\*1501–3 (0.31), DQA1\*0102 (0.50), DQB1\*0602 (0.42) and DPB1\*0402 (0.29). The estimation of the haplotype frequencies as well as the observation of the segregation of several haplotypes using additional HLA typing of relatives, revealed that the three-locus haplotype DRB1\*1501–3-DQA1\*0102- DQB1\*0602 was found at the highest frequency (0.31) among these individuals. This haplotype is not typically African and has already been described in Caucasians, but its presence at high frequency is exclusive to populations originating from Central Africa, and can thus be designated as a particular genetic marker of these populations. On the other hand, the absence in the Gabonese Banzabi group of DRB1\*04 and the concomitant predominance at equal prevalence rates of DRB1\*02 and DRB1\*05, conforms to the other sub-Saharan population groups which have already been typed for their DR1-DR10 allospecificities [4]. Similarly, the predominant alleles observed at the DQA1, DQB1 and DPB1 loci studied have already been described in other sub-Saharan populations [5]. As an example, the determination of DRB1- DQA1-DQB1 haplotype frequencies for 230 Gabonese individuals belonging to tribes as different as Fang, Kele, Myene, Punu, Sira and Tsogo, revealed, as for the Banzabi group, the highest frequency (0.24) for the DRB1\*15/16-DQA1\*0102-DQB1\*0602 haplotype [6]. The same predominant haplotype was observed with a high frequency of 0.27 among 126 healthy individuals in Cameroon, by means of a determination by high-resolution PCR using

sequence-specific oligonucleotide probes (PCR-SSOP) and/or DNA sequencing [7].

that characterize populations from Central Africa.

**2.1 HLA characterization** 

stem cell allogeneic transplants.

Red blood cell polymorphisms are frequently found in areas where malaria is currently or was historically endemic. This observation led to the idea that some of these polymorphisms might provide a relative advantage for survival [11]. The best-characterized polymorphism in this context is the sickle cell trait (HbAS), comprising heterozygous carriage of hemoglobin (Hb) S, which results from a valine substitution for glutamic acid at position 6 of the hemoglobin β chain. HbAS provides carriers with a high degree of protection against severe *Plasmodium falciparum* malaria during early life, which explains the relatively high penetrance of this mutation— in some areas reaching 30%—in sub-Saharan African communities exposed to high rates of infection with *P. falciparum* [12]. The mutation in the homozygous state (HbSS) leads to the disease referred to as "sickle cell anemia," a lifethreatening condition that usually results in early death [13, 14]. HbAS in such populations thus exemplifies a balanced polymorphism that confers a selective advantage to the heterozygote [15]. Molecular determination of the HbS carriage is assessed by PCR-RFLP, where a 369-bp segment of the codon 6 in the beta-globine gene, encompassing the A>T substitution, is amplified, before being digested with the restriction endonuclease *DdeI*.

In sub-Saharan populations, the ABO blood group distribution is in large part dominated by the O blood group, with prevalence rates of at least 50%. Strong hypotheses favor a selection pressure exerted by the plasmodial parasite on its host cell, and include i) the worldwide distribution of the ABO blood groups with a type O predominance in malarious regions of the world [16], ii) the fact that *Plasmodium falciparum* has substantially affected the human genome and was present when the ABO polymorphisms arose [17], iii) the associations of ABO blood groups and clinical outcome of malaria with the observation of a degree of protection conferred by blood group O against severe courses of the disease [18] and iv) the potential role that erythrocyte surface antigens may play in cytoadhesion of infected erythrocytes to micro vessel endothelia and in parasite invasion [19]. No molecular method is used for the determination of ABO blood groups, as hematological methods (Beth-Vincent and Simonin techniques) are both simple and robust.

G6PD is a cytoplasmic enzyme allowing cells to withstand oxidant stress. It is encoded by one of the most polymorphic genes in humans, located on the X chromosome. In Africa, G6PD is represented by three major variants, G6PD B (normal), G6PD A (90% enzyme activity) and G6PD A- (12% enzyme activity) [20]. The location of the G6PD gene on the X chromosome and the subsequent variable X-chromosome inactivation implies that the expression of G6PD

Application of PCR Technologies

**2.3 Innate immunity** 

to Humans, Animals, Plants and Pathogens from Central Africa 313

Other erythrocyte polymorphisms characterize the sub-Saharan populations, including Central Africans. It is the case of the alpha-thalassemia, which consists in the deletion of 1, 2, 3 or the 4 genes encoding the alpha chain of the globin. Several forms of alpha-thalassemia are distributed worldwide, and the form encountered in sub-Saharan Africa resides in a gene deletion of 3.7 kb (-α3.7 type), which generates the formation of a functional hybrid gene. A PCR amplification strategy using three primers allows to determine the normal (αα/αα), heterozygous (-α3.7 /αα) and homozygous (-α3.7/-α3.7) state as well as the - -/-α3.7 form (H haemoglobin) [30]. The prevalence of α+-thalassemia in Africa ranges from 5 to 50%, according to a gradient from North Africa to equatorial Africa and from South Africa to equatorial Africa: so, the highest prevalence rates are reached in the Central African Republic [31] and in a Bantu population from the republic of Congo [32]. Different erythrocyte polymorphisms may coexist in the same individual, as the results of advantageous interactions. Namely, a beneficial effect of α+-thalassemia on the hematological characteristics of sickle-cell anemia patients has been found, in accordance with the observation in HbAS individuals of decreasing values of HbS quantification

For the needs of malaria-linked studies, polymorphisms of some products of the inflammatory

Mannose binding lectin (MBL) is a member of the collectin family of proteins, which are components of the innate immune system, acting therefore against multiple pathogenic organisms. MBL is thought to be more effective at an early age, before effective acquired immune responses have developed, and low plasma concentrations of non-functional MBL have been attributed to mutations in the first exon of the MBL gene: MBLIVS-I-5 G>A, MBL54 G>A and MBL57 G>A. PCR-RFLP determination may be performed, using *NlaIII (*codon 52), *BanI* (codon 54) and *MboII* (codon 57) endonucleases. At least one MBP gene mutation was present in 34% of a Gabonese population sample (Banzabi), with an overall gene frequency of 0.03, 0.02 and 0.18 mutations at codons 52, 54 and 57, respectively [22, 25]. There are other published MBL2 genotyping techniques, based on sequence-specific PCR, denaturing gradient gel electrophoresis of PCR-amplified fragments, real-time PCR with the hybridization of sequence-specific probes and sequence-based typing. A new strategy that combines sequence-specific PCR and sequence-based typing (Haplotype Specific Sequencing or HSS) was recently improved and allowed identification of 14 MBL allelespecific fragments (located in the promoter and exon 1) among Gabonese individuals [33]. Inducible nitric oxide synthase 2 (NOS2) is the critical enzyme involved in the synthesis of nitric oxide, a short-lived molecule with diverse functions including antimalarial activity, that can also cause damage to the host cell. The most investigated polymorphism is located in the promoter region of NOS2, and concerns the point mutation NOS2-954 G>C, which is associated with an increased production of NOS2. By the means of a PCR amplification followed by enzymatic digestion with *BsaI*, this point mutation was found in 18% of Gabonese individuals from the Banzabi ethnic group, mainly in the heterozygous state [22, 25]. A similar high prevalence was found in another Gabonese population group, recruited in Lambaréné [34].

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that provides rapid host defense against infection but is detrimental or fatal in excess. The main studied

response have been investigated among populations from Central African countries.

accompanying decreasing numbers of α-globin genes (from 4 to 2) [32].

deficiency differs markedly among heterozygous females and therefore that these females do not constitute a homogeneous group [21]. PCR-RFLP is used for the molecular determination of the predominant G6PD A- variant in sub-Saharan Africa: mutation 376 A>G responsible for the G6PD A electrophoretic mobility and mutation 202 G>A responsible for the A- deficiency, are determined by PCR amplification of exons 5 and 4 respectively, followed by restriction enzyme analysis, using *FokI* (376 A>G mutation) and *NlaIII* (202 G>A mutation). However, the 376 A>G mutation may also be associated with other deleterious mutations such as 542 A>T (G6PD Santamaria), 680 G>T or 968 T>C, revealed after electrophoretic migration of digested amplified products with *BspEI*, *BstNI* and *NciI* respectively.

Table 1 presents data obtained among healthy individuals in order to avoid distribution bias due to selection of genetic traits by secularly settled diseases such as malaria. No HbSS individual was recorded in the studies gathered in this Table, because of an age range beyond the life expectancy of most HbSS patients in developing countries. Since the G6PD A and B variants have almost the same enzyme activity, the patients were stratified into groups with normal (female BB, AB, AA and male B and A genotypes), heterozygous (female A-B and A-A genotypes) and homo-/hemi-zygous (female A-A- and male Agenotypes) state, corresponding to decreasing levels of G6PD enzymatic activity. Some research teams have extensively studied erythrocyte polymorphisms in relation to malaria morbidity, among children hospitalized at the Albert Schweitzer Hospital from Lambaréné, in the Moyen Ogooué province of Gabon. As these genetic traits strongly influence the distribution of the clinical pattern of malaria, their frequency distribution is not representative of the whole population, and therefore they could not be reported in Table 1.


M: males; F: females.

Table 1. Erythrocyte polymorphisms among healthy individuals from Central Africa

Other erythrocyte polymorphisms characterize the sub-Saharan populations, including Central Africans. It is the case of the alpha-thalassemia, which consists in the deletion of 1, 2, 3 or the 4 genes encoding the alpha chain of the globin. Several forms of alpha-thalassemia are distributed worldwide, and the form encountered in sub-Saharan Africa resides in a gene deletion of 3.7 kb (-α3.7 type), which generates the formation of a functional hybrid gene. A PCR amplification strategy using three primers allows to determine the normal (αα/αα), heterozygous (-α3.7 /αα) and homozygous (-α3.7/-α3.7) state as well as the - -/-α3.7 form (H haemoglobin) [30]. The prevalence of α+-thalassemia in Africa ranges from 5 to 50%, according to a gradient from North Africa to equatorial Africa and from South Africa to equatorial Africa: so, the highest prevalence rates are reached in the Central African Republic [31] and in a Bantu population from the republic of Congo [32]. Different erythrocyte polymorphisms may coexist in the same individual, as the results of advantageous interactions. Namely, a beneficial effect of α+-thalassemia on the hematological characteristics of sickle-cell anemia patients has been found, in accordance with the observation in HbAS individuals of decreasing values of HbS quantification accompanying decreasing numbers of α-globin genes (from 4 to 2) [32].

## **2.3 Innate immunity**

312 Polymerase Chain Reaction

deficiency differs markedly among heterozygous females and therefore that these females do not constitute a homogeneous group [21]. PCR-RFLP is used for the molecular determination of the predominant G6PD A- variant in sub-Saharan Africa: mutation 376 A>G responsible for the G6PD A electrophoretic mobility and mutation 202 G>A responsible for the A- deficiency, are determined by PCR amplification of exons 5 and 4 respectively, followed by restriction enzyme analysis, using *FokI* (376 A>G mutation) and *NlaIII* (202 G>A mutation). However, the 376 A>G mutation may also be associated with other deleterious mutations such as 542 A>T (G6PD Santamaria), 680 G>T or 968 T>C, revealed after electrophoretic migration of digested

Table 1 presents data obtained among healthy individuals in order to avoid distribution bias due to selection of genetic traits by secularly settled diseases such as malaria. No HbSS individual was recorded in the studies gathered in this Table, because of an age range beyond the life expectancy of most HbSS patients in developing countries. Since the G6PD A and B variants have almost the same enzyme activity, the patients were stratified into groups with normal (female BB, AB, AA and male B and A genotypes), heterozygous (female A-B and A-A genotypes) and homo-/hemi-zygous (female A-A- and male Agenotypes) state, corresponding to decreasing levels of G6PD enzymatic activity. Some research teams have extensively studied erythrocyte polymorphisms in relation to malaria morbidity, among children hospitalized at the Albert Schweitzer Hospital from Lambaréné, in the Moyen Ogooué province of Gabon. As these genetic traits strongly influence the distribution of the clinical pattern of malaria, their frequency distribution is not representative of the whole population, and therefore they could not be reported in Table 1.

**Prevalence rate (%)** 

**Cameroun (Ebolowa)** 

N = 1,007 [24] 51 24 19 6

N = 240 [25] 81 19 0

N = 561 M [26] 93

0

7

**Republic of Congo (Brazzaville)** 

N = 13,045 [27] 53 22 21 4

N = 868 [28] 80 20 0

N = 398 M & F [29] 68

21

11

amplified products with *BspEI*, *BstNI* and *NciI* respectively.

**Erythrocyte polymorphisms** 

Group O Group A Group B Group AB

Hb AA Hb AS Hb SS

G6PD state:


M: males; F: females.

ABO blood groups:

HbS genotypes:

 **Gabon** 

**(Dienga)** 

N = 279 [22] [23] 54 27 17 2

N = 279 [22] [23] 77 23 0

N = 271 M & F [22] [23] 78

13

9

Table 1. Erythrocyte polymorphisms among healthy individuals from Central Africa

For the needs of malaria-linked studies, polymorphisms of some products of the inflammatory response have been investigated among populations from Central African countries.

Mannose binding lectin (MBL) is a member of the collectin family of proteins, which are components of the innate immune system, acting therefore against multiple pathogenic organisms. MBL is thought to be more effective at an early age, before effective acquired immune responses have developed, and low plasma concentrations of non-functional MBL have been attributed to mutations in the first exon of the MBL gene: MBLIVS-I-5 G>A, MBL54 G>A and MBL57 G>A. PCR-RFLP determination may be performed, using *NlaIII (*codon 52), *BanI* (codon 54) and *MboII* (codon 57) endonucleases. At least one MBP gene mutation was present in 34% of a Gabonese population sample (Banzabi), with an overall gene frequency of 0.03, 0.02 and 0.18 mutations at codons 52, 54 and 57, respectively [22, 25]. There are other published MBL2 genotyping techniques, based on sequence-specific PCR, denaturing gradient gel electrophoresis of PCR-amplified fragments, real-time PCR with the hybridization of sequence-specific probes and sequence-based typing. A new strategy that combines sequence-specific PCR and sequence-based typing (Haplotype Specific Sequencing or HSS) was recently improved and allowed identification of 14 MBL allelespecific fragments (located in the promoter and exon 1) among Gabonese individuals [33].

Inducible nitric oxide synthase 2 (NOS2) is the critical enzyme involved in the synthesis of nitric oxide, a short-lived molecule with diverse functions including antimalarial activity, that can also cause damage to the host cell. The most investigated polymorphism is located in the promoter region of NOS2, and concerns the point mutation NOS2-954 G>C, which is associated with an increased production of NOS2. By the means of a PCR amplification followed by enzymatic digestion with *BsaI*, this point mutation was found in 18% of Gabonese individuals from the Banzabi ethnic group, mainly in the heterozygous state [22, 25]. A similar high prevalence was found in another Gabonese population group, recruited in Lambaréné [34].

Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine that provides rapid host defense against infection but is detrimental or fatal in excess. The main studied

Application of PCR Technologies

**P450 Tissue location** 

CYP8A1 Ovary, heart, skeletal muscle, lung and prostate

CYP2A13 Lung tissue

CYP3A5 Liver

CYP2F1 Lung tissue

CYP4A11 Liver and kidney

untranslated region

**Clinical implication** 

Pathogenesis of vascular diseases

Susceptibility of tobacco-related tumorigenesis

Metabolism of chemotherapeutic agents and toxins

Metabolism of pneumotoxicants with carcinogenic

Regulation of blood pressure in the kidney

effects

diverse populations including Gabonese

to Humans, Animals, Plants and Pathogens from Central Africa 315

synthesis of a truncated protein of the CYP2F1, which activity in lung tissue is linked to carcinogenic effects, was mostly represented in the Gabonese population sample [40]. The genetic polymorphism of the CYP3A5 enzyme, implicated in the metabolism of chemotherapeutic agents but also toxins, was analyzed using a PCR-SSCP strategy, leading to the observation of great inter-ethnic differences in the distribution of a maximum of 17 alleles, some of them being linked to the synthesis of a non functional enzyme. According to the determination of the CYP3A5 predicted phenotype, Gabonese individuals were the most numerous (90.0%) to express a complete and functional CYP3A5 protein compared to French Caucasians (10.4%) and Tunisians (30.0%) [41]. The CYP4A11 enzyme is involved in the regulation of the blood pressure in the kidney, and an 8590T>C mutation has been associated to an increased prevalence of hypertension. Using PCR-SSCP and nucleotide sequence analysis, the frequency of this mutation was found lower in Gabonese compared to other investigated African population groups (Tunisians, Senegalese) [42]. Lastly, 3 single nucleotide polymorphisms (SNPs) affecting the human type II inosine monophosphate dehydrogenase (IMPDH2) gene have been determined by PCR-SSCP. This enzyme participates in the metabolism of purines and constitutes a target for antiviral drugs. It resulted that African

**Gene** 

9 VNTRs in the 5'-

regulatory region of the *CYP8A1*

3 SNPs: 578C>T (exon 2), 3375C>T (exon 5) and 720C>G (3'UTR)

17 SNPs on the 13 exons of the *CYP3A5* gene

Frameshift mutation in *CYP2F1* exon 2 (c.14\_15insC)

1 SNP on

8590T>C

VNTR: variable number of tandem repeats; SNP: single nucleotide polymorphism; 3'UTR: 3'

*CYP4A22*-exon 11:

Table 2. Genetic polymorphisms in enzymes of the cytochrome P450 superfamily (CYP), in

proximal

gene

**polymorphism DNA samples origin (n) Reference** 

European (78 French Caucasians); African (50 Gabonese and 50 Tunisians)

European (52 French Caucasians); African (36 Gabonese and 48 Tunisians)

European (51 French Caucasians); African (36 Gabonese and 36 Tunisians)

European (90 French Caucasians); African (32 Gabonese, 37 Tunisians and 75 Senegalese)

European (99 French Caucasians); African (36 Gabonese, 53 Tunisians and 50 Senegalese); South American (60 Peruvians)

[38]

[39]

[41]

[40]

[42]

polymorphisms are located in the promoter region of the gene and are TNFα-308 G>A and TNFα-238 G>A base substitutions. These two polymorphisms have not been related to any variation in cytokine production, but may serve as markers for a functional polymorphism elsewhere in the TNF-α gene. Indeed, the TNFα376 A allele (G>A substitution), which is frequently found in linkage disequilibrium with TNFα-238 A allele, is related to enhanced secretion of TNF and might be responsible for increased antigen- or T-cell mediated B-cell stimulation and proliferation [35]. Molecular determination is assessed by PCR-RFLP using *NcoI* (-308), *AlwI* (-238) and *FokI* (376) restriction endonucleases. Prevalence rates of 22% (TNFα-308 A allele) and 17% (TNFα-238 A allele) were found in a Gabonese population (Banzabi), mainly in the heterozygous state [22, 25].

Haptoglobin (Hp) is an acute-phase protein that binds irreversibly to hemoglobin (Hb), enabling its safe and rapid clearance. Therefore, Hp has an important protective role in hemolytic disease because it greatly reduces the oxidative and peroxidative potential of free Hb. Haptoglobin exists in three phenotypic forms: Hp1-1, 2-1, and 2-2, which are encoded by two co-dominant alleles, *Hp1* and *Hp2*. A fourth phenotype HpO, referred to as hypo- or an-haptoglobinaemia has been reported to be the predominant phenotype in West Africa. Functional differences between the different Hp phenotypes have been reported, the ability to bind Hb being in the order of 1-1 > 2-1 > 2-2. The gene frequencies of different Hp phenotypes show marked geographical differences as well as large variations among different ethnic groups. Hp genotypes determined by PCR in 511 Gabonese children from the village of Bakoumba (South-East of Gabon), distributed into 36.5%, 47.6% and 15.9% for Hp1- 1, Hp2-1 and Hp2-2 respectively [36]. In South-West Cameroon, the genotype distribution among 98 pregnant women was 53% for Hp1-1, 22% for Hp2-1 and 25% for Hp2-2 [37].

#### **2.4 Polymorphism of the cytochrome P450 superfamily**

The DNA samples of the Gabonese individuals from the Banzabi ethnic group already described [2] entered a dataset of DNA samples from European (French Caucasians), African (Senegalese), South American (Peruvians) and North African (Tunisians) populations, in order to evaluate the inter-ethnic variations in the genetic polymorphism of several components of the cytochrome P450 superfamily (CYP) which gathers a large and diverse group of enzymes (Table 2). The function of most CYP enzymes is to catalyze the oxidation of organic substances. Their substrates include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. The investigation of the variable number of tandem repeat (VNTR) polymorphism of the human prostacyclin synthase gene (CYP8A1) revealed a particular distribution of the nine characterized alleles in the Gabonese population group, which may be associated with a more frequent and severe form of hypertension found in some Black populations [38]. The frequencies of three single nucleotide polymorphisms occurring in the CYP2A13 were determined by PCR-single strand conformational polymorphism (PCR-SSCP) (578C>T (Arg101Stop)) and PCR-RFLP (3375C>T (Arg257Cys) and 720C>G (3'-untranslated region)) and were respectively 0, 15.3 and 20.8 among the Gabonese group, differing from those of other groups under comparison: these marked inter-ethnic variations in an enzyme involved in the metabolism of compounds provided by the use of tobacco, have consequences on the susceptibility to lung cancer [39]. More precisely, it appears that black populations could present a higher deficit in CYP2A13 activity compared with other population groups, compatible with a reduced risk for smokingrelated lung adenocarcinoma. In the same way, a frameshift mutation, responsible for the

polymorphisms are located in the promoter region of the gene and are TNFα-308 G>A and TNFα-238 G>A base substitutions. These two polymorphisms have not been related to any variation in cytokine production, but may serve as markers for a functional polymorphism elsewhere in the TNF-α gene. Indeed, the TNFα376 A allele (G>A substitution), which is frequently found in linkage disequilibrium with TNFα-238 A allele, is related to enhanced secretion of TNF and might be responsible for increased antigen- or T-cell mediated B-cell stimulation and proliferation [35]. Molecular determination is assessed by PCR-RFLP using *NcoI* (-308), *AlwI* (-238) and *FokI* (376) restriction endonucleases. Prevalence rates of 22% (TNFα-308 A allele) and 17% (TNFα-238 A allele) were found in a Gabonese population

Haptoglobin (Hp) is an acute-phase protein that binds irreversibly to hemoglobin (Hb), enabling its safe and rapid clearance. Therefore, Hp has an important protective role in hemolytic disease because it greatly reduces the oxidative and peroxidative potential of free Hb. Haptoglobin exists in three phenotypic forms: Hp1-1, 2-1, and 2-2, which are encoded by two co-dominant alleles, *Hp1* and *Hp2*. A fourth phenotype HpO, referred to as hypo- or an-haptoglobinaemia has been reported to be the predominant phenotype in West Africa. Functional differences between the different Hp phenotypes have been reported, the ability to bind Hb being in the order of 1-1 > 2-1 > 2-2. The gene frequencies of different Hp phenotypes show marked geographical differences as well as large variations among different ethnic groups. Hp genotypes determined by PCR in 511 Gabonese children from the village of Bakoumba (South-East of Gabon), distributed into 36.5%, 47.6% and 15.9% for Hp1- 1, Hp2-1 and Hp2-2 respectively [36]. In South-West Cameroon, the genotype distribution

among 98 pregnant women was 53% for Hp1-1, 22% for Hp2-1 and 25% for Hp2-2 [37].

The DNA samples of the Gabonese individuals from the Banzabi ethnic group already described [2] entered a dataset of DNA samples from European (French Caucasians), African (Senegalese), South American (Peruvians) and North African (Tunisians) populations, in order to evaluate the inter-ethnic variations in the genetic polymorphism of several components of the cytochrome P450 superfamily (CYP) which gathers a large and diverse group of enzymes (Table 2). The function of most CYP enzymes is to catalyze the oxidation of organic substances. Their substrates include metabolic intermediates such as lipids and steroidal hormones, as well as xenobiotic substances such as drugs and other toxic chemicals. The investigation of the variable number of tandem repeat (VNTR) polymorphism of the human prostacyclin synthase gene (CYP8A1) revealed a particular distribution of the nine characterized alleles in the Gabonese population group, which may be associated with a more frequent and severe form of hypertension found in some Black populations [38]. The frequencies of three single nucleotide polymorphisms occurring in the CYP2A13 were determined by PCR-single strand conformational polymorphism (PCR-SSCP) (578C>T (Arg101Stop)) and PCR-RFLP (3375C>T (Arg257Cys) and 720C>G (3'-untranslated region)) and were respectively 0, 15.3 and 20.8 among the Gabonese group, differing from those of other groups under comparison: these marked inter-ethnic variations in an enzyme involved in the metabolism of compounds provided by the use of tobacco, have consequences on the susceptibility to lung cancer [39]. More precisely, it appears that black populations could present a higher deficit in CYP2A13 activity compared with other population groups, compatible with a reduced risk for smokingrelated lung adenocarcinoma. In the same way, a frameshift mutation, responsible for the

(Banzabi), mainly in the heterozygous state [22, 25].

**2.4 Polymorphism of the cytochrome P450 superfamily** 

synthesis of a truncated protein of the CYP2F1, which activity in lung tissue is linked to carcinogenic effects, was mostly represented in the Gabonese population sample [40]. The genetic polymorphism of the CYP3A5 enzyme, implicated in the metabolism of chemotherapeutic agents but also toxins, was analyzed using a PCR-SSCP strategy, leading to the observation of great inter-ethnic differences in the distribution of a maximum of 17 alleles, some of them being linked to the synthesis of a non functional enzyme. According to the determination of the CYP3A5 predicted phenotype, Gabonese individuals were the most numerous (90.0%) to express a complete and functional CYP3A5 protein compared to French Caucasians (10.4%) and Tunisians (30.0%) [41]. The CYP4A11 enzyme is involved in the regulation of the blood pressure in the kidney, and an 8590T>C mutation has been associated to an increased prevalence of hypertension. Using PCR-SSCP and nucleotide sequence analysis, the frequency of this mutation was found lower in Gabonese compared to other investigated African population groups (Tunisians, Senegalese) [42]. Lastly, 3 single nucleotide polymorphisms (SNPs) affecting the human type II inosine monophosphate dehydrogenase (IMPDH2) gene have been determined by PCR-SSCP. This enzyme participates in the metabolism of purines and constitutes a target for antiviral drugs. It resulted that African


VNTR: variable number of tandem repeats; SNP: single nucleotide polymorphism; 3'UTR: 3' untranslated region

Table 2. Genetic polymorphisms in enzymes of the cytochrome P450 superfamily (CYP), in diverse populations including Gabonese

Application of PCR Technologies

**4. Plants** 

**4.1 Methods and approaches** 

fragmentation, logging activities, etc.

genetic diversity of current Gorilla populations [45].

**3.3 Central African elephants: Forest or savannah elephants?** 

elephant split inapplicable to modern African elephant populations.

to Humans, Animals, Plants and Pathogens from Central Africa 317

separates populations in Cameroon and northern Gabon from those in southern Gabon [47]. For Gorilla, rivers are more permeable and allow limited admixture among populations separated by waterways [45]. Anthony et al. also showed that like for plant species (see section 4) past vicariance events and Pleistocene refugia played an important role in shaping

Despite their morphology typical from forest elephants, a genetic study based on mtDNA [48] shows that Central African elephants are sharing their history with both forest and savannah elephants from Western Africa. It also gives evidence that Central African forest populations show lower genetic diversity than those in savannahs, and infers a recent population expansion. These results do not support the separation of African elephants into two evolutionary lineages (forest and savannah). The demographic history of African elephants seems more complex, with a combination of multiple refugial mitochondrial lineages and recurrent hybridization among them rendering a simple forest/savannah

This paragraph is giving on overview of approaches and methods related to the molecular ecology field and used to study natural or human-induced dynamic of plant species in Central Africa. Acknowledging the past history of the Central African forest domain is crucial for our understanding of spatial and temporal evolution of species living throughout the region.

Historical processes responsible for the contemporary distributions of individuals can be studied within the field of historical biogeography or phylogeography. For phylogeographic studies the distribution of genetic lineages within or among closely related species is considered throughout the geographical space and current patterns are interpreted in light of past vicariance events, population bottleneck, survival in glacial refugia and/or colonization routes [49, 50, 51]. This approach can be combined with landscape genetic methods to respectively infer impact of historical and environmental processes on the distribution of the genetic diversity. Landscape genetic methods allow to correlate the distribution of the genetic diversity with environmental parameters and to reveal, for example, the impact of topographic features on gene flow or the role of soil heterogeneity in structuring the genetic diversity [52]. At finer scales, classical population genetic approaches address the role of additional evolutionary forces (drift, dispersal, mutation, mating system, etc.) in shaping current patterns. All these genetic-based approaches belong to the molecular ecology field and are combined to address questions linked to the natural species dynamic or more importantly, questions linked to the survival of threatened species facing forest

All these approaches primary necessitate analyses of the genetic diversity at individual level. In this purpose, various techniques based on PCR are used. Different genetic markers can be chosen based on their respective evolutionary properties. For analyses of large-scale patterns, sequences of cytoplasmic DNA (ctDNA) like chloroplastic DNA (cpDNA) for plants are chosen. Cytoplamic DNA are haploid, non-recombining (or recombination events are rare) and generally characterized by uniparental inheritance (chloroplasts are generally

population groups (Tunisians, Gabonese, and Senegalese) presented a higher IMPDH2 activity than Caucasians, with implications for the dose requirement of IMPDH2 inhibitors administered to patients [43].

This compilation of genetic data on populations from Central Africa is far from being exhaustive. As an example, the genetic polymorphism of Toll-Like Receptors (TLR) is to date extensively explored in order to deepen the understanding of the first steps of the immune recognition. Also, cytokines that regulate adaptive immune responses (humoral immunity and cell-mediated immunity) may present inter-individual genetic variations such as it is the case for IL-2, IL-4, IL-5, IFN-gamma, TGF-beta, LT-alpha or IL-13. Finally, increasing information is generated every day thanks to equipments (such as real-time PCR systems or DNA sequencers) that allow handling simultaneously a great number of biological samples. Altogether, this review of genetic data gathered during the last twenty years among Central African populations, illustrates in which point Africa, which is thought to be the homeland of all modern humans, is the most genetically diverse region of the world.

## **3. Animals**

Methods used to infer the respective role of historical, environmental and evolutionary processes on animal distribution are related to the molecular ecology field and, as such, very similar to those employed to study plant dynamic (see section 4.). For animal, sequence of genes of mitochondrial DNA (mtDNA) such as cytochrome b or control region genes are largely used in phylogenetic and phylogeographic studies. The evolutionary pace of mitochondrial genomes being relatively fast, mtDNA sequences can also be used in population genetics study even if nuclear markers (microsatellites, SNP, etc.) provide a higher level of information.

## **3.1 Species identification from fecal pellets**

The inability to correctly identify species and determine their proportional abundance in the wild is of real conservation concern, not only for species management but also in the regulation of illegal trade. However, estimating species abundance using classical ecological methods based on direct observation is very challenging in Central Africa. Indirect methods based on animal tracks, especially fecal pellets have been proposed; however pellets of parapatric related species are sometimes very similar and difficult to use to reliably differentiate species in the field. To address this problem, a PCR-based method has been proposed to differentiate Central African artiodactyls species and especially duikers (*Cephalophus*) from their fecal pellets [44]. In this purpose, a mtDNA sequence database was compiled from all forest *Cephalophus* species and other similarly sized, sympatric *Tragelaphus, Neotragus* and *Hyemoschus* species. The tree-based approach proposed by the authors is reliable to recover most species identity from Central African duikers.

#### **3.2 Rivers are playing a major role in genetic differentiation for large primates in central Africa**

For both Gorillas (*Gorilla gorilla*; [45, 46]) and Mandrills (*Mandrillus sphinx*; [47]) phylogeographic studies based on mtDNA (for both species) and microsatellite (only for Gorilla) markers have shown that rivers hamper gene flow among populations and have a major role in partitioning the species diversity. For Mandrills, the Ogooué river (Gabon) separates populations in Cameroon and northern Gabon from those in southern Gabon [47]. For Gorilla, rivers are more permeable and allow limited admixture among populations separated by waterways [45]. Anthony et al. also showed that like for plant species (see section 4) past vicariance events and Pleistocene refugia played an important role in shaping genetic diversity of current Gorilla populations [45].

## **3.3 Central African elephants: Forest or savannah elephants?**

Despite their morphology typical from forest elephants, a genetic study based on mtDNA [48] shows that Central African elephants are sharing their history with both forest and savannah elephants from Western Africa. It also gives evidence that Central African forest populations show lower genetic diversity than those in savannahs, and infers a recent population expansion. These results do not support the separation of African elephants into two evolutionary lineages (forest and savannah). The demographic history of African elephants seems more complex, with a combination of multiple refugial mitochondrial lineages and recurrent hybridization among them rendering a simple forest/savannah elephant split inapplicable to modern African elephant populations.

## **4. Plants**

316 Polymerase Chain Reaction

population groups (Tunisians, Gabonese, and Senegalese) presented a higher IMPDH2 activity than Caucasians, with implications for the dose requirement of IMPDH2 inhibitors

This compilation of genetic data on populations from Central Africa is far from being exhaustive. As an example, the genetic polymorphism of Toll-Like Receptors (TLR) is to date extensively explored in order to deepen the understanding of the first steps of the immune recognition. Also, cytokines that regulate adaptive immune responses (humoral immunity and cell-mediated immunity) may present inter-individual genetic variations such as it is the case for IL-2, IL-4, IL-5, IFN-gamma, TGF-beta, LT-alpha or IL-13. Finally, increasing information is generated every day thanks to equipments (such as real-time PCR systems or DNA sequencers) that allow handling simultaneously a great number of biological samples. Altogether, this review of genetic data gathered during the last twenty years among Central African populations, illustrates in which point Africa, which is thought to be the homeland of all modern humans, is the most genetically diverse region of the world.

Methods used to infer the respective role of historical, environmental and evolutionary processes on animal distribution are related to the molecular ecology field and, as such, very similar to those employed to study plant dynamic (see section 4.). For animal, sequence of genes of mitochondrial DNA (mtDNA) such as cytochrome b or control region genes are largely used in phylogenetic and phylogeographic studies. The evolutionary pace of mitochondrial genomes being relatively fast, mtDNA sequences can also be used in population genetics study even if nuclear markers (microsatellites, SNP, etc.) provide a

The inability to correctly identify species and determine their proportional abundance in the wild is of real conservation concern, not only for species management but also in the regulation of illegal trade. However, estimating species abundance using classical ecological methods based on direct observation is very challenging in Central Africa. Indirect methods based on animal tracks, especially fecal pellets have been proposed; however pellets of parapatric related species are sometimes very similar and difficult to use to reliably differentiate species in the field. To address this problem, a PCR-based method has been proposed to differentiate Central African artiodactyls species and especially duikers (*Cephalophus*) from their fecal pellets [44]. In this purpose, a mtDNA sequence database was compiled from all forest *Cephalophus* species and other similarly sized, sympatric *Tragelaphus, Neotragus* and *Hyemoschus* species. The tree-based approach proposed by the

authors is reliable to recover most species identity from Central African duikers.

**3.2 Rivers are playing a major role in genetic differentiation for large primates in** 

For both Gorillas (*Gorilla gorilla*; [45, 46]) and Mandrills (*Mandrillus sphinx*; [47]) phylogeographic studies based on mtDNA (for both species) and microsatellite (only for Gorilla) markers have shown that rivers hamper gene flow among populations and have a major role in partitioning the species diversity. For Mandrills, the Ogooué river (Gabon)

administered to patients [43].

**3. Animals** 

**central Africa** 

higher level of information.

**3.1 Species identification from fecal pellets** 

## **4.1 Methods and approaches**

This paragraph is giving on overview of approaches and methods related to the molecular ecology field and used to study natural or human-induced dynamic of plant species in Central Africa. Acknowledging the past history of the Central African forest domain is crucial for our understanding of spatial and temporal evolution of species living throughout the region.

Historical processes responsible for the contemporary distributions of individuals can be studied within the field of historical biogeography or phylogeography. For phylogeographic studies the distribution of genetic lineages within or among closely related species is considered throughout the geographical space and current patterns are interpreted in light of past vicariance events, population bottleneck, survival in glacial refugia and/or colonization routes [49, 50, 51]. This approach can be combined with landscape genetic methods to respectively infer impact of historical and environmental processes on the distribution of the genetic diversity. Landscape genetic methods allow to correlate the distribution of the genetic diversity with environmental parameters and to reveal, for example, the impact of topographic features on gene flow or the role of soil heterogeneity in structuring the genetic diversity [52]. At finer scales, classical population genetic approaches address the role of additional evolutionary forces (drift, dispersal, mutation, mating system, etc.) in shaping current patterns. All these genetic-based approaches belong to the molecular ecology field and are combined to address questions linked to the natural species dynamic or more importantly, questions linked to the survival of threatened species facing forest fragmentation, logging activities, etc.

All these approaches primary necessitate analyses of the genetic diversity at individual level. In this purpose, various techniques based on PCR are used. Different genetic markers can be chosen based on their respective evolutionary properties. For analyses of large-scale patterns, sequences of cytoplasmic DNA (ctDNA) like chloroplastic DNA (cpDNA) for plants are chosen. Cytoplamic DNA are haploid, non-recombining (or recombination events are rare) and generally characterized by uniparental inheritance (chloroplasts are generally

Application of PCR Technologies

**5. Pathogens** 

**5.1.1 Parasites** 

**5.1 Pathogens in humans** 

and to find new therapeutic strategies.

**5.1.1.1 PCR and diagnostic for human parasites in Central Africa** 

contribute to the maintenance of the genetic diversity.

to Humans, Animals, Plants and Pathogens from Central Africa 319

insect-pollinated and its seeds are dispersed by animals, including elephants. Using spatial genetic structure analyses, Ndiade-Bourobou et al. were able to demonstrate that dispersal distances were uncommonly high and able to connect trees present in very low density throughout forest [61]. This process allows the maintenance of high genetic diversity in reducing inbreeding effect and assures as such the survival of the species. This equilibrium is very vulnerable as both tree and animal-vectors densities have dramatically dropped due to additional effects of logging, hunting and poaching activities. For *Aucoumea klaineanea* Pierre Burseraceae, a highly logged tree species in Gabon, Born et al. show that dispersal distance is very limited and that founder effects associated with colonization processes are avoided by the homogeneity in reproductive success in adults [62]. Their results also suggest [63] that reduced density of trees and/or forest opening is balanced by higher gene dispersal distances. This result is linked with dispersal syndromes of the species that locally

A lot of diseases of animal origin and their rapid spread and possible transmission to humans (HIV/AIDS, Ebola, Avian Influenza, etc.) can pose a threat to human health. Tools have evolved from simple serological screenings to specific amplification using conventional or Real Time PCR methods, hence allowing more suitable diagnostic methods for early stage detection, identification and characterization of emerging or re-emerging pathogens. We'll successively take examples of pathogens infecting i) humans (parasites, viruses, bacteria, in section 5.1), nonhuman primates and other animals (section 5.2), and finally pathogens of plants (section 5.3).

Health in Central Africa is triggered by malaria, the most studied human parasite. Malaria transmission remains holoendemic in Central Africa in spite of decades of efforts in implementation/operational research. Other parasitic diseases are of utmost importance in term of public health, as human African trypanosomiasis (or sleeping sickness), filariasis, intestinal parasites, schistosomiasis, toxoplasmosis and amibiasis; however, they are all considered as neglected diseases. The PCR techniques contribute to the diagnostic of these infections. These techniques also improve our understanding of the physiopathology of these diseases through basic research. PCR indubitably helps to diagnose more efficiently

The Table 3 shows a few examples of PCR-based diagnostics for human parasites, although these techniques are not the gold standard for diagnosis of human parasites. The high cost of the PCR-based techniques is mainly mentioned as inconvenient. New diagnostic techniques should be implemented once it's demonstrated that the balance cost/benefit is lower than 1. First, the technique must be feasible in routine laboratories in terms of equipment and training of local agents. Secondly, the new technique has to offer a benefit in terms of clinical treatment of the patients. This clinical benefit may result in a better specificity and sensitivity, and in a reduced time to diagnosis. The improvement of sensitivity allows the detection of sub-microscopic infections, as detailed in the chapter of this book titled "Submicroscopic infections of human parasitic diseases" by Touré-Ndouo.

maternally inherited for angiosperm, paternally for gymnosperm plant species). These markers allow inference in genealogical histories of individuals, populations and/or species. It is however highly recommended to combine cytoplasmic with nuclear markers for intraspecific phylogeographic studies because of the uniparental inheritance of ctDNA. It is especially true for species with sex-biased dispersal capacities. For instance, cpDNA would show a very strong spatial structure for tree with heavy barochore (dispersed by gravity) seeds whereas nuclear genes dispersed by both seed and anemophilous (transported by wind) pollen, would not reveal any spatial structure. Therefore, sequences from nuclear genes could provide valuable information in phylogeographic assessments. They are nonetheless more complicated to analyze because of i) the difficulty to isolate haplotype from diploid organisms, ii) intragenic recombination and iii) the relatively slow pace of sequence evolution at most nuclear loci. Other nuclear PCR-based genetic markers such as microsatellites, AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA) or SNPs (described in section 2.4) are also used for phylogeographic studies, most of them being particularly valuable for population genetic studies.

#### **4.2 Importance of the past climatic changes in shaping pattern of genetic diversity in Central Africa**

The Lower Guinea forest domain (the Atlantic coastal forest distributed from Nigeria to Congo) has undergone major distribution range shifts during the Quaternary, but few studies have investigated their impact on the genetic diversity of plant species. Several phylogeographic studies using either cpDNA polymorphism [52, 53, 54, 55, 56, 57] and/or nuclear markers such as RAPD [58] and microsatellite markers [53, 59, 60] have recently been published, considering Central African trees as model species, to give insight into the historical biogeography of the region. For most of the studied species, the genetic diversity is very spatially structured throughout the species distribution giving strong phylogeographic signals. These results show that the Central African rainforest domain was very fragmented during the cool and dry periods from the Last Glacial Maximum period at the end of the Pleistocene (20000-13000 years before present) and more recently during the Little Ice Age (about 4000-2500 years before present). During these periods, most tree species and probably forest species in general, only survived in a reduced number of isolated populations in areas where environmental conditions remained suitable. The question is now to test for the presence of forest refugia in Central Africa, in other words: did forestspecies all survived in the same areas? In this case, effort for the conservation of these areas must be treated with the highest priority as refugia may play a major role in the survival of forest-species, while climate is changing, probably in buffering effect of the fluctuations. First results show that some refugia were shared among several tree species with one main refugium in the North and one in the South of the thermal equator (e.g. *Milicia excelsa* in [53], *Erythrophleum suaveolens* in [55], *Irvingia gabonensis* in [56], *Distemonanthus benthamianus* in [60]. Other species managed to survive in additional areas with at least four remaining populations for *Aucoumea klaineana* in Gabon [59]. More species covering all functional groups (pioneer, understorey, long-lived, etc.) must be studied to be able to infer general trends to allow predictions about impact of the Global Climate Change on species distribution.

#### **4.3 Importance of species life history traits in the maintenance of genetic diversity**

At finer scale, microsatellite loci were used to infer species dispersal ability of threatened tree species. *Baillonella toxisperma* Pierre Sapotaceae is a very low-density tree. The species is insect-pollinated and its seeds are dispersed by animals, including elephants. Using spatial genetic structure analyses, Ndiade-Bourobou et al. were able to demonstrate that dispersal distances were uncommonly high and able to connect trees present in very low density throughout forest [61]. This process allows the maintenance of high genetic diversity in reducing inbreeding effect and assures as such the survival of the species. This equilibrium is very vulnerable as both tree and animal-vectors densities have dramatically dropped due to additional effects of logging, hunting and poaching activities. For *Aucoumea klaineanea* Pierre Burseraceae, a highly logged tree species in Gabon, Born et al. show that dispersal distance is very limited and that founder effects associated with colonization processes are avoided by the homogeneity in reproductive success in adults [62]. Their results also suggest [63] that reduced density of trees and/or forest opening is balanced by higher gene dispersal distances. This result is linked with dispersal syndromes of the species that locally contribute to the maintenance of the genetic diversity.

## **5. Pathogens**

318 Polymerase Chain Reaction

maternally inherited for angiosperm, paternally for gymnosperm plant species). These markers allow inference in genealogical histories of individuals, populations and/or species. It is however highly recommended to combine cytoplasmic with nuclear markers for intraspecific phylogeographic studies because of the uniparental inheritance of ctDNA. It is especially true for species with sex-biased dispersal capacities. For instance, cpDNA would show a very strong spatial structure for tree with heavy barochore (dispersed by gravity) seeds whereas nuclear genes dispersed by both seed and anemophilous (transported by wind) pollen, would not reveal any spatial structure. Therefore, sequences from nuclear genes could provide valuable information in phylogeographic assessments. They are nonetheless more complicated to analyze because of i) the difficulty to isolate haplotype from diploid organisms, ii) intragenic recombination and iii) the relatively slow pace of sequence evolution at most nuclear loci. Other nuclear PCR-based genetic markers such as microsatellites, AFLP (Amplified Fragment Length Polymorphism), RAPD (Random Amplification of Polymorphic DNA) or SNPs (described in section 2.4) are also used for phylogeographic studies, most of

**4.2 Importance of the past climatic changes in shaping pattern of genetic diversity in** 

The Lower Guinea forest domain (the Atlantic coastal forest distributed from Nigeria to Congo) has undergone major distribution range shifts during the Quaternary, but few studies have investigated their impact on the genetic diversity of plant species. Several phylogeographic studies using either cpDNA polymorphism [52, 53, 54, 55, 56, 57] and/or nuclear markers such as RAPD [58] and microsatellite markers [53, 59, 60] have recently been published, considering Central African trees as model species, to give insight into the historical biogeography of the region. For most of the studied species, the genetic diversity is very spatially structured throughout the species distribution giving strong phylogeographic signals. These results show that the Central African rainforest domain was very fragmented during the cool and dry periods from the Last Glacial Maximum period at the end of the Pleistocene (20000-13000 years before present) and more recently during the Little Ice Age (about 4000-2500 years before present). During these periods, most tree species and probably forest species in general, only survived in a reduced number of isolated populations in areas where environmental conditions remained suitable. The question is now to test for the presence of forest refugia in Central Africa, in other words: did forestspecies all survived in the same areas? In this case, effort for the conservation of these areas must be treated with the highest priority as refugia may play a major role in the survival of forest-species, while climate is changing, probably in buffering effect of the fluctuations. First results show that some refugia were shared among several tree species with one main refugium in the North and one in the South of the thermal equator (e.g. *Milicia excelsa* in [53], *Erythrophleum suaveolens* in [55], *Irvingia gabonensis* in [56], *Distemonanthus benthamianus* in [60]. Other species managed to survive in additional areas with at least four remaining populations for *Aucoumea klaineana* in Gabon [59]. More species covering all functional groups (pioneer, understorey, long-lived, etc.) must be studied to be able to infer general trends to allow

predictions about impact of the Global Climate Change on species distribution.

**4.3 Importance of species life history traits in the maintenance of genetic diversity**  At finer scale, microsatellite loci were used to infer species dispersal ability of threatened tree species. *Baillonella toxisperma* Pierre Sapotaceae is a very low-density tree. The species is

them being particularly valuable for population genetic studies.

**Central Africa** 

A lot of diseases of animal origin and their rapid spread and possible transmission to humans (HIV/AIDS, Ebola, Avian Influenza, etc.) can pose a threat to human health. Tools have evolved from simple serological screenings to specific amplification using conventional or Real Time PCR methods, hence allowing more suitable diagnostic methods for early stage detection, identification and characterization of emerging or re-emerging pathogens. We'll successively take examples of pathogens infecting i) humans (parasites, viruses, bacteria, in section 5.1), nonhuman primates and other animals (section 5.2), and finally pathogens of plants (section 5.3).

#### **5.1 Pathogens in humans**

## **5.1.1 Parasites**

Health in Central Africa is triggered by malaria, the most studied human parasite. Malaria transmission remains holoendemic in Central Africa in spite of decades of efforts in implementation/operational research. Other parasitic diseases are of utmost importance in term of public health, as human African trypanosomiasis (or sleeping sickness), filariasis, intestinal parasites, schistosomiasis, toxoplasmosis and amibiasis; however, they are all considered as neglected diseases. The PCR techniques contribute to the diagnostic of these infections. These techniques also improve our understanding of the physiopathology of these diseases through basic research. PCR indubitably helps to diagnose more efficiently and to find new therapeutic strategies.

#### **5.1.1.1 PCR and diagnostic for human parasites in Central Africa**

The Table 3 shows a few examples of PCR-based diagnostics for human parasites, although these techniques are not the gold standard for diagnosis of human parasites. The high cost of the PCR-based techniques is mainly mentioned as inconvenient. New diagnostic techniques should be implemented once it's demonstrated that the balance cost/benefit is lower than 1. First, the technique must be feasible in routine laboratories in terms of equipment and training of local agents. Secondly, the new technique has to offer a benefit in terms of clinical treatment of the patients. This clinical benefit may result in a better specificity and sensitivity, and in a reduced time to diagnosis. The improvement of sensitivity allows the detection of sub-microscopic infections, as detailed in the chapter of this book titled "Submicroscopic infections of human parasitic diseases" by Touré-Ndouo.

Application of PCR Technologies

Central African routine laboratories.

**Fundamental research** 

malaria field related to PCR-based techniques.

to Humans, Animals, Plants and Pathogens from Central Africa 321

day, cost/benefit may be convincing and PCR-based diagnostic may be implemented. However, the benefits linked to PCR-based diagnosis for malaria are the identification of the different *Plasmodium* species and a lower detection limit. This is not necessarily clinically relevant. In addition, the existence of alternative diagnostic techniques as rapid diagnostic tests (RDTs) based on immunochromatographic assays to detect specific *Plasmodium* antigens that

Finally, PCR-based diagnosis is a very good tool for epidemiological survey. It still needs improvement in terms of cost, feasibility and quickness to deserve an implementation in

As malaria is the most prevalent infection in Central Africa with the higher mortality incidence, this part will focus on malaria. The aim of this part is to point out the central role of PCR techniques in malaria research performed in Central Africa, without providing an exhausting list of its applications. The Figure 1 summarizes the research applications in the

The link between fundamental and operational research is tight, particularly for pathologies like malaria that need field studies to confirm hypotheses. Molecular epidemiology for malaria parasite is an example of this tight link. The study of SNPs related to drug resistance in *P. falciparum* on a genome-wide scale in a diversity of strains from Africa provides information on the frequency of the studied SNPs. If drug resistance requires several SNPs and those naturally occurring SNPs are rare in most genes, it may last years for the parasite to acquire a drug resistant phenotype. So, it is important to know whether *P. falciparum* genome presents low or high level of SNPs in endemic areas. However, the generation of new *P. falciparum* variants encoding for different levels of SNPs can result of tandem repeats of similar sequences (called RATs) that could undergo slip-strand mispairing. Replication slippage or deletion mechanisms lead to the apparition or lost of different RATs. Interestingly, the high frequency of RATs close to drug resistance or immune response

target sequences can result in a fast increase of important SNPs (reviewed in [71]).

the parasites and their diversity in the different geographic endemic areas.

The development of new diagnostics for malaria is also dependant of PCR-based techniques. The first RDTs for malaria were supplied more than 15 years ago. Some of them are based on immunochromatographic detection of *P. falciparum* histidine-rich protein 2 (PfHRP2), using monoclonal antibodies. PfHRP2 is an abundant circulating protein easily detectable in the blood of patients. However, some studies reported variable test performances. In that way, complementary studies were necessary to compare the PfHRP2 sequences from several parasite strains and the potential consequences on the performance of PfHR2-based RDTs. The genetic diversity of the *pfhrp2* gene was studied in isolates originating from 19 countries including Central African countries and the relationship between the *pfhrp2* diversities and the sensitivities of PfHRP2-based RDTs was assessed [72]. The results indicated that 2 types of repeats in the DNA sequence of PfHRP2 were predictive of RDT detection sensitivity with 87.5% accuracy. These results pointed out the importance of the genetic background of

Parasite antigen diversity studies at the molecular level are also performed for vaccine research. *P. falciparum* erythrocyte membrane protein 1 (PfEMP1) is a major vaccine target as

are recommended by the WHO, increases the cost/benefit ratio for PCR [68, 69].

**5.1.1.2 PCR and research on human parasites in Central Africa** 

The main advantages of diagnosis by PCR for human parasites from Central Africa are both the higher specificity and the small amounts of blood or tissue required. The specificity of DNA sequences offers a simple tool to distinguish species. As an example, the species spectrum of intestinal parasites involved in hospitalized AIDS patients was determined in the Democratic Republic of the Congo [64]. Opportunistic infections were detected by PCR, as *Cryptosporidium* sp., *Enterocytozoon bieneusi*, *Isospora belli* and *Encephalitozoon intestinalis*. The other intestinal parasites detected by PCR were *Entamoeba histolytica*, *Entamoeba dispar*, *Ascoris lumbricoides*, *Giardia intestinalis*, hookworm, *Trichiuris trichiura*, *Enterobius vermicularis*, and *Schistosoma mansoni*. Furthermore, the PCR-based diagnostic is quite more sensitive than microscopic examination, which is sometimes not sufficient to differentiate various parasite species. This is clearly the case for filariasis [65] and schistosomiasis [66]. In human sleeping sickness, PCR on blood allows avoiding painful lumbar punctures and was proposed as a less-invasive alternative to replace the cerebrospinal fluid examination. However, in this case, PCR is a good tool for primodiagnostic but cannot be used for posttreatment follow-up. Indeed, the high sensitivity of PCR leads to detection of persisting DNA in blood of patients even after successful treatment [67].


\* Se. sensitivity, Spe. Specificity, CSF cerebrospinal fluid

§ qPCR, quantitative polymerase chain reaction

\$ 30% of samples not done by PCR

Table 3. Efficiency and characteristics of PCR-based diagnostic in several endemic human parasitosis that are prevalent in Central Africa

Malaria constitutes one of the major public health problems in Central Africa. As *Plasmodium falciparum* infection is deadly when untreated in children and pregnant women, its diagnostic has to be accurate and fast. At hospital level, where many malaria diagnostics are performed a day, cost/benefit may be convincing and PCR-based diagnostic may be implemented. However, the benefits linked to PCR-based diagnosis for malaria are the identification of the different *Plasmodium* species and a lower detection limit. This is not necessarily clinically relevant. In addition, the existence of alternative diagnostic techniques as rapid diagnostic tests (RDTs) based on immunochromatographic assays to detect specific *Plasmodium* antigens that

Finally, PCR-based diagnosis is a very good tool for epidemiological survey. It still needs improvement in terms of cost, feasibility and quickness to deserve an implementation in Central African routine laboratories.

are recommended by the WHO, increases the cost/benefit ratio for PCR [68, 69].

#### **5.1.1.2 PCR and research on human parasites in Central Africa**

As malaria is the most prevalent infection in Central Africa with the higher mortality incidence, this part will focus on malaria. The aim of this part is to point out the central role of PCR techniques in malaria research performed in Central Africa, without providing an exhausting list of its applications. The Figure 1 summarizes the research applications in the malaria field related to PCR-based techniques.

#### **Fundamental research**

320 Polymerase Chain Reaction

The main advantages of diagnosis by PCR for human parasites from Central Africa are both the higher specificity and the small amounts of blood or tissue required. The specificity of DNA sequences offers a simple tool to distinguish species. As an example, the species spectrum of intestinal parasites involved in hospitalized AIDS patients was determined in the Democratic Republic of the Congo [64]. Opportunistic infections were detected by PCR, as *Cryptosporidium* sp., *Enterocytozoon bieneusi*, *Isospora belli* and *Encephalitozoon intestinalis*. The other intestinal parasites detected by PCR were *Entamoeba histolytica*, *Entamoeba dispar*, *Ascoris lumbricoides*, *Giardia intestinalis*, hookworm, *Trichiuris trichiura*, *Enterobius vermicularis*, and *Schistosoma mansoni*. Furthermore, the PCR-based diagnostic is quite more sensitive than microscopic examination, which is sometimes not sufficient to differentiate various parasite species. This is clearly the case for filariasis [65] and schistosomiasis [66]. In human sleeping sickness, PCR on blood allows avoiding painful lumbar punctures and was proposed as a less-invasive alternative to replace the cerebrospinal fluid examination. However, in this case, PCR is a good tool for primodiagnostic but cannot be used for posttreatment follow-up. Indeed, the high sensitivity of PCR leads to detection of persisting

**Se.\* Spe.\* Advantage Inconvenient Ref. technique Reference** 

High cost

for follow-up

Cost

High cost; Not intended for routine diagnostic

Microscopy examination of thick and thin blood smears

Microscopic analysis of the CSF

Knott's concentration and microscopic examination

Microscopic examination of Kato

Microscopic examination of filtrated urine samples

[70]

[67]

[65]

DNA in blood of patients even after successful treatment [67].

Limit of detection greatly reduced

88.40% 99.20% Non invasive Not suitable

High se. and spe. for 3 filariosis coendemic

High spe. to distinguish species

[66] *S. haematobium*

Table 3. Efficiency and characteristics of PCR-based diagnostic in several endemic human

Malaria constitutes one of the major public health problems in Central Africa. As *Plasmodium falciparum* infection is deadly when untreated in children and pregnant women, its diagnostic has to be accurate and fast. At hospital level, where many malaria diagnostics are performed a

*Plasmodium* spp

*T. brucei gambiense* in blood by PCR

*L. loa*, *M. perstans* and *W. bancrofti* by nested PCR

*S. mansoni* in fecal samples by qPCR

in fecal samples by qPCR

\$ 30% of samples not done by PCR

(qPCR)§ 99.40% 90.90%

100%\$ 100%\$

86.50% 100%

82.80% 100%

\* Se. sensitivity, Spe. Specificity, CSF cerebrospinal fluid § qPCR, quantitative polymerase chain reaction

parasitosis that are prevalent in Central Africa

The link between fundamental and operational research is tight, particularly for pathologies like malaria that need field studies to confirm hypotheses. Molecular epidemiology for malaria parasite is an example of this tight link. The study of SNPs related to drug resistance in *P. falciparum* on a genome-wide scale in a diversity of strains from Africa provides information on the frequency of the studied SNPs. If drug resistance requires several SNPs and those naturally occurring SNPs are rare in most genes, it may last years for the parasite to acquire a drug resistant phenotype. So, it is important to know whether *P. falciparum* genome presents low or high level of SNPs in endemic areas. However, the generation of new *P. falciparum* variants encoding for different levels of SNPs can result of tandem repeats of similar sequences (called RATs) that could undergo slip-strand mispairing. Replication slippage or deletion mechanisms lead to the apparition or lost of different RATs. Interestingly, the high frequency of RATs close to drug resistance or immune response target sequences can result in a fast increase of important SNPs (reviewed in [71]).

The development of new diagnostics for malaria is also dependant of PCR-based techniques. The first RDTs for malaria were supplied more than 15 years ago. Some of them are based on immunochromatographic detection of *P. falciparum* histidine-rich protein 2 (PfHRP2), using monoclonal antibodies. PfHRP2 is an abundant circulating protein easily detectable in the blood of patients. However, some studies reported variable test performances. In that way, complementary studies were necessary to compare the PfHRP2 sequences from several parasite strains and the potential consequences on the performance of PfHR2-based RDTs. The genetic diversity of the *pfhrp2* gene was studied in isolates originating from 19 countries including Central African countries and the relationship between the *pfhrp2* diversities and the sensitivities of PfHRP2-based RDTs was assessed [72]. The results indicated that 2 types of repeats in the DNA sequence of PfHRP2 were predictive of RDT detection sensitivity with 87.5% accuracy. These results pointed out the importance of the genetic background of the parasites and their diversity in the different geographic endemic areas.

Parasite antigen diversity studies at the molecular level are also performed for vaccine research. *P. falciparum* erythrocyte membrane protein 1 (PfEMP1) is a major vaccine target as

Application of PCR Technologies

[80, 81].

fundamental research

**5.1.2 Viruses** 

to Humans, Animals, Plants and Pathogens from Central Africa 323

fundamental research. Sulfadoxine-pyrimethamine (SP) treatment has been used for a long time as second-line treatment for uncomplicated malaria in case of chloroquine treatment failure. The parasite mechanisms of resistance to SP have been well described and result in SNPs located on *Pfdhfr* and *Pfdhps* genes that appear in a few years following the implementation of such molecules. PCR followed by sequencing is the usual technique to study the rate of these mutations. In Gabon, Congo and Cameroon, the rate of *Pfdhfr* and *Pfdhps* mutations has been followed for years and constituted serious arguments to search other alternative treatments to chloroquine [77, 78, 79]. Since the era of ACT has begun, research teams based in Central Africa also use PCR-based techniques to follow the emergence of molecular markers related to the resistance to artemisinin-based molecules

Malaria prevention is also carried out through the use of insecticide treated materials or indoor residual spraying in Central Africa. This strategy has some implications on the spread of pyrethroid resistance in *Anopheles gambiae* and this has become a major concern in Africa. A PCR-RFLP assay was developed in Cameroon to follow two SNPs in the gene encoding subunit 2 of the sodium channel, also called the knockdown (*kdr*) mutations [82]. Since that time, studies to follow the situation of insecticide resistance are performed. In Gabon, both *kdr-e* and *kdr-w* alleles were shown to be present at high frequency in the *Anopheles gambiae* population. Of course, these results have implications for the effectiveness of the current

vector control programmes that are based on pyrethroid-impregnated bed nets [83].

Fig. 1. The use of PCR-based techniques in the malaria field for operational and

This part will describe how the PCR-based techniques have been applied to many viruses infecting humans living in Central Africa, such as Human Immunodeficiency Virus (HIV), Human T cell Leukemia Virus (HTLV), Influenza virus, Hepatitis virus, and Ebola virus, for their origin, circulation, diversity, diagnosis, surveillance, and/or monitoring. Table 4 gives

evidence supports the central role of PfEMP1 in the development of a protective acquired immunity in children and pregnant women living in high level endemic areas. However, PfEMP1 undergoes a serious problem. PfEMP1 is highly polymorphic and encoded by a gene family of 50-60 *var* genes. To identify specific *var* genes or domain structuring these genes and related to protective immunity, many molecular studies were done and are still currently performed, all based on the basic molecular technique, PCR. In pregnancyassociated malaria, some studies showed that the *var* gene expressed called *var2csa* is relatively conserved. A comparative study showed that Duffy binding–like domains from placental parasites from Gabon and Cameroon shared 85%–99% amino-acid identities, confirming the conserved nature of placental variants [73]. This demonstration of sequence conservation in PfEMP1 DNA and its implication in the binding to chondroitin sulfate A (CSA) and to the pathology was clearly relevant to vaccine development for pregnancyassociated malaria. Today, it is largely recognized that the parasite ligand mediating CSA binding and causing malaria in pregnancy is VAR2CSA, a member of PfEMP1 family, and that it is a promising target for vaccine design. Recent researches focus on the molecular variability of *var2csa* in field isolates and on the immune response induced by different domains of the protein. Vaccine research largely depends on immunological studies, as this is clearly the case with the example of PfEMP1. However, PCR is not the favorite technique for such studies unlike flow cytometry or Enzyme Linked Immunosorbent Assay (ELISA). For immunological topics related to malaria, PCR is mainly used in studies on human genetic markers linked to malaria protection (see section 2 of this chapter).

#### **Operational research**

The evaluation of the therapeutic and control strategies implemented to fight against malaria constitutes operational research. First, PCR has become an essential technique for the evaluation of antimalarial treatment efficiency. Historically, *in vivo* resistance of *P. falciparum* to antimalarial drugs was classified into three grades, RI (low), RII (intermediate), and RIII (high) [74]. Since 2002, therapeutic failures are divided in early and late treatment failures (ETF, LTF), and LTF includes late clinical failures and late parasitological failures [75]. Both classifications are based on follow-up studies of parasitemia in patients treated with antimalarial treatments. Usually, follow-ups last 28 days, but are now extended to 42 days with the use of artemisinin-based treatment combinations (ACT) [75]. The classification relies on the reappearance or not of parasites during the follow-up. In highly endemic areas for malaria, the reappearance of parasites may be linked to the persistence of the initial infection, or to a new infection that occurred during the follow-up (the incubation time for *P. falciparum* is 7 to 10 days). A first study was performed in Central Africa in Gabon to demonstrate the great advantage of PCR to distinguish recrudescent *P. falciparum* clones from new ones, in studies of antimalarial treatment efficacy [76]. The technique involves amplification by PCR of regions of 3 highly polymorphic parasite genes, merozoïte surface protein-1 (*msp-1*), *msp-2* and glutamate-rich protein (*glurp*). Through this study, the authors showed that 39% of RI resistant cases were in fact due to new infections. Today, PCR genotyping is systematically included in treatment efficacy studies [75].

The implementation of therapeutic strategies for malaria in a specific area has an impact on the deployment of parasite resistance to the drug used. It is of high importance to study the development of parasite resistance in malaria endemic areas, in order to suggest new policies once treatments become inefficient. PCR is definitely the basic tool to perform such studies once molecular mechanisms of resistance have been demonstrated through more

evidence supports the central role of PfEMP1 in the development of a protective acquired immunity in children and pregnant women living in high level endemic areas. However, PfEMP1 undergoes a serious problem. PfEMP1 is highly polymorphic and encoded by a gene family of 50-60 *var* genes. To identify specific *var* genes or domain structuring these genes and related to protective immunity, many molecular studies were done and are still currently performed, all based on the basic molecular technique, PCR. In pregnancyassociated malaria, some studies showed that the *var* gene expressed called *var2csa* is relatively conserved. A comparative study showed that Duffy binding–like domains from placental parasites from Gabon and Cameroon shared 85%–99% amino-acid identities, confirming the conserved nature of placental variants [73]. This demonstration of sequence conservation in PfEMP1 DNA and its implication in the binding to chondroitin sulfate A (CSA) and to the pathology was clearly relevant to vaccine development for pregnancyassociated malaria. Today, it is largely recognized that the parasite ligand mediating CSA binding and causing malaria in pregnancy is VAR2CSA, a member of PfEMP1 family, and that it is a promising target for vaccine design. Recent researches focus on the molecular variability of *var2csa* in field isolates and on the immune response induced by different domains of the protein. Vaccine research largely depends on immunological studies, as this is clearly the case with the example of PfEMP1. However, PCR is not the favorite technique for such studies unlike flow cytometry or Enzyme Linked Immunosorbent Assay (ELISA). For immunological topics related to malaria, PCR is mainly used in studies on human

genetic markers linked to malaria protection (see section 2 of this chapter).

genotyping is systematically included in treatment efficacy studies [75].

The evaluation of the therapeutic and control strategies implemented to fight against malaria constitutes operational research. First, PCR has become an essential technique for the evaluation of antimalarial treatment efficiency. Historically, *in vivo* resistance of *P. falciparum* to antimalarial drugs was classified into three grades, RI (low), RII (intermediate), and RIII (high) [74]. Since 2002, therapeutic failures are divided in early and late treatment failures (ETF, LTF), and LTF includes late clinical failures and late parasitological failures [75]. Both classifications are based on follow-up studies of parasitemia in patients treated with antimalarial treatments. Usually, follow-ups last 28 days, but are now extended to 42 days with the use of artemisinin-based treatment combinations (ACT) [75]. The classification relies on the reappearance or not of parasites during the follow-up. In highly endemic areas for malaria, the reappearance of parasites may be linked to the persistence of the initial infection, or to a new infection that occurred during the follow-up (the incubation time for *P. falciparum* is 7 to 10 days). A first study was performed in Central Africa in Gabon to demonstrate the great advantage of PCR to distinguish recrudescent *P. falciparum* clones from new ones, in studies of antimalarial treatment efficacy [76]. The technique involves amplification by PCR of regions of 3 highly polymorphic parasite genes, merozoïte surface protein-1 (*msp-1*), *msp-2* and glutamate-rich protein (*glurp*). Through this study, the authors showed that 39% of RI resistant cases were in fact due to new infections. Today, PCR

The implementation of therapeutic strategies for malaria in a specific area has an impact on the deployment of parasite resistance to the drug used. It is of high importance to study the development of parasite resistance in malaria endemic areas, in order to suggest new policies once treatments become inefficient. PCR is definitely the basic tool to perform such studies once molecular mechanisms of resistance have been demonstrated through more

**Operational research** 

fundamental research. Sulfadoxine-pyrimethamine (SP) treatment has been used for a long time as second-line treatment for uncomplicated malaria in case of chloroquine treatment failure. The parasite mechanisms of resistance to SP have been well described and result in SNPs located on *Pfdhfr* and *Pfdhps* genes that appear in a few years following the implementation of such molecules. PCR followed by sequencing is the usual technique to study the rate of these mutations. In Gabon, Congo and Cameroon, the rate of *Pfdhfr* and *Pfdhps* mutations has been followed for years and constituted serious arguments to search other alternative treatments to chloroquine [77, 78, 79]. Since the era of ACT has begun, research teams based in Central Africa also use PCR-based techniques to follow the emergence of molecular markers related to the resistance to artemisinin-based molecules [80, 81].

Malaria prevention is also carried out through the use of insecticide treated materials or indoor residual spraying in Central Africa. This strategy has some implications on the spread of pyrethroid resistance in *Anopheles gambiae* and this has become a major concern in Africa. A PCR-RFLP assay was developed in Cameroon to follow two SNPs in the gene encoding subunit 2 of the sodium channel, also called the knockdown (*kdr*) mutations [82]. Since that time, studies to follow the situation of insecticide resistance are performed. In Gabon, both *kdr-e* and *kdr-w* alleles were shown to be present at high frequency in the *Anopheles gambiae* population. Of course, these results have implications for the effectiveness of the current vector control programmes that are based on pyrethroid-impregnated bed nets [83].

Fig. 1. The use of PCR-based techniques in the malaria field for operational and fundamental research

#### **5.1.2 Viruses**

This part will describe how the PCR-based techniques have been applied to many viruses infecting humans living in Central Africa, such as Human Immunodeficiency Virus (HIV), Human T cell Leukemia Virus (HTLV), Influenza virus, Hepatitis virus, and Ebola virus, for their origin, circulation, diversity, diagnosis, surveillance, and/or monitoring. Table 4 gives

Application of PCR Technologies

to Humans, Animals, Plants and Pathogens from Central Africa 325

countries worldwide have been offered early infant diagnosis (EID) and antiretroviral treatment thanks to the Clinton HIV/AIDS initiative (CHAI) and UNICEF, both managing the funds from UNITAID. The Amplicor HIV-1 DNA commercial test is currently used in several laboratories throughout Africa, and Cameroon is probably the leading country in Central Africa with a well-developed national EID programme, implemented by the

Two main tests employing PCR techniques are useful for the biological follow-up of HIV-1 infected individuals i) the viral load (VL), which uses RNA PCR and ii) the resistance testing, which consists in amplification of specific viral fragments and sequencing. Viral load is mostly useful to follow the progression of the disease and for therapeutic monitoring as well. According to the commercial kits that are currently available, products of amplification can either be detected at the end of the reaction or while they accumulate in a real time manner. The lack of a commercially available viral load assay for HIV-2 is a concern for the proper management of patients infected with HIV-2 strains [106]. The resistance testing is actually an HIV-1 genotyping assay where the protease and the reverse transcriptase conserved regions of the *pol* gene are amplified and sequenced, as described by Fokam et al. [107]. Only two commercial tests approved by the Food and Drug Administration are currently available, and have been used widely to follow-up patients under antiretroviral treatment [108, 109, 110] and to report drug resistance mutations in HIV-1 reverse transcriptase or protease [109, 111, 112]. However, such commercial kits are very expensive for resource-limited countries like those of Central Africa and also their performance is questionable because of the great diversity of strains found in that region. For these reasons, an in-house genotyping assay has been developed in Cameroon recently and it is considered as more performant and cost effective than commercial kits [107].

Implementation research is essential for the control of infectious diseases of poverty [113]. Although PCR technologies are sophisticated and require a certain level of technical

The heteroduplex mobility analysis (HMA) is a molecular biology technique based on PCR amplification then followed by polyacrylamide gel electrophoresis analysis. This method has been first used for the subtype determination of HIV-1 group M envelope sequences,

Heteroduplexes are formed with uncharacterized DNA fragments and known DNA sequences (as reference) included in the kit. Importantly, *env* gene fragments of uncharacterized DNA fragments are amplified by nested PCR whereas the reference

Mobility of such heteroduplexes is analyzed on polyacrylamide gels. The closest is the unknown DNA sequence with the reference sequence; the fastest is the mobility of the

The HMA technique has been used to characterize HIV strains from Cameroon [1].

sequences are obtained by direct amplification of plasmids from the kit.

Ministry of Public Health in the 10 regions of the country since 2007 [105].

**PCR allows the management of HIV infection** 

Box 1. Heteroduplex Mobility Analysis

Principle of the HMA test:

**The use of PCR in implementation research** 

heteroduplex on the polyacrylamide gel.

but has been recently developed for *gag* gene sequences.

several examples of pathogens infecting humans in Central Africa, which have benefited from PCR technologies, with a particular emphasis on viruses.

#### **5.1.2.1 Human Immunodeficiency Virus (HIV)**

Central Africa has been described as the "epicenter of the HIV pandemic"[84]. Scores of articles have used PCR methods to report findings related to the viral diversity of HIV in this region, emergence of new strains [85] and recombinant forms [86], emergence of resistance to antiretroviral drugs [87], and challenges encountered for the genotyping tests because of the broad diversity of HIV strains [88]. In this section we'll explain the usefulness of PCR in i) the identification of various HIV strains found in Central Africa, ii) the early diagnosis of HIV, especially in exposed infants, iii) the management of infected patients, iv) implementation research and finally, we'll underline the need of an African AIDS vaccine.

#### **PCR has help in the discovery and description of the virus**

Since the discovery of HIV in the early 80s by Montagnier and Gallo, many strains, types, subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been described and characterized in patients from the Central African region. The discovery of new HIV variants occurred by atypical serological reaction, and confirmation was obtained by simple PCR, nested PCR, heteroduplex mobility assay (HMA) (see Box 1) or sequencing. Particularly, full-length genomes sequencing has been instrumental in the characterization of new HIV CRFs, such as HIV-1 CRF 25\_cpx [89] and CRF 22\_01A1 [86, 90] in Cameroon. Obviously, the characterization of all these variants has an impact on HIV diagnosis, treatment and vaccine development, especially for the HIV-infected individuals leaving in Central Africa. The genetic diversity of HIV-1 group M in the republic of Congo was described and documented [91]. This was achieved using specific PCR coupled to HMA techniques of the *env* and *gag* genes (see Box 1). In Equatorial Guinea, Hunt et al. described the variability of HIV-1 group O, while Peeters et al. performed a wider study of HIV-1 group O distribution in Africa [92, 93, 94]. Although ELISA was mainly used in this latter study, indeterminate cases were solved using PCR. In Gabon, a great quantity of HIV strains collected from 1986 to 1994 was characterized by molecular biology techniques (PCR, sequencing); then phylogenetic trees were constructed [95]. A high prevalence of HIV-1 recombinant forms has been reported in Gabon [96]. In Cameroon, many studies have been carried out on genotyping subtypes of HIV-1 [86, 97, 98, 99]. Recently, new HIV-1 groups named group N and group P have been identified from Cameroonian patients [100, 101, 102, 103].

### **PCR is used routinely for the diagnosis of HIV**

Despite antibody testing being commonly used in HIV RDTs, this methodology is not suitable in children born of HIV seropositive mothers during the first 15 to 18 months of life. The reason is that maternal antibodies transferred to the infant during pregnancy or breastfeeding persist up to 18 months and could give false positive results. Therefore, detection of proviral DNA by PCR is recommended for the early diagnosis in HIV-exposed infants. Detection of HIV proviral DNA is performed using the Roche Amplicor HIV-1 DNA commercial test, which is so far considered as the gold standard. This test reveals an HIV-1 infection within neonates and infants from 6 weeks of life and beyond. This test targets the *gag* gene during amplification where a fragment of 120bp is amplified and then, detection is based on ELISA. The kit is stored at 4°C and was especially designed for HIV-1 group M. Blood samples are collected as Dried Blood Spots (DBS), which have already been used for nationwide HIV prevalence survey in Africa [104]. More than 305,000 children in 34

several examples of pathogens infecting humans in Central Africa, which have benefited

Central Africa has been described as the "epicenter of the HIV pandemic"[84]. Scores of articles have used PCR methods to report findings related to the viral diversity of HIV in this region, emergence of new strains [85] and recombinant forms [86], emergence of resistance to antiretroviral drugs [87], and challenges encountered for the genotyping tests because of the broad diversity of HIV strains [88]. In this section we'll explain the usefulness of PCR in i) the identification of various HIV strains found in Central Africa, ii) the early diagnosis of HIV, especially in exposed infants, iii) the management of infected patients, iv) implementation research and finally, we'll underline the need of an African AIDS vaccine.

Since the discovery of HIV in the early 80s by Montagnier and Gallo, many strains, types, subtypes, circulating recombinant forms (CRFs) and unique recombinant forms (URFs) have been described and characterized in patients from the Central African region. The discovery of new HIV variants occurred by atypical serological reaction, and confirmation was obtained by simple PCR, nested PCR, heteroduplex mobility assay (HMA) (see Box 1) or sequencing. Particularly, full-length genomes sequencing has been instrumental in the characterization of new HIV CRFs, such as HIV-1 CRF 25\_cpx [89] and CRF 22\_01A1 [86, 90] in Cameroon. Obviously, the characterization of all these variants has an impact on HIV diagnosis, treatment and vaccine development, especially for the HIV-infected individuals leaving in Central Africa. The genetic diversity of HIV-1 group M in the republic of Congo was described and documented [91]. This was achieved using specific PCR coupled to HMA techniques of the *env* and *gag* genes (see Box 1). In Equatorial Guinea, Hunt et al. described the variability of HIV-1 group O, while Peeters et al. performed a wider study of HIV-1 group O distribution in Africa [92, 93, 94]. Although ELISA was mainly used in this latter study, indeterminate cases were solved using PCR. In Gabon, a great quantity of HIV strains collected from 1986 to 1994 was characterized by molecular biology techniques (PCR, sequencing); then phylogenetic trees were constructed [95]. A high prevalence of HIV-1 recombinant forms has been reported in Gabon [96]. In Cameroon, many studies have been carried out on genotyping subtypes of HIV-1 [86, 97, 98, 99]. Recently, new HIV-1 groups named group N and group P have been

Despite antibody testing being commonly used in HIV RDTs, this methodology is not suitable in children born of HIV seropositive mothers during the first 15 to 18 months of life. The reason is that maternal antibodies transferred to the infant during pregnancy or breastfeeding persist up to 18 months and could give false positive results. Therefore, detection of proviral DNA by PCR is recommended for the early diagnosis in HIV-exposed infants. Detection of HIV proviral DNA is performed using the Roche Amplicor HIV-1 DNA commercial test, which is so far considered as the gold standard. This test reveals an HIV-1 infection within neonates and infants from 6 weeks of life and beyond. This test targets the *gag* gene during amplification where a fragment of 120bp is amplified and then, detection is based on ELISA. The kit is stored at 4°C and was especially designed for HIV-1 group M. Blood samples are collected as Dried Blood Spots (DBS), which have already been used for nationwide HIV prevalence survey in Africa [104]. More than 305,000 children in 34

from PCR technologies, with a particular emphasis on viruses.

**PCR has help in the discovery and description of the virus** 

identified from Cameroonian patients [100, 101, 102, 103].

**PCR is used routinely for the diagnosis of HIV** 

**5.1.2.1 Human Immunodeficiency Virus (HIV)** 

countries worldwide have been offered early infant diagnosis (EID) and antiretroviral treatment thanks to the Clinton HIV/AIDS initiative (CHAI) and UNICEF, both managing the funds from UNITAID. The Amplicor HIV-1 DNA commercial test is currently used in several laboratories throughout Africa, and Cameroon is probably the leading country in Central Africa with a well-developed national EID programme, implemented by the Ministry of Public Health in the 10 regions of the country since 2007 [105].

#### **PCR allows the management of HIV infection**

Two main tests employing PCR techniques are useful for the biological follow-up of HIV-1 infected individuals i) the viral load (VL), which uses RNA PCR and ii) the resistance testing, which consists in amplification of specific viral fragments and sequencing. Viral load is mostly useful to follow the progression of the disease and for therapeutic monitoring as well. According to the commercial kits that are currently available, products of amplification can either be detected at the end of the reaction or while they accumulate in a real time manner. The lack of a commercially available viral load assay for HIV-2 is a concern for the proper management of patients infected with HIV-2 strains [106]. The resistance testing is actually an HIV-1 genotyping assay where the protease and the reverse transcriptase conserved regions of the *pol* gene are amplified and sequenced, as described by Fokam et al. [107]. Only two commercial tests approved by the Food and Drug Administration are currently available, and have been used widely to follow-up patients under antiretroviral treatment [108, 109, 110] and to report drug resistance mutations in HIV-1 reverse transcriptase or protease [109, 111, 112]. However, such commercial kits are very expensive for resource-limited countries like those of Central Africa and also their performance is questionable because of the great diversity of strains found in that region. For these reasons, an in-house genotyping assay has been developed in Cameroon recently and it is considered as more performant and cost effective than commercial kits [107].

The heteroduplex mobility analysis (HMA) is a molecular biology technique based on PCR amplification then followed by polyacrylamide gel electrophoresis analysis. This method has been first used for the subtype determination of HIV-1 group M envelope sequences, but has been recently developed for *gag* gene sequences.

#### Principle of the HMA test:

Heteroduplexes are formed with uncharacterized DNA fragments and known DNA sequences (as reference) included in the kit. Importantly, *env* gene fragments of uncharacterized DNA fragments are amplified by nested PCR whereas the reference sequences are obtained by direct amplification of plasmids from the kit. Mobility of such heteroduplexes is analyzed on polyacrylamide gels. The closest is the unknown DNA sequence with the reference sequence; the fastest is the mobility of the heteroduplex on the polyacrylamide gel.

The HMA technique has been used to characterize HIV strains from Cameroon [1].

#### Box 1. Heteroduplex Mobility Analysis

#### **The use of PCR in implementation research**

Implementation research is essential for the control of infectious diseases of poverty [113]. Although PCR technologies are sophisticated and require a certain level of technical

Application of PCR Technologies

**5.1.2.3 Influenza virus** 

**5.1.2.4 Hepatitis viruses** 

GB-C/HG virus and TT virus in Gabon [141].

**5.1.2.5 Ebola virus** 

to Humans, Animals, Plants and Pathogens from Central Africa 327

few as 10 copies of HTLV-3 or HTLV-4 sequences of the gene *pol* in a small amount of DNA from human peripheral blood lymphocytes [125]. However, a new method using a single tube, multiplex, real time PCR has been developed at the Centre International de Recherches Médicales de Franceville (CIRMF), Gabon, which allows detecting HTLV-1, HTLV-2 and HTLV-3 simultaneously [126]. This new PCR-based technique could be of valuable use for

Despite influenza surveillance was increasing worldwide, developing countries in general and Central Africa in particular paid very little attention to the 2009 pandemic. Very recently however, samples from patients living with influenza-like illness in Yaounde, Cameroon were analyzed with various techniques including real time reverse transcription-polymerase chain reaction (RT-PCR) thus allowing the detection and subtyping of influenza A (H1N1 and H3N2) and B viruses from these patients [127]. Because of the H1N1 influenza A pandemic, Cameroon entered in a global surveillance network and received a laboratory equipped with a

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are endemic in the Central African region. Since the last two decades, the use of PCR techniques and phylogenetic analysis has led to characterize the genotype distribution of HCV in this area. The RNA is amplified by RT-PCR and nested PCR and the primers commonly used are specific to the 5'UTR and NS5B regions. In Cameroon, genotypes 1 and 4 are the most prevalent, but highly heterogeneous, with 5 subtypes 1, 4 subtypes 4 and unclassified subtypes, while the genotype 2 prevalence is low, with homogeneous sequences [129, 130]. Further work has help to understand the history of the HCV epidemic in Cameroon, where mass therapeutic or vaccine campaigns would have contributed to the spread of this infection during the colonial era [131]. In Gabon and Central African Republic, the predominance of the heterogeneous genotype 4 has been reported [132, 133, 134]. Equally, few HBV genotype studies have been conducted Central Africa. Makuwa et al. reported the identification of HBV-A3 in rural Gabon [135], while this genotype is co-circulating with HBV-E among Pygmies in Cameroon [136]. More recently, a pilot study was conducted in the village of Dienga, Gabon (previously described in section 2.1) with the aim of looking at potential interactions between HBV, HCV and *P. falciparum* infections, which are all very prevalent in this region [137]. In this study, HCV chronic carrier were identified by ELISA and by qualitative RT-PCR amplification of the 5' non coding region, and *P. falciparum* infection were assessed by microscopic examination and in case of negative result, by PCR targeting the gene encoding *P. falciparum* SSUrRNA, previously described by Snounou et al. [138]. Interestingly, these results showed that HCV infection may lead to slower emergence of *P. falciparum* in blood [137]. Other studies have demonstrated the usefulness of the PCR as a tool for the description of the molecular diversity of other less known/marginal viruses in this region, such as hepatitis delta virus in Cameroon [139] and in Gabon [140], or hepatitis

Since the first declaration of deaths due to Ebola virus in Zaïre in 1976, the Central African region has been particularly affected by repeated Ebola outbreaks, which affected

epidemiological studies in countries where those viruses are prevalent.

robust PCR platform for diagnosing influenza viruses in remote settings [128].

expertise and facilities that are usually not available and not affordable in poor-resources settings, implementation research studies can help to find alternative solutions. For example, the fact that DBS can replace blood samples advantageously has been instrumental in increasing access to HIV diagnosis in exposed infants living in remote settings, through the implementation and scale-up of the EID program [105]. Equally, DBS can improve the biological follow-up of HIV-1-infected individuals, both for the VL quantification and the resistance testing. Indeed, DBS, which can be collected on sites, transported and tested after a long-term storage, are suitable for the differed quantification of HIV-1 RNA, thus allowing people living with HIV/AIDS in rural areas to have access to this sophisticated test [114]. On another hand, implementation of resistance testing on DBS is in progress in Africa [115, 116] and will soon benefit HIV-1-infected patients living far from urban areas in Central Africa [108]. While waiting for the development of point of care assays, DBS appear to be a good alternative for the monitoring of HIV-1-infected people in remote settings (reviewed in [117]). However, the transport of samples and the return of results remains challenging, and need additional implementation research.

#### **Back to the sites**

Central Africa could be the ideal place where an AIDS vaccine could be designed, because of the great diversity of strains that are found in this region. However, when the scientific community is reflecting on how simian immunodeficiency virus infections hosted by African nonhuman primates could help in designing an AIDS vaccine for example, Central African scientists are absent [118]. This situation should change and African institutions, supported by their government, should advocate strongly for and invest in an African AIDS vaccine. To this end, the African AIDS Vaccine Partnership (AAVP) intends to promote cutting-edge research for the development of an African HIV vaccine [119]. In addition, the European Developing Clinical Trial Partnership (EDCTP) is supporting several African institutions from Gabon, Congo and Cameroon to build capacity for the conduct of future HIV/AIDS clinical trials [120] and is advocating for support from governments.

#### **5.1.2.2 Human T cell Lymphotropic Virus (HTLV)**

Central Africa is one of the few regions of the world where HTLV type 1 (HTLV-1) is highly endemic, as reviewed by Gessain & Mahieux [121]. Sequencing of HTLV-1 focuses on the gene *env* and the long terminal repeat fragments [122]. Molecular studies have demonstrated that the several molecular subtypes (genotypes) are related to the geographical origin and not to the disease. For example, while the subtype A is considered as cosmopolitan, the subtype B is mainly found in Central Africa (Democratic Republic of Congo, Gabon, and Cameroon). The subtype D has also been described in individuals from Cameroon, Gabon, Central African Republic, but less frequently than the subtype B, and more specifically in Pygmies. New subtypes (E and F) would be equally present in this region [121]. Interestingly, the first complete nucleotide sequence of HTLV type 2 (HTLV-2) has been obtained in a 44-year-old male living in a rural area of Gabon, by using nested PCR [123]. However, HTLV-2 does not seem to be as prevalent as HTLV-1 in this region since in a recent epidemiological survey performed on 907 pregnant women, only one case of HTLV-2 was reported [122]. In Cameroon however, HTLV-2 seroprevalence was 2.5% in Bakola Pygmies, but no HTLV-2 infection was found in Bantus [124]. HTLV type 3 (HTLV-3) and HTLV type 4 (HTLV-4) have been recently identified in primate hunters in Central Africa. Real-time PCR quantitative assays have been developed in the USA and allow detecting as few as 10 copies of HTLV-3 or HTLV-4 sequences of the gene *pol* in a small amount of DNA from human peripheral blood lymphocytes [125]. However, a new method using a single tube, multiplex, real time PCR has been developed at the Centre International de Recherches Médicales de Franceville (CIRMF), Gabon, which allows detecting HTLV-1, HTLV-2 and HTLV-3 simultaneously [126]. This new PCR-based technique could be of valuable use for epidemiological studies in countries where those viruses are prevalent.

## **5.1.2.3 Influenza virus**

326 Polymerase Chain Reaction

expertise and facilities that are usually not available and not affordable in poor-resources settings, implementation research studies can help to find alternative solutions. For example, the fact that DBS can replace blood samples advantageously has been instrumental in increasing access to HIV diagnosis in exposed infants living in remote settings, through the implementation and scale-up of the EID program [105]. Equally, DBS can improve the biological follow-up of HIV-1-infected individuals, both for the VL quantification and the resistance testing. Indeed, DBS, which can be collected on sites, transported and tested after a long-term storage, are suitable for the differed quantification of HIV-1 RNA, thus allowing people living with HIV/AIDS in rural areas to have access to this sophisticated test [114]. On another hand, implementation of resistance testing on DBS is in progress in Africa [115, 116] and will soon benefit HIV-1-infected patients living far from urban areas in Central Africa [108]. While waiting for the development of point of care assays, DBS appear to be a good alternative for the monitoring of HIV-1-infected people in remote settings (reviewed in [117]). However, the transport of samples and the return of results remains challenging, and

Central Africa could be the ideal place where an AIDS vaccine could be designed, because of the great diversity of strains that are found in this region. However, when the scientific community is reflecting on how simian immunodeficiency virus infections hosted by African nonhuman primates could help in designing an AIDS vaccine for example, Central African scientists are absent [118]. This situation should change and African institutions, supported by their government, should advocate strongly for and invest in an African AIDS vaccine. To this end, the African AIDS Vaccine Partnership (AAVP) intends to promote cutting-edge research for the development of an African HIV vaccine [119]. In addition, the European Developing Clinical Trial Partnership (EDCTP) is supporting several African institutions from Gabon, Congo and Cameroon to build capacity for the conduct of future

Central Africa is one of the few regions of the world where HTLV type 1 (HTLV-1) is highly endemic, as reviewed by Gessain & Mahieux [121]. Sequencing of HTLV-1 focuses on the gene *env* and the long terminal repeat fragments [122]. Molecular studies have demonstrated that the several molecular subtypes (genotypes) are related to the geographical origin and not to the disease. For example, while the subtype A is considered as cosmopolitan, the subtype B is mainly found in Central Africa (Democratic Republic of Congo, Gabon, and Cameroon). The subtype D has also been described in individuals from Cameroon, Gabon, Central African Republic, but less frequently than the subtype B, and more specifically in Pygmies. New subtypes (E and F) would be equally present in this region [121]. Interestingly, the first complete nucleotide sequence of HTLV type 2 (HTLV-2) has been obtained in a 44-year-old male living in a rural area of Gabon, by using nested PCR [123]. However, HTLV-2 does not seem to be as prevalent as HTLV-1 in this region since in a recent epidemiological survey performed on 907 pregnant women, only one case of HTLV-2 was reported [122]. In Cameroon however, HTLV-2 seroprevalence was 2.5% in Bakola Pygmies, but no HTLV-2 infection was found in Bantus [124]. HTLV type 3 (HTLV-3) and HTLV type 4 (HTLV-4) have been recently identified in primate hunters in Central Africa. Real-time PCR quantitative assays have been developed in the USA and allow detecting as

HIV/AIDS clinical trials [120] and is advocating for support from governments.

need additional implementation research.

**5.1.2.2 Human T cell Lymphotropic Virus (HTLV)** 

**Back to the sites** 

Despite influenza surveillance was increasing worldwide, developing countries in general and Central Africa in particular paid very little attention to the 2009 pandemic. Very recently however, samples from patients living with influenza-like illness in Yaounde, Cameroon were analyzed with various techniques including real time reverse transcription-polymerase chain reaction (RT-PCR) thus allowing the detection and subtyping of influenza A (H1N1 and H3N2) and B viruses from these patients [127]. Because of the H1N1 influenza A pandemic, Cameroon entered in a global surveillance network and received a laboratory equipped with a robust PCR platform for diagnosing influenza viruses in remote settings [128].

## **5.1.2.4 Hepatitis viruses**

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are endemic in the Central African region. Since the last two decades, the use of PCR techniques and phylogenetic analysis has led to characterize the genotype distribution of HCV in this area. The RNA is amplified by RT-PCR and nested PCR and the primers commonly used are specific to the 5'UTR and NS5B regions. In Cameroon, genotypes 1 and 4 are the most prevalent, but highly heterogeneous, with 5 subtypes 1, 4 subtypes 4 and unclassified subtypes, while the genotype 2 prevalence is low, with homogeneous sequences [129, 130]. Further work has help to understand the history of the HCV epidemic in Cameroon, where mass therapeutic or vaccine campaigns would have contributed to the spread of this infection during the colonial era [131]. In Gabon and Central African Republic, the predominance of the heterogeneous genotype 4 has been reported [132, 133, 134]. Equally, few HBV genotype studies have been conducted Central Africa. Makuwa et al. reported the identification of HBV-A3 in rural Gabon [135], while this genotype is co-circulating with HBV-E among Pygmies in Cameroon [136]. More recently, a pilot study was conducted in the village of Dienga, Gabon (previously described in section 2.1) with the aim of looking at potential interactions between HBV, HCV and *P. falciparum* infections, which are all very prevalent in this region [137]. In this study, HCV chronic carrier were identified by ELISA and by qualitative RT-PCR amplification of the 5' non coding region, and *P. falciparum* infection were assessed by microscopic examination and in case of negative result, by PCR targeting the gene encoding *P. falciparum* SSUrRNA, previously described by Snounou et al. [138]. Interestingly, these results showed that HCV infection may lead to slower emergence of *P. falciparum* in blood [137]. Other studies have demonstrated the usefulness of the PCR as a tool for the description of the molecular diversity of other less known/marginal viruses in this region, such as hepatitis delta virus in Cameroon [139] and in Gabon [140], or hepatitis GB-C/HG virus and TT virus in Gabon [141].

#### **5.1.2.5 Ebola virus**

Since the first declaration of deaths due to Ebola virus in Zaïre in 1976, the Central African region has been particularly affected by repeated Ebola outbreaks, which affected

Application of PCR Technologies

**Pathogen- genotype Group/ Subtype Regions (specific group) Technique Zone of amplification References Reviews** HIV-1 M/A,C, D, G, H, F, J, K, CRF01-AE DRC PCR & HMA *env* V3-V5 region [91] [93] [146] [117] M/CRFs Cameroon Nested PCR *gag*, *pol*, *env* genes [86] M/CRFs South Est Gabon PCR *pol* gene [147] N Cameroon PCR LTR-*gag*, *pol*-*vif*, *env* genes, entire genome [101] O Cameroon, Equatorial Guinea PCR Nested PCR LTR-*gag*, *pol*-*vif*, *env* genes, entire genome [94, 148] P Cameroon RT PCR *pol* integrase and *env*

to Humans, Animals, Plants and Pathogens from Central Africa 329

fragments [100, 102] HIV-2 Equatorial Guinea nested PCR *pol* gene [149] HTLV-1 A Congo, DRC, Chad nested PCR, PCR multiplex, real time PCR gene *env* and LTR, gene *tax*

[150] [122] [126] [121] B DRC, Gabon, Cameroon, CAR D Cameroon, Gabon (Pygmies) E DRC F Gabon HTLV-2 Gab, B Gabon Cameroon (Bakola Pygmies) nested PCR, PCR, multiplex, real time PCR entire proviral genome, gene *env* and LTR, gene *tax*, Long Terminal Repeats [123] [122] [126] [124] HTLV-3 Gabon, Cameroon multiplex, real time PCR, nested PCR gene *tax*

genes *tax* and *pol*

[126]

[151] [152]

populations from Gabon and Republic of Congo in addition to the Democratic Republic of Congo. However, publications on the detection of Ebola virus in humans by molecular studies such as RT-PCR are scarce. The first reason is that infected patients have been reluctant to any type of invasive sampling method. The second is that for cultural reasons, families strongly refuse that researchers collect postmortem skin biopsies [142]. By analyzing few serum samples and less invasive specimens such as oral fluid samples, Formenty et al. could detect Ebola virus by RT-PCR and compare the two types of specimens [142]. This RT-PCR method has been developed, implemented and evaluated for diagnostics purposes at the CIRMF in Gabon, where a tremendous work is being done in the field of Ebola and other hemorrhagic fevers [143]. It is clear that the RT-PCR is the most appropriate tool not only to diagnose the infection in patients at a very early stage, but also to follow-up recovering patients [144]. Of note, studies were more easily carried out in animals, where important findings using PCR technologies were reported (see section 5.2).

In conclusion to this section on viruses, it is important to mention that new random priming methods adapted from the sequence independent single primer amplification (SISPA) technology are now available, and could be used to sequence whole genomes of all sorts of (known or unknown) RNA and DNA viruses [145]. This methodology, together with molecular clock analyses are needed to better understand the origin, circulation and diversity of all the viruses present in Central African populations.

## **5.1.3 Bacteria**

In a review on the molecular epidemiology of bacterial infections in sub-Saharan infections, almost no information is reported from Central Africa [156]. Recently, molecular epidemiology methods have been applied to the genetic typing of *Mycobacterium tuberculosis* complex strains, the etiologic agents of tuberculosis, whose incidence is increasing dramatically in sub-Saharan Africa [157]. In 1993, a novel typing method called spoligotyping has been described [158]. This PCR-based method uses the DNA polymorphism of *M. tuberculosis* complex strains to detect and differentiate clinical isolates simultaneously, and allows their genotypic classification [159]. Briefly, this method aims at analyzing the so called DVR regions, which is composed of direct repeat (DR) regions, in which variable repeat sequences are inserted [160]. Spoligotyping, which is frequently compared to the conventional and more powerful RFLP method, remains a useful tool for genotyping clinical isolates in various epidemiological settings. In Cameroon, Niobe-Eyangoh et al. have used spoligotyping for analysis of hundreds of *M. tuberculosis* complex isolates from patients living in the West region [155]. This technique, which is considered as rapid, simple, and cost-effective, has been found accurate and easy to implement in that country, where the distribution of *M. tuberculosis* complex strains remains however still poorly documented, as well as the rest of Central Africa (see Table 4).

#### **5.2 Pathogens in animals**

Non-human primates from Central Africa have been extensively studied because it has been found that they are naturally infected with viruses or parasites similar to those affecting humans. The fact that humans are living in permanent contact with wild animals through hunting and butchering can explain transmission of pathogens from animals to humans.


populations from Gabon and Republic of Congo in addition to the Democratic Republic of Congo. However, publications on the detection of Ebola virus in humans by molecular studies such as RT-PCR are scarce. The first reason is that infected patients have been reluctant to any type of invasive sampling method. The second is that for cultural reasons, families strongly refuse that researchers collect postmortem skin biopsies [142]. By analyzing few serum samples and less invasive specimens such as oral fluid samples, Formenty et al. could detect Ebola virus by RT-PCR and compare the two types of specimens [142]. This RT-PCR method has been developed, implemented and evaluated for diagnostics purposes at the CIRMF in Gabon, where a tremendous work is being done in the field of Ebola and other hemorrhagic fevers [143]. It is clear that the RT-PCR is the most appropriate tool not only to diagnose the infection in patients at a very early stage, but also to follow-up recovering patients [144]. Of note, studies were more easily carried out in animals, where important findings using PCR technologies were reported (see section 5.2). In conclusion to this section on viruses, it is important to mention that new random priming methods adapted from the sequence independent single primer amplification (SISPA) technology are now available, and could be used to sequence whole genomes of all sorts of (known or unknown) RNA and DNA viruses [145]. This methodology, together with molecular clock analyses are needed to better understand the origin, circulation and

In a review on the molecular epidemiology of bacterial infections in sub-Saharan infections, almost no information is reported from Central Africa [156]. Recently, molecular epidemiology methods have been applied to the genetic typing of *Mycobacterium tuberculosis* complex strains, the etiologic agents of tuberculosis, whose incidence is increasing dramatically in sub-Saharan Africa [157]. In 1993, a novel typing method called spoligotyping has been described [158]. This PCR-based method uses the DNA polymorphism of *M. tuberculosis* complex strains to detect and differentiate clinical isolates simultaneously, and allows their genotypic classification [159]. Briefly, this method aims at analyzing the so called DVR regions, which is composed of direct repeat (DR) regions, in which variable repeat sequences are inserted [160]. Spoligotyping, which is frequently compared to the conventional and more powerful RFLP method, remains a useful tool for genotyping clinical isolates in various epidemiological settings. In Cameroon, Niobe-Eyangoh et al. have used spoligotyping for analysis of hundreds of *M. tuberculosis* complex isolates from patients living in the West region [155]. This technique, which is considered as rapid, simple, and cost-effective, has been found accurate and easy to implement in that country, where the distribution of *M. tuberculosis* complex strains remains however still

Non-human primates from Central Africa have been extensively studied because it has been found that they are naturally infected with viruses or parasites similar to those affecting humans. The fact that humans are living in permanent contact with wild animals through hunting and butchering can explain transmission of pathogens from animals to

diversity of all the viruses present in Central African populations.

poorly documented, as well as the rest of Central Africa (see Table 4).

**5.1.3 Bacteria** 

**5.2 Pathogens in animals** 

humans.


HIV: Human Immunodeficiency Virus, HTLV: Human T cell Leukemia Virus, HCV: Hepatitis Virus C, HBV: Hepatitis Virus B, LTR: Long Terminal Repeats, CAR: the Central African Republic, DRC: Democratic Republic of Congo

Table 4. Examples of pathogens infecting humans in Central Africa, which have benefited from PCR technologies

Application of PCR Technologies

Viruses (HBV) [169].

**5.2.1 Pathogens in non-human primates** 

to Humans, Animals, Plants and Pathogens from Central Africa 331

A substantial proportion of wild-living primates in Central Africa are naturally infected with Simian Immunodeficiency Viruses (SIVs) [161, 162, 163], Simian T-cell Lymphotropic Viruses (STLVs) [164, 165, 166, 167], Simian Foamy Viruses (SFV) [168] and also Hepatitis B

SIVs are lentiviruses that are found naturally in a great variety of nonhuman primates from Equatorial Africa, including but no restricted to chimpanzees (SIVcpz), mandrills, (SIVmnd-1 and SIVmnd-2), drills (SIVdrl), talapoin monkeys (SIVtal), sun tailed monkeys (SIVsun), African green monkeys (SIVagm), red-capped mangabeys (SIVrcm) (see [162, 163, 170] and [171] for review). The evolutionary origins of these related viruses have been studied by amplification of the *gag*, *pol*, and *env* genes, and by construction and analysis of evolutionary trees. Sequence analysis of the entire genome and phylogenetic analyses have led to the identification of distinct primate lentivirus lineages in which most of the SIV strains described so far can be classified (see [171] and Table 5). The example of SIVs illustrates how the PCR techniques have been instrumental in the characterization of new strains of pathogens in non-human primates of Central Africa. As previously mentioned for animals (see section 3) phylogeographic studies have been equally carried out for pathogens. In mandrills for example, the two types of viruses appear to be geographically distributed, since SIVmnd-1 was found in mandrills from central and southern Gabon whereas SIVmnd-

2 was identified in northern and western Gabon, as well as in Cameroon [163].

number of species, especially in case of wild living primates.

of emergence of new viral diseases in Central Africa is still latent.

view of the phylogenetic relationships among *Plasmodium* species [173].

simian viruses and their counterpart in humans.

Other examples of pathogens in non-human primates from Central Africa could have been used, like the STLVs (the simian counterpart of HTLVs), the SFVs and/or HBV, which similarly to SIVs have been described and characterized with molecular techniques including PCR. With no pretention of being exhaustive, the Table 5 summarizes several examples of pathogens found in animals from this region, with the technique used, the gene amplified, and appropriate references for more details. Of note, molecular techniques adapted to non-invasive fecal samples have been pivotal to identify simian viruses in quite a

These findings from Central Africa on pathogens in non-human primates together with those reported in humans, give a more comprehensive picture of the relationship between

Indeed, the use of PCR related technologies and the clustering of sequences has helped to understand that i) cross species transmission of viruses (from non-human primates to humans) occurred in Central Africa through highly exposed population such as hunters and people handling primates as bush meat [164] and ii) species barriers could be easier to cross over than geographic barriers [165]. Taken together, these observations reveal that the risk

Similarly, various species of *Plasmodium*, including *P. falciparum* have been found in great apes (chimpanzees and gorillas) in Central Africa [172, 173]. If blood samples are not suitable for systematical analyses in primates, especially in case of wild primates; the identification of *Plasmodium* by PCR has been facilitated by the use of fecal primate samples, which are also broadly collected for the identification of simian viruses (see above). The identification of new species of *Plasmodium*, such as *P. gaboni*, which infects chimpanzees and *P. GorA* and *P. GorB*, which infect gorillas, has help to obtain a more comprehensive

#### **5.2.1 Pathogens in non-human primates**

330 Polymerase Chain Reaction

HIV: Human Immunodeficiency Virus, HTLV: Human T cell Leukemia Virus, HCV: Hepatitis Virus C, HBV: Hepatitis Virus B, LTR: Long Terminal Repeats, CAR: the Central African Republic, DRC:

HCV-4

HBV

Table 4. Examples of pathogens infecting humans in Central Africa, which have benefited

Democratic Republic of Congo

from PCR technologies

**Pathogen- genotype Group/ Subtype Regions (specific group) Technique Zone of amplification References Reviews** HTLV-4 South East Cameroon nested PCR gene *tax*

[151] Influenza A H1N1 Cameroon RT PCR HA NA and M

H3N2

B/Victoria/2/

87 lineage and

B/Yagamata/1

6/88 linea

ge

HCV-1 1a, 1b, 1c, 1e, 1h, 1l Cameroon South-West CAR

HCV-2 2f Cameroon South-West CAR

4e, 4f, 4k, 4c

Cameroon,

South-West CAR,

Gabon

Gabon, DRC,

Cameroon

E Cameroon (Pygmies)

Ebola DRC, Gabon, Congo RT PCR RNA polymerase L and NP genes [142, 153] [154] [143]

*Mycobacterium tuberculosis* Cameroon spoligotyping DVR region [155]

Touré-Ndouo,

2011

(chapter in

this book)

*Plasmodium falciparum* Gabon PCR SSUrRNA gene [137, 138]

(Pygmies) Semi nested PCR HBs (surface) gene [135, 136]

4r, 4t, 4p,

unclassified

A3

RT PCR & nested

NS5b gene

[129, 131,

132, 133]

NS5b and E2 regions

5'UTR region

PCR

Influenza

B

 genes *tax* and *pol*

sequences [127]

A substantial proportion of wild-living primates in Central Africa are naturally infected with Simian Immunodeficiency Viruses (SIVs) [161, 162, 163], Simian T-cell Lymphotropic Viruses (STLVs) [164, 165, 166, 167], Simian Foamy Viruses (SFV) [168] and also Hepatitis B Viruses (HBV) [169].

SIVs are lentiviruses that are found naturally in a great variety of nonhuman primates from Equatorial Africa, including but no restricted to chimpanzees (SIVcpz), mandrills, (SIVmnd-1 and SIVmnd-2), drills (SIVdrl), talapoin monkeys (SIVtal), sun tailed monkeys (SIVsun), African green monkeys (SIVagm), red-capped mangabeys (SIVrcm) (see [162, 163, 170] and [171] for review). The evolutionary origins of these related viruses have been studied by amplification of the *gag*, *pol*, and *env* genes, and by construction and analysis of evolutionary trees. Sequence analysis of the entire genome and phylogenetic analyses have led to the identification of distinct primate lentivirus lineages in which most of the SIV strains described so far can be classified (see [171] and Table 5). The example of SIVs illustrates how the PCR techniques have been instrumental in the characterization of new strains of pathogens in non-human primates of Central Africa. As previously mentioned for animals (see section 3) phylogeographic studies have been equally carried out for pathogens. In mandrills for example, the two types of viruses appear to be geographically distributed, since SIVmnd-1 was found in mandrills from central and southern Gabon whereas SIVmnd-2 was identified in northern and western Gabon, as well as in Cameroon [163].

Other examples of pathogens in non-human primates from Central Africa could have been used, like the STLVs (the simian counterpart of HTLVs), the SFVs and/or HBV, which similarly to SIVs have been described and characterized with molecular techniques including PCR. With no pretention of being exhaustive, the Table 5 summarizes several examples of pathogens found in animals from this region, with the technique used, the gene amplified, and appropriate references for more details. Of note, molecular techniques adapted to non-invasive fecal samples have been pivotal to identify simian viruses in quite a number of species, especially in case of wild living primates.

These findings from Central Africa on pathogens in non-human primates together with those reported in humans, give a more comprehensive picture of the relationship between simian viruses and their counterpart in humans.

Indeed, the use of PCR related technologies and the clustering of sequences has helped to understand that i) cross species transmission of viruses (from non-human primates to humans) occurred in Central Africa through highly exposed population such as hunters and people handling primates as bush meat [164] and ii) species barriers could be easier to cross over than geographic barriers [165]. Taken together, these observations reveal that the risk of emergence of new viral diseases in Central Africa is still latent.

Similarly, various species of *Plasmodium*, including *P. falciparum* have been found in great apes (chimpanzees and gorillas) in Central Africa [172, 173]. If blood samples are not suitable for systematical analyses in primates, especially in case of wild primates; the identification of *Plasmodium* by PCR has been facilitated by the use of fecal primate samples, which are also broadly collected for the identification of simian viruses (see above). The identification of new species of *Plasmodium*, such as *P. gaboni*, which infects chimpanzees and *P. GorA* and *P. GorB*, which infect gorillas, has help to obtain a more comprehensive view of the phylogenetic relationships among *Plasmodium* species [173].


Application of PCR Technologies

Congo

PCR technologies

to Humans, Animals, Plants and Pathogens from Central Africa 333

SIV: Simian Immunodeficiency Virus, STLV: Simian T cell Lymphotropic Virus, SFV: Simian Foamy Virus, LTR: Long Terminal Repeats, CAR: the Central African Republic, DRC: Democratic Republic of

SFV SFVcpz

**Pathogen-**

**genotype Subtype/ lineage Regions (animals) Technique Zone of amplification References Reviews** 

Gabon, Cameroon (chimpanzees);

Cameroon, CAR, Gabon, Republic

of Congo, DRC (wild

nested PCR

integrase and LTR

[184]

[185]

[168]

region *gag*, *pol*-RT

and *pol*-IN LTR

RT PCR

chimpanzees); Gabon (wild and

semi-free ranging captive

mandrills)

Ebola Gabon (Fruit bats) PCR RNA polymerase [153]

Influenza H5N1 Northern Cameroon (ducks) PCR NA sequences [176]

Table 5. Examples of pathogens infecting animals of Central Africa that have benefited from


SIV: Simian Immunodeficiency Virus, STLV: Simian T cell Lymphotropic Virus, SFV: Simian Foamy Virus, LTR: Long Terminal Repeats, CAR: the Central African Republic, DRC: Democratic Republic of Congo

Table 5. Examples of pathogens infecting animals of Central Africa that have benefited from PCR technologies

332 Polymerase Chain Reaction

**Pathogen- genotype Subtype/ lineage Regions (animals) Technique Zone of amplification References Reviews** *Plasmodium gaboni* Gabon (chimpanzees) PCR complete mitochondrial genome (including *Cyt b*, *Cox I* and *Cox III* genes) [172] [177] *GorA GorB* Gabon (wild chimpanzees, wild gorilla, captive wild-born gorilla) Plasmodium-

specific PCR assay mitochondrial *cytochrome b*

*falciparum* Gabon (wild chimpanzees, gorilla) nuclear and mitochondrial genomes [177] SIV SIVmnd-1 SIVmnd-2 Gabon (mandrills), Cameroon (mandrills) PCR entire genome [178] [163] [171] SIVtal Cameroon (talapoin monkeys) PCR entire genome [162] SIVsun Gabon (wild-caught sun tailed monke

y) PCR entire genome [161]

SIVrcm

Gabon (red capped mangabeys);

Nigeria/Cameroon border (red-

SIVcpz Cameroon, Gabon, DRC (chimpanzees) PCR entire genome

Cameroon (agile mangabeys,

mustached monkeys, talapoins, gorilla,

mandrills, African green monkeys,

Discriminatory

PCR LTR & *env* sequences [164] [165]

agile mangabeys, and crested mona

and greater spot-nosed monkeys);

Gabon (mandrills)

STLV-2 DRC (wild-living bonobos) Generic PCR *tax* gene [183]

STLV-3 Cameroon (agile mangabeys) Discriminatory PCR LTR & *env* sequences [164]

STLV-1 D, F

capped mangabeys) PCR entire genome [170] [179]

[180]

[181]

[182]

 gene [173]

Application of PCR Technologies

living in remote areas [117].

resources, capacity building and ethics–related issues.

to Humans, Animals, Plants and Pathogens from Central Africa 335

is equipped with BSL3 and BSL4 facility, and the CIRCB (Yaounde, Cameroon), among others. Despite the amount of work and publications that have been generated from the Central African region, institutions and scientists involved in molecular biology research in Central Africa are facing several problems including procurement, maintenance, human

Obtaining the valuable results depends on multiple factors including methodology of sampling, processing, storage and shipment of samples to laboratory with respect of maintain of the cold chain. As described above, problems related to sampling were well circumvented with animals. Indeed, by using shed hair or feces, which are non invasive methods of sampling, phylogenetic analyses have allowed a better understanding of the evolutionary history of gorillas [46] mandrills [47] or elephants [48]. Equally, a number of simian viruses have been characterized in fecal samples, which is more convenient, especially in case of wild-living primates. In these contexts, new reagents such as the RNA later® have been very helpful to stabilize and protect RNA in fresh collected specimens, hence allowing an extended period of storage before processing the samples. In humans, the collection of samples via DBS is simple, convenient, and cost effective. Transportation does not require any cold chain, and storage is easier than samples obtained from whole blood. In the field of HIV, DBS are advantageous for the biological follow-up of infected patients

Another issue, which has to be taken into consideration, is related to the issue of the quality control and quality assurance, which need permanent improvement and capacity building efforts. Due to limited resources and equipment, and possibly because the culture of research is still dramatically lacking in most of sub-Saharan African countries [188], only few laboratories have obtained certification and the roadmap to accreditation is still far ahead. Therefore there is an urgent need that institutions from Central Africa participate more in laboratory accreditation programs, with the goal of seeking lab accreditation and excellence in general. For example, the World Health Organization (WHO)-AFRO and the Center for Disease Control Global AIDS program have launched recently an accreditation program for quality improvement of African laboratories for HIV monitoring. However, such programs will also improve the monitoring of HIV-TB coinfected patients, and by extension, the follow-up of patients suffering from other diseases, such as malaria or any neglected disease. Equally, support from the EDCTP is currently helping African institutions -grouped in regional Networks of Excellence- to conduct future clinical trials in the four regions of sub-Saharan Africa. To achieve this goal, a lot of efforts have been put into building capacity of young African scientists and laboratories, which have to meet

international standards and respect good clinical and laboratory practices [120].

Studies reported here have been carried out mainly in the framework of collaborative research with institutions from the North. However, DNA samples are often kept abroad, with the partners, without any signed material transfer agreement. In some other cases, African scientists and institutions from the region are not associated to the work and/or publications. The researcher's community has to be aware of avoiding the "banking" of DNA from African populations outside from Africa, mutualising benefits with the concerned populations and scientific partners as well as respecting ethical issues, such as establishing a fair partnership between African scientists and scientist from the North. The lack of these aspects have been demonstrated in a recent bibliometric review on human genetic studies performed during the two last decades in Cameroon [189]. Recently, the

By sequencing the complete mitochondrial gene or at least a part of the cytochrome b, and Bayesian or maximum-likelihood methods, phylogenetic analyses can be performed, hence allowing a better understanding of the origins and evolution of malaria parasites and possibly transmission between apes and humans [172].

## **5.2.2 Pathogens in other animal species**

Apart from non-human primates, other animals from the Central African region have been studied for their possible implication in the life cycle of viruses causing hemorrhagic fever like Ebola or Marburg, which are both affecting great apes and humans. For example, sequences of Ebola were detected by PCR in small rodents and shrews, suggesting that common terrestrial small mammals living in peripheral forest areas may play a role in the life cycle of the Ebola virus [174]. More recently, Ebola and Marburg viruses were found in symptomless infected fruit bats in Central Africa, indicating that these animals could therefore act as the natural reservoir of these both viruses [153, 175].

In the context of outbreaks of highly pathogenic avian influenza, ducks from the far north region of Cameroon were found to host a highly pathogenic avian influenza subtype H5N1, whose sequence was closely related to H5N1 isolates reported in other African countries [176].

## **5.3 Pathogens in plants**

For plant pathogen, PCR-based techniques are essentially used in two purposes: i) to identify pathogen species, comparing pathogen sequences to known pathogen sequence libraries or ii) to characterize pathogen colonization dynamic. One example of each application is summarized below.

## **5.3.1 Which fungi are attacking Central African** *Terminalia* **species?**

Begoude et al. collected fungal inoculum on *Terminalia* in Cameroon to identify which pathogens are threatening these highly logged tree species. They compared DNA sequence for the ITS and tef 1-alpha gene regions to known pathogen libraries and showed that the majority of isolates are from the *Lasiodiplodia* genus [186].

## **5.3.2 The colonization dynamic of** *Mycosphaerella fijiensis* **in Cameroon**

Dispersal processes of fungal plant pathogens can be inferred from analyses of spatial genetic structures resulting from recent range expansions. The fungus *Mycosphaerella fijiensis*, pathogenic on banana, is an example of a recent worldwide epidemic and is currently threatening Cameroonian banana plantations. Halkett et al. collected fungal isolates in Cameroon and analyzed them using 19 microsatellite markers. They demonstrated that large gene flows are linking populations even separated by long distances, through dense banana plantations, and so ensuring stable demographic regime and promoting efficient colonization dynamic of the fungus [187].

## **6. Opportunities and challenges**

Some of the few research institutes and molecular biology laboratories that have been mainly involved in the findings reported above are the CIRMF (Franceville, Gabon), which

By sequencing the complete mitochondrial gene or at least a part of the cytochrome b, and Bayesian or maximum-likelihood methods, phylogenetic analyses can be performed, hence allowing a better understanding of the origins and evolution of malaria parasites and

Apart from non-human primates, other animals from the Central African region have been studied for their possible implication in the life cycle of viruses causing hemorrhagic fever like Ebola or Marburg, which are both affecting great apes and humans. For example, sequences of Ebola were detected by PCR in small rodents and shrews, suggesting that common terrestrial small mammals living in peripheral forest areas may play a role in the life cycle of the Ebola virus [174]. More recently, Ebola and Marburg viruses were found in symptomless infected fruit bats in Central Africa, indicating that these animals could

In the context of outbreaks of highly pathogenic avian influenza, ducks from the far north region of Cameroon were found to host a highly pathogenic avian influenza subtype H5N1, whose sequence was closely related to H5N1 isolates reported in other African countries [176].

For plant pathogen, PCR-based techniques are essentially used in two purposes: i) to identify pathogen species, comparing pathogen sequences to known pathogen sequence libraries or ii) to characterize pathogen colonization dynamic. One example of each

Begoude et al. collected fungal inoculum on *Terminalia* in Cameroon to identify which pathogens are threatening these highly logged tree species. They compared DNA sequence for the ITS and tef 1-alpha gene regions to known pathogen libraries and showed that the

Dispersal processes of fungal plant pathogens can be inferred from analyses of spatial genetic structures resulting from recent range expansions. The fungus *Mycosphaerella fijiensis*, pathogenic on banana, is an example of a recent worldwide epidemic and is currently threatening Cameroonian banana plantations. Halkett et al. collected fungal isolates in Cameroon and analyzed them using 19 microsatellite markers. They demonstrated that large gene flows are linking populations even separated by long distances, through dense banana plantations, and so ensuring stable demographic regime

Some of the few research institutes and molecular biology laboratories that have been mainly involved in the findings reported above are the CIRMF (Franceville, Gabon), which

possibly transmission between apes and humans [172].

therefore act as the natural reservoir of these both viruses [153, 175].

**5.3.1 Which fungi are attacking Central African** *Terminalia* **species?** 

**5.3.2 The colonization dynamic of** *Mycosphaerella fijiensis* **in Cameroon** 

majority of isolates are from the *Lasiodiplodia* genus [186].

and promoting efficient colonization dynamic of the fungus [187].

**5.2.2 Pathogens in other animal species** 

**5.3 Pathogens in plants** 

application is summarized below.

**6. Opportunities and challenges** 

is equipped with BSL3 and BSL4 facility, and the CIRCB (Yaounde, Cameroon), among others. Despite the amount of work and publications that have been generated from the Central African region, institutions and scientists involved in molecular biology research in Central Africa are facing several problems including procurement, maintenance, human resources, capacity building and ethics–related issues.

Obtaining the valuable results depends on multiple factors including methodology of sampling, processing, storage and shipment of samples to laboratory with respect of maintain of the cold chain. As described above, problems related to sampling were well circumvented with animals. Indeed, by using shed hair or feces, which are non invasive methods of sampling, phylogenetic analyses have allowed a better understanding of the evolutionary history of gorillas [46] mandrills [47] or elephants [48]. Equally, a number of simian viruses have been characterized in fecal samples, which is more convenient, especially in case of wild-living primates. In these contexts, new reagents such as the RNA later® have been very helpful to stabilize and protect RNA in fresh collected specimens, hence allowing an extended period of storage before processing the samples. In humans, the collection of samples via DBS is simple, convenient, and cost effective. Transportation does not require any cold chain, and storage is easier than samples obtained from whole blood. In the field of HIV, DBS are advantageous for the biological follow-up of infected patients living in remote areas [117].

Another issue, which has to be taken into consideration, is related to the issue of the quality control and quality assurance, which need permanent improvement and capacity building efforts. Due to limited resources and equipment, and possibly because the culture of research is still dramatically lacking in most of sub-Saharan African countries [188], only few laboratories have obtained certification and the roadmap to accreditation is still far ahead. Therefore there is an urgent need that institutions from Central Africa participate more in laboratory accreditation programs, with the goal of seeking lab accreditation and excellence in general. For example, the World Health Organization (WHO)-AFRO and the Center for Disease Control Global AIDS program have launched recently an accreditation program for quality improvement of African laboratories for HIV monitoring. However, such programs will also improve the monitoring of HIV-TB coinfected patients, and by extension, the follow-up of patients suffering from other diseases, such as malaria or any neglected disease. Equally, support from the EDCTP is currently helping African institutions -grouped in regional Networks of Excellence- to conduct future clinical trials in the four regions of sub-Saharan Africa. To achieve this goal, a lot of efforts have been put into building capacity of young African scientists and laboratories, which have to meet international standards and respect good clinical and laboratory practices [120].

Studies reported here have been carried out mainly in the framework of collaborative research with institutions from the North. However, DNA samples are often kept abroad, with the partners, without any signed material transfer agreement. In some other cases, African scientists and institutions from the region are not associated to the work and/or publications. The researcher's community has to be aware of avoiding the "banking" of DNA from African populations outside from Africa, mutualising benefits with the concerned populations and scientific partners as well as respecting ethical issues, such as establishing a fair partnership between African scientists and scientist from the North. The lack of these aspects have been demonstrated in a recent bibliometric review on human genetic studies performed during the two last decades in Cameroon [189]. Recently, the

Application of PCR Technologies

will complement each other.

**8. Acknowledgments** 

Capacity in Africa (ISHReCA).

**9. References** 

diagnostics and monitoring of infected individuals.

to Humans, Animals, Plants and Pathogens from Central Africa 337

pathogens), and particularly the inter relationship between species. Indubitably, this will be of help for a better management of resources at the global level. In addition, progresses have been made in fundamental research, operational research, and research applied to

Challenges in conducting PCR-based research are procurement and storage of reagents and blood samples due to the cold chain, maintenance of equipment, as well as human resources, capacity-building and ethics-related issues. However, new initiatives such as those launched by the African Society of Human Genetics (H3 Africa), the AAVP (promoting an African AIDS Vaccine), and the EDCTP (supporting regional Networks of Excellence for the future conduct of clinical trials) are real opportunities for the scientific community that is working in Africa, to perform cutting-edge research where sophisticated molecular biology laboratories and bioinformatics platforms will be created/renovated and

In conclusion, despite a challenging research environment and though the paucity of facilities, scientists from Central Africa have brought a significant contribution to the scientific community, through PCR-related technologies. Collaborative research with northern partners has been fruitful and must be always conducted while keeping in mind a fair partnership and authorship. PCR-based research is increasing significantly in Central

This paper has voluntarily been written by female scientists only, who have personally contributed to some of the findings presented in this chapter. All authors and individuals acknowledged below have been working or are currently working in Central Africa, particularly in Cameroon (at the CIRCB, Yaoundé and/or University of Buea) and Gabon (at the CIRMF, Franceville). We acknowledge Dr Mireille Bawe Johnson, Cardiff University, Biodiversity and Ecological Processes Group, Cardiff, UK, Dr Maria Makuwa, Laboratory Coordinator and Administrator at the Global Viral Forecasting Initiative (GVFI)/Institut National de Recherche Biomedicale (INRB), Kinshasa, Democratic Republic of Congo and Dr Lucy M. Ndip, head of the laboratory for Emerging Infectious Diseases, University of Buea, Cameroon for their contribution during the pre submission of this chapter. We also want to thank Dr Michaela Müller-Trutwin for her advice and Dr Sandrine Souquiere, for the critical reading of the manuscript. Finally, we are grateful to Mrs Clemence Rochelle Akoumba for her kind assistance in collecting some of the full papers referenced below, and Mrs Nchangwi Syntia Munung for her great help in managing references in Endnote®. Odile Ouwe Missi Oukem-Boyer is member of the Central Africa Network for Tuberculosis, HIV/AIDS, and Malaria (CANTAM) and of the Initiative to Strengthen Health Research

[1] Pasquier, C., et al. (2001). HIV-1 subtyping using phylogenetic analysis of pol gene

[2] Migot-Nabias, F., et al. (1999). HLA class II polymorphism in a Gabonese Banzabi

sequences. *J Virol Methods*, 94, (1-2), 45-54, ISSN 0166-0934 (Print).

population. *Tissue Antigens*, 53, (6), 580-585, ISSN 0001-2815 (Print).

Africa and must be recognized at the level of the scientific community.

African Society of Human Genetics launched the Human Heredity and Health in Africa (H3Africa) initiative, with the support of the National Institutes of Health and the Wellcome Trust (see http://h3africa.org/). The aim of this initiative, which was first discussed at the Yaoundé meeting in March 2009, is to conduct genomics-based research projects in Africa in order to better understand health and diseases in various African populations and to identify possible populations that are at risk of developing a specific disease. To this end, various calls for proposals have been launched, in which African institutions will take the leading role. One of these calls is the H3 Africa biorepository grant, which will address the need of biobanking samples in Africa for Africa. This H3Africa programme gives a lot of hope that capacity building and ethics-related will be soon addressed in favor of African institutions and African scientists and other scientists living in Africa, and that partnerships will eventually result in true win-win collaborations.

## **7. Conclusion**

The contribution of PCR technologies to humans, animals, plants and pathogens from Central Africa is considerable, hence allowing the discovery of new species of plants and pathogens in this region, particularly in Gabon (see http://www.cirmf.org/en/publications). The richness of animals, plants, and pathogens is unquestionable and the Central African region is notorious for its great biodiversity.

In this chapter, a great number of PCR-based techniques have been described, including but not limited to PCR-restriction fragment length polymorphism, PCR using sequence-specific oligonucleotide probes, combination of sequence-specific PCR and sequence-based typing also called Haplotype Specific Sequencing, PCR-single strand conformational polymorphism, reverse transcriptase PCR, sequence independent single primer amplification technology, nested and semi-nested PCR, quantitative PCR, real time PCR, PCR multiplex, Heteroduplex Mobility Analysis, and spoligotyping. Applied to humans, these techniques have contributed significantly to increase the knowledge on human genetics, through immunogenetics and genetics epidemiology of infectious diseases. Particularly, a great number of molecular studies describe the genetic polymorphism of the various populations and ethnic groups living in this region (section 2). Applied to wild animals and non-invasive samples such as shed hair or feces, PCR technologies have for example facilitated the identification of related species, which are not easy to differentiate just by direct observation as done by ecologists, by using mitochondrial DNA (section 3). Applied to plants, PCR-based methods have contributed to a better understanding of spatial and temporal evolution of species found in that region, including colonization routes, and tree densities than can be modified because of activities of humans in that region (section 4). Finally, application of PCR technologies has been reported for pathogens infecting humans, animals and plants (section 5). Parasites, viruses, and bacteria that are prevalent in humans, non-human primates and other animal species, and fungal plant pathogens have been discovered and characterized through PCR-based techniques.

The PCR-generated knowledge is benefiting to a broad range of disciplines, such as genetics, molecular ecology, phylogeography, botany, evolution, molecular epidemiology, and infectious diseases, amongst others.

Altogether, these finding have contributed to a better understanding of the relationship between humans from Central Africa and their environment (animals, plants and pathogens), and particularly the inter relationship between species. Indubitably, this will be of help for a better management of resources at the global level. In addition, progresses have been made in fundamental research, operational research, and research applied to diagnostics and monitoring of infected individuals.

Challenges in conducting PCR-based research are procurement and storage of reagents and blood samples due to the cold chain, maintenance of equipment, as well as human resources, capacity-building and ethics-related issues. However, new initiatives such as those launched by the African Society of Human Genetics (H3 Africa), the AAVP (promoting an African AIDS Vaccine), and the EDCTP (supporting regional Networks of Excellence for the future conduct of clinical trials) are real opportunities for the scientific community that is working in Africa, to perform cutting-edge research where sophisticated molecular biology laboratories and bioinformatics platforms will be created/renovated and will complement each other.

In conclusion, despite a challenging research environment and though the paucity of facilities, scientists from Central Africa have brought a significant contribution to the scientific community, through PCR-related technologies. Collaborative research with northern partners has been fruitful and must be always conducted while keeping in mind a fair partnership and authorship. PCR-based research is increasing significantly in Central Africa and must be recognized at the level of the scientific community.

## **8. Acknowledgments**

336 Polymerase Chain Reaction

African Society of Human Genetics launched the Human Heredity and Health in Africa (H3Africa) initiative, with the support of the National Institutes of Health and the Wellcome Trust (see http://h3africa.org/). The aim of this initiative, which was first discussed at the Yaoundé meeting in March 2009, is to conduct genomics-based research projects in Africa in order to better understand health and diseases in various African populations and to identify possible populations that are at risk of developing a specific disease. To this end, various calls for proposals have been launched, in which African institutions will take the leading role. One of these calls is the H3 Africa biorepository grant, which will address the need of biobanking samples in Africa for Africa. This H3Africa programme gives a lot of hope that capacity building and ethics-related will be soon addressed in favor of African institutions and African scientists and other scientists living in Africa, and that partnerships

The contribution of PCR technologies to humans, animals, plants and pathogens from Central Africa is considerable, hence allowing the discovery of new species of plants and pathogens in this region, particularly in Gabon (see http://www.cirmf.org/en/publications). The richness of animals, plants, and pathogens is unquestionable and the Central African

In this chapter, a great number of PCR-based techniques have been described, including but not limited to PCR-restriction fragment length polymorphism, PCR using sequence-specific oligonucleotide probes, combination of sequence-specific PCR and sequence-based typing also called Haplotype Specific Sequencing, PCR-single strand conformational polymorphism, reverse transcriptase PCR, sequence independent single primer amplification technology, nested and semi-nested PCR, quantitative PCR, real time PCR, PCR multiplex, Heteroduplex Mobility Analysis, and spoligotyping. Applied to humans, these techniques have contributed significantly to increase the knowledge on human genetics, through immunogenetics and genetics epidemiology of infectious diseases. Particularly, a great number of molecular studies describe the genetic polymorphism of the various populations and ethnic groups living in this region (section 2). Applied to wild animals and non-invasive samples such as shed hair or feces, PCR technologies have for example facilitated the identification of related species, which are not easy to differentiate just by direct observation as done by ecologists, by using mitochondrial DNA (section 3). Applied to plants, PCR-based methods have contributed to a better understanding of spatial and temporal evolution of species found in that region, including colonization routes, and tree densities than can be modified because of activities of humans in that region (section 4). Finally, application of PCR technologies has been reported for pathogens infecting humans, animals and plants (section 5). Parasites, viruses, and bacteria that are prevalent in humans, non-human primates and other animal species, and fungal plant pathogens have been

The PCR-generated knowledge is benefiting to a broad range of disciplines, such as genetics, molecular ecology, phylogeography, botany, evolution, molecular epidemiology, and

Altogether, these finding have contributed to a better understanding of the relationship between humans from Central Africa and their environment (animals, plants and

will eventually result in true win-win collaborations.

region is notorious for its great biodiversity.

discovered and characterized through PCR-based techniques.

infectious diseases, amongst others.

**7. Conclusion** 

This paper has voluntarily been written by female scientists only, who have personally contributed to some of the findings presented in this chapter. All authors and individuals acknowledged below have been working or are currently working in Central Africa, particularly in Cameroon (at the CIRCB, Yaoundé and/or University of Buea) and Gabon (at the CIRMF, Franceville). We acknowledge Dr Mireille Bawe Johnson, Cardiff University, Biodiversity and Ecological Processes Group, Cardiff, UK, Dr Maria Makuwa, Laboratory Coordinator and Administrator at the Global Viral Forecasting Initiative (GVFI)/Institut National de Recherche Biomedicale (INRB), Kinshasa, Democratic Republic of Congo and Dr Lucy M. Ndip, head of the laboratory for Emerging Infectious Diseases, University of Buea, Cameroon for their contribution during the pre submission of this chapter. We also want to thank Dr Michaela Müller-Trutwin for her advice and Dr Sandrine Souquiere, for the critical reading of the manuscript. Finally, we are grateful to Mrs Clemence Rochelle Akoumba for her kind assistance in collecting some of the full papers referenced below, and Mrs Nchangwi Syntia Munung for her great help in managing references in Endnote®. Odile Ouwe Missi Oukem-Boyer is member of the Central Africa Network for Tuberculosis, HIV/AIDS, and Malaria (CANTAM) and of the Initiative to Strengthen Health Research Capacity in Africa (ISHReCA).

## **9. References**


Application of PCR Technologies

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**17** 

*México* 

**Study of Mycobacterium Tuberculosis by** 

H. W. Araujo-Torres1,3, J. A. Narváez-Zapata2, M. G. Castillo-Álvarez1, MS. Puga-Hernández4, J. Flores-Gracia5 and M. A. Reyes-López1,\*

**Molecular Methods in Northeast Mexico** 

*1Conservation Medicine Lab., Centro de Biotecnología Genómica del* 

*2Industrial Biotechnology Lab., Centro de Biotecnología Genómica* 

*5Instituto Tecnológico de Ciudad Victoria, Cd. Victoria, Tamps* 

*3Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada* 

*4Laboratorio Estatal de Salud Pública de Tamaulipas, Cd. Victoria, Tamps,* 

One third of the world population is afected by TB and one million people did die this year 2011 in undeveloped countries (Venkatesh et al., 2011). In Tamaulipas, a Northern State of Mexico and a border state between USA and Mexico, frequency is 26.9 new TB cases per 100,000 people, twice of national rate of 12.85 cases per 100,000 people (Ferrer et al., 2010). Only on the border of Tamaulipas about 320 cases are diagnosed each year. Many of these cases correspond to people from other states of Mexico, probably by geographic position and by migration problematic of this study zone (Fitchett et al., 2011). Only 92% of the treated population are cured mainly because much of these people are poor and whose

The long presence of this disease has increased the need to know specifically which *Mycobacterium tuberculosis* strains are circulating in the region. Additionally, it is necessary to know the antibiotic/susceptibility profile of these strains since many of them acquire

In general, the diagnostic of this disease is traditionally conducted by using gold standard techniques focused to identify the presence of *M. tuberculosis* in clinical specimen of humans or cattle. These techniques included the strain of microorganism in Ziehl-Neelsen and culture in Lowenstein-Jensen medium (Cadmus et al., 2011), both regarded as reference techniques in the diagnosis of TB. Differentiation among mycobacteria of the *M. tuberculosis* complex (MTC) and other than MTC (NMTC) is accomplished by applying biochemical tests: niacin production, catalase activity, thermostable at 68 ° C and reduction of nitrate.

nutritional status directly affects the possibility of quick recovery (SSA, 2009).

resistance against the traditional antibiotics along time.

**1. Introduction** 

 \*

Corresponding Author

*Instituto Politécnico Nacional, Cd. Reynosa, Tamps,* 

*del Instituto Politécnico Nacional, Altamira, Tamps,* 

*del Instituto Politécnico Nacional, Cd. Reynosa, Tamps,* 


## **Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico**

H. W. Araujo-Torres1,3, J. A. Narváez-Zapata2, M. G. Castillo-Álvarez1, MS. Puga-Hernández4, J. Flores-Gracia5 and M. A. Reyes-López1,\* *1Conservation Medicine Lab., Centro de Biotecnología Genómica del Instituto Politécnico Nacional, Cd. Reynosa, Tamps, 2Industrial Biotechnology Lab., Centro de Biotecnología Genómica del Instituto Politécnico Nacional, Cd. Reynosa, Tamps, 3Centro de Investigación en Ciencia Aplicada y Tecnología Avanzada del Instituto Politécnico Nacional, Altamira, Tamps, 4Laboratorio Estatal de Salud Pública de Tamaulipas, Cd. Victoria, Tamps, 5Instituto Tecnológico de Ciudad Victoria, Cd. Victoria, Tamps México* 

## **1. Introduction**

348 Polymerase Chain Reaction

[173] Prugnolle, F., et al. (2010). African great apes are natural hosts of multiple related

[175] Towner, J. S., et al. (2007). Marburg virus infection detected in a common African bat.

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[183] Ahuka-Mundeke, S., et al. (2011). Identification and Molecular Characterization of

[184] Calattini, S., et al. (2006). Detection and molecular characterization of foamy viruses in

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[187] Halkett, F., et al. (2010). Genetic discontinuities and disequilibria in recently

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1-related lentivirus isolated from a wild captured chimpanzee. *Virology*, 221, (2),

New Simian T Cell Lymphotropic Viruses in Nonhuman Primates Bushmeat from the Democratic Republic of Congo. *AIDS Res Hum Retroviruses*, 2011 Sep 14. [Epub

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infection in wild-living chimpanzees. *PLoS Pathog*, 4, (7), e1000097, ISSN 1553-7366

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established populations of the plant pathogenic fungus Mycosphaerella fijiensis.

The Case of Cameroon through a Bibliometric Analysis. *Dev World Bioeth*, 11(3):

One third of the world population is afected by TB and one million people did die this year 2011 in undeveloped countries (Venkatesh et al., 2011). In Tamaulipas, a Northern State of Mexico and a border state between USA and Mexico, frequency is 26.9 new TB cases per 100,000 people, twice of national rate of 12.85 cases per 100,000 people (Ferrer et al., 2010). Only on the border of Tamaulipas about 320 cases are diagnosed each year. Many of these cases correspond to people from other states of Mexico, probably by geographic position and by migration problematic of this study zone (Fitchett et al., 2011). Only 92% of the treated population are cured mainly because much of these people are poor and whose nutritional status directly affects the possibility of quick recovery (SSA, 2009).

The long presence of this disease has increased the need to know specifically which *Mycobacterium tuberculosis* strains are circulating in the region. Additionally, it is necessary to know the antibiotic/susceptibility profile of these strains since many of them acquire resistance against the traditional antibiotics along time.

In general, the diagnostic of this disease is traditionally conducted by using gold standard techniques focused to identify the presence of *M. tuberculosis* in clinical specimen of humans or cattle. These techniques included the strain of microorganism in Ziehl-Neelsen and culture in Lowenstein-Jensen medium (Cadmus et al., 2011), both regarded as reference techniques in the diagnosis of TB. Differentiation among mycobacteria of the *M. tuberculosis* complex (MTC) and other than MTC (NMTC) is accomplished by applying biochemical tests: niacin production, catalase activity, thermostable at 68 ° C and reduction of nitrate.

<sup>\*</sup> Corresponding Author

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 351

Veracruz, Coahuila, Mexico city, among others) and other Central America countries (i.e.

One of the reasons of this migration may be high number of manufacturing factories that offer a high number of jobs and that appeal and wait to travel. In addition, many of these people only remain of 4 to 5 years here and wait to travel to U.S.A. As it was previously mentioned, many of these people are poor and with low nutritional status and their lifestyle (i.e. drug or alcohol consumes) could prompted the TB disease (Wagner et al., 2011). Therefore, TB will become a big issue between Mexico and USA. From here, both countries have agreements on health and security cooperation. These agreements include the fast detection of this disease and the discrimination among *M. tuberculosis* strains (Fitchett et al,

Recently, it has been reported an increase in the TB cases around of the world, particularly due to generation of new *M. tuberculosis* strains with resistance to traditional antibiotics, or multidrug resistance (Sougakoff, 2011). In 2007, the 14th edition of the Merck list shows 30 different anti-TB drugs, many analogues or prodrugs of antibiotics, as the first line of defense against this disease. In Mexico, the antibiotics most commonly used are the rifampicin (RIF), the isoniazid (INH), the pyrazinamide (PZA), the streptomycin (STR) and

Worldwide, rifampicin is the drug mostly in the control of this bacterium (Connell et al., 2011). These antibiotics are not enough to halt the emergence and spread of multidrug resistant (MDR) strains causing a serious problem for the TB control and increasing public health problems (Zumi, et al. 2001). This have prompts the development of fast and reliable diagnostic process to detect, to discriminate, and to evaluate resistance of *M. tuberculosis*

Molecular biology has allowed detection of DNA or RNA sequence of different mycobacteria. An example of these approaches is using probes. Probes were prepared from nucleic acid sequences complementary to the DNA or RNA sequences from different species (including *M. tuberculosis, M. avium, M. kansasii, M. gordonae*.), which may be labeled with radioactive isotopes (hot probes) or chromogenic substances (cold probes). The gene probe is capable of binding or hybridizing with a homologous fragment of the study sample, which has been previously denatured by physical means. Hybridization of the probe to its complementary fragment is easily detected with addition of a marker. The main advantages of these techniques are fast and specific. Its disadvantages high cost and that many probes

Typing techniques based on amplification of nucleic acids by PCR provide a fast and reliable approach to obtain genetic information about bacteria or microorganism groups. Molecular typing methods for tuberculosis are based on that those infected by strains of *M. tuberculosis* have the same genotype (genetic fingerprinting) and are epidemiologically

related, while those infected with different genotypes (unique patterns) are not.

Guatemala, Honduras, among others).

**1.3 Multidrug resistant in Mycobacterium tuberculosis** 

the ethambutol (EMB) (Borrell, Gagneux., 2011).

strains against main drugs.

**1.4 Molecular approaches** 

cannot identify species within the MTC.

2011).

Actually, detection for *M. tuberculosis* has been shorted due mainly to application of molecular methods directly to clinical samples. Usually, the detection of this bacterium takes 2 to 4 weeks (Marhöfer et al., 2011). Some molecular techniques are already on the market, being the most commonly used *AMPLICOR M. tuberculosis PCR test* (Roche), *M. tuberculosis* Direct test (MTDT) (GenProbe) and LCX *M. tuberculosis* assay (Abbott). In addition, PCR amplification of ribosomal sequences (Ribotyping) or amplification of repetitive intragenic consensus sequences (i.e. ERIC-PCR, spoligotyping, MIRU-VNTR), among others, are the usual methods used to specifically discriminate among different *M. tuberculosis* strains (Rodwell et al., 2010; Pang et al., 2011).

The use of molecular approach is also applied in the analysis of the antibiotic resistance of these isolates. In this sense, molecular detection of specific mutations in genes involved with drug resistance has successfully been applied in the identification of these (i.e. detection of mutations on *rpo*B, *kat*G, *mab*A, etc.) (Sala & Hartkoorn, 2011).

As an example of the conjunction of the background described above, this chapter briefly presents a work of potential tuberculosis patient samples, to which mycobacteria were isolated to determine whether any of them were resistant to some antibiotics and if it could be grouped by health districts of Tamaulipas and also grouped the isolates strains in MTC and NMTC, and identify them, potentially.

Therefore, the main aim of this study was determinated by using a molecular approach the specific *M. tuberculosis* strains presents in the region and identify specific mutations in these strains related with drug resistance. The information here generated helps to take epidemiological decisions aimed to control and to prevent this disease in the Northeast of Tamaulipas.

## **1.1 TB statistical**

This main issue is due to produce a resistance against antibiotics used traditionally to control the disease. The first antibiotic against TB was created in the 40's decade. Consequently, the incidence of this disease declined in the following decades, especially in developed countries. However, in the last 20 years it has been observed an increase in the TB cases around of the world, particularly due to generation of new *M. tuberculosis* strains with resistance of the traditional antibiotics, or multidrug resistance (Yew et al., 2010). The death associated to TB may increase in undeveloped countries since some others diseases as the HIV may duplicate the death frequency in patients with both diseases (Havlir & Barnes 1999; Sonnenberg et al., 2005). Given this situation the TB was declared as global emergency by WHO (WHO 1993).

#### **1.2 Situation of TB in northern Mexico**

The Northeast state of Tamaulipas exhibits a high peak of occurrence of TB with regarding at Mexican rate and the frequency of this disease has remained stable during the last 10 years (Ferrer et al., 2010). Besides, this region is a natural corridor for exporting of cattle between Mexico and EE.UU. Therefore, both countries have commitment to keep safe their borders (Fitchett et al., 2011). Among the factors that may partially explain it, the high migrations rate reported in this region with people from other Mexican states (mainly Veracruz, Coahuila, Mexico city, among others) and other Central America countries (i.e. Guatemala, Honduras, among others).

One of the reasons of this migration may be high number of manufacturing factories that offer a high number of jobs and that appeal and wait to travel. In addition, many of these people only remain of 4 to 5 years here and wait to travel to U.S.A. As it was previously mentioned, many of these people are poor and with low nutritional status and their lifestyle (i.e. drug or alcohol consumes) could prompted the TB disease (Wagner et al., 2011). Therefore, TB will become a big issue between Mexico and USA. From here, both countries have agreements on health and security cooperation. These agreements include the fast detection of this disease and the discrimination among *M. tuberculosis* strains (Fitchett et al, 2011).

## **1.3 Multidrug resistant in Mycobacterium tuberculosis**

Recently, it has been reported an increase in the TB cases around of the world, particularly due to generation of new *M. tuberculosis* strains with resistance to traditional antibiotics, or multidrug resistance (Sougakoff, 2011). In 2007, the 14th edition of the Merck list shows 30 different anti-TB drugs, many analogues or prodrugs of antibiotics, as the first line of defense against this disease. In Mexico, the antibiotics most commonly used are the rifampicin (RIF), the isoniazid (INH), the pyrazinamide (PZA), the streptomycin (STR) and the ethambutol (EMB) (Borrell, Gagneux., 2011).

Worldwide, rifampicin is the drug mostly in the control of this bacterium (Connell et al., 2011). These antibiotics are not enough to halt the emergence and spread of multidrug resistant (MDR) strains causing a serious problem for the TB control and increasing public health problems (Zumi, et al. 2001). This have prompts the development of fast and reliable diagnostic process to detect, to discriminate, and to evaluate resistance of *M. tuberculosis* strains against main drugs.

#### **1.4 Molecular approaches**

350 Polymerase Chain Reaction

Actually, detection for *M. tuberculosis* has been shorted due mainly to application of molecular methods directly to clinical samples. Usually, the detection of this bacterium takes 2 to 4 weeks (Marhöfer et al., 2011). Some molecular techniques are already on the market, being the most commonly used *AMPLICOR M. tuberculosis PCR test* (Roche), *M. tuberculosis* Direct test (MTDT) (GenProbe) and LCX *M. tuberculosis* assay (Abbott). In addition, PCR amplification of ribosomal sequences (Ribotyping) or amplification of repetitive intragenic consensus sequences (i.e. ERIC-PCR, spoligotyping, MIRU-VNTR), among others, are the usual methods used to specifically discriminate among different *M.* 

The use of molecular approach is also applied in the analysis of the antibiotic resistance of these isolates. In this sense, molecular detection of specific mutations in genes involved with drug resistance has successfully been applied in the identification of these (i.e. detection of

As an example of the conjunction of the background described above, this chapter briefly presents a work of potential tuberculosis patient samples, to which mycobacteria were isolated to determine whether any of them were resistant to some antibiotics and if it could be grouped by health districts of Tamaulipas and also grouped the isolates strains in MTC

Therefore, the main aim of this study was determinated by using a molecular approach the specific *M. tuberculosis* strains presents in the region and identify specific mutations in these strains related with drug resistance. The information here generated helps to take epidemiological decisions aimed to control and to prevent this disease in the Northeast of

This main issue is due to produce a resistance against antibiotics used traditionally to control the disease. The first antibiotic against TB was created in the 40's decade. Consequently, the incidence of this disease declined in the following decades, especially in developed countries. However, in the last 20 years it has been observed an increase in the TB cases around of the world, particularly due to generation of new *M. tuberculosis* strains with resistance of the traditional antibiotics, or multidrug resistance (Yew et al., 2010). The death associated to TB may increase in undeveloped countries since some others diseases as the HIV may duplicate the death frequency in patients with both diseases (Havlir & Barnes 1999; Sonnenberg et al., 2005). Given this situation the TB was declared as global emergency

The Northeast state of Tamaulipas exhibits a high peak of occurrence of TB with regarding at Mexican rate and the frequency of this disease has remained stable during the last 10 years (Ferrer et al., 2010). Besides, this region is a natural corridor for exporting of cattle between Mexico and EE.UU. Therefore, both countries have commitment to keep safe their borders (Fitchett et al., 2011). Among the factors that may partially explain it, the high migrations rate reported in this region with people from other Mexican states (mainly

*tuberculosis* strains (Rodwell et al., 2010; Pang et al., 2011).

mutations on *rpo*B, *kat*G, *mab*A, etc.) (Sala & Hartkoorn, 2011).

and NMTC, and identify them, potentially.

Tamaulipas.

**1.1 TB statistical** 

by WHO (WHO 1993).

**1.2 Situation of TB in northern Mexico** 

Molecular biology has allowed detection of DNA or RNA sequence of different mycobacteria. An example of these approaches is using probes. Probes were prepared from nucleic acid sequences complementary to the DNA or RNA sequences from different species (including *M. tuberculosis, M. avium, M. kansasii, M. gordonae*.), which may be labeled with radioactive isotopes (hot probes) or chromogenic substances (cold probes). The gene probe is capable of binding or hybridizing with a homologous fragment of the study sample, which has been previously denatured by physical means. Hybridization of the probe to its complementary fragment is easily detected with addition of a marker. The main advantages of these techniques are fast and specific. Its disadvantages high cost and that many probes cannot identify species within the MTC.

Typing techniques based on amplification of nucleic acids by PCR provide a fast and reliable approach to obtain genetic information about bacteria or microorganism groups. Molecular typing methods for tuberculosis are based on that those infected by strains of *M. tuberculosis* have the same genotype (genetic fingerprinting) and are epidemiologically related, while those infected with different genotypes (unique patterns) are not.

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 353

of 126 bp that appear to be restricted only to transcribed regions of chromosome. Its position in the genome appears to be different in different species. As any technique, ERIC-PCR is used as typing, to study the clonal relationship in various Gram-negative bacteria such as *Acinetobacter baumannii*. The DNA patterns obtained with the ERIC-PCR are usually less complex than those generated by other techniques such as REP-PCR. The technique is quick and easy to perform, and provides highly reproducible results (Gillings & Holley 1997.).

Additionally, the presence of ERIC sequences has been detected in genome of *M. tuberculosis* (Sechi et al, 1998). Studies showed that the level of differentiation obtained by ERIC-PCR is superior to that obtained by the RFLP-IS6110 genetic profile comparable to that obtained by (*GTG*) *5*-*PCR* fingerprinting (PCR-GTG). The use of the PCR-GTG, a repetitive marker in the *M. tuberculosis* chromosome with an IS6110 sequence has been successfully applied to a PCR-based fingerprinting method. This method is fast and sensitive and can be applied to the study of the epidemiology of infections caused by *M. tuberculosis* and therapeutic implications for health, particularly when the IS6110 RFLP-DNA profile does not provide

The aim of this study was to conduct a molecular characterization of mycobacteria strains by typing and drug resistance gene mutations from samples of potential TB patients in

Two strategies were conducted to study the samples isolated from patients with probable TB clinical diagnosis from the State Public Health Laboratory of Tamaulipas (LESPT) from The State of Tamaulipas, MX. The first one was the identification of strains as belonged or not to MTC. Second one was to detect mutations on the genes related to drug resistance to

Specimens included in this study were collected over a period of 16 months (October 2008 to January 2010) from acid-fast bacilli AFB-positive sputum obtained from the State Public Health Laboratory (LESPT). Basically, LESPT concentrates most of the TB cases from Tamaulipas. All the samples were taken under the informed consent of the patients. . In addition, a structured test was used to obtain standard demographic and epidemiologic data of the patients. Two sputum consecutive specimens were collected from each individual. These samples were mixed with 1% cetylpyridinium chloride and immediately transported to the LESPT where they were stored at 4o C (Kent and Kubica, 1985). All strains cultured were identified to species level by standard microbiological procedures in

Samples were first lysed (tissue samples were mechanically disrupted) and proteins simultaneously denatured in the appropriate lysis buffer. QIAGEN Proteinase K was then

any help.

**2. Objective** 

**3. Methods** 

the LESPT.

**3.2 DNA extraction** 

Northeast Mexico.

major antibiotics against *M. tuberculosis.*

**3.1 Samples and cultures** 

Among the techniques of molecular biology that are currently used, as: ribotyping, the PCR amplification of repetitive extragenic palindromic sequences (REP-PCR) and the repetitive intragenic consensus sequences of *Enterobacteriaceae* (ERIC-PCR). These techniques can also be used in clinical studies to establish patterns of colonization and to identify sources of transmission of infectious microorganisms, which may contribute to a better understanding of the epidemiology and pathogenesis thereby helping to develop disease prevention strategies (Struelens, MESGEM, 1996).

## **1.5 Ribotyping**

Ribotyping technique applied in the diagnostic of diseases has been used for differentiation of bacterial serotypes involved with the occurrence of outbreaks. Additionally, this technique has an extended use in the study of nosocomial fungus (Pavlic and Griffiths, 2009). Ribotyping is also used to study the ecology, the genotypic variation and the transmission of Streptococcus mutants from person to person (Alam et al., 1999). The patterns are simplified to ribotyping, making visible the DNA fragments containing parts or all of ribosomal genes, sometimes detected bacterial serotypes (Pavlic and Griffiths, 2009).

Because of the epidemiological and clinical importance of some bacterial strains such as *M. tuberculosis*, it is interesting the application of related techniques like typing by PCR, in breaking through in a better understanding of the ecology and epidemiology of these bacteria. Some studies show this approach to evaluate the discriminatory power of different methods for genotyping of MTC isolates, they compared the performance of i) IS6110 DNA fingerprint, ii) spoligotyping and iii) 24-loci MIRU-VNTR (mycobacterial interspersed repetitive units - variable number of tandem repeats) typing in a long term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the most-northern federal state of Germany (Roetzer et al, 2011), other group studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variablenumber tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period (Pérez-Lago et al, 2011), another study was to compare polymerase chain reaction (PCR)-based methods- spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India (Varma-Basil et al 2011) and finally, a study where Forty three isoniazid (INH)-resistant *M. tuberculosis* isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction (Soudani et al, 2011).

#### **1.6 ERIC-PCR**

Amplification of enterobacterial repetitive intergenic consensus by PCR (ERIC-PCR) has only been used sporadically to detect mycobacteria. ERIC sequences are repetitive elements of 126 bp that appear to be restricted only to transcribed regions of chromosome. Its position in the genome appears to be different in different species. As any technique, ERIC-PCR is used as typing, to study the clonal relationship in various Gram-negative bacteria such as *Acinetobacter baumannii*. The DNA patterns obtained with the ERIC-PCR are usually less complex than those generated by other techniques such as REP-PCR. The technique is quick and easy to perform, and provides highly reproducible results (Gillings & Holley 1997.).

Additionally, the presence of ERIC sequences has been detected in genome of *M. tuberculosis* (Sechi et al, 1998). Studies showed that the level of differentiation obtained by ERIC-PCR is superior to that obtained by the RFLP-IS6110 genetic profile comparable to that obtained by (*GTG*) *5*-*PCR* fingerprinting (PCR-GTG). The use of the PCR-GTG, a repetitive marker in the *M. tuberculosis* chromosome with an IS6110 sequence has been successfully applied to a PCR-based fingerprinting method. This method is fast and sensitive and can be applied to the study of the epidemiology of infections caused by *M. tuberculosis* and therapeutic implications for health, particularly when the IS6110 RFLP-DNA profile does not provide any help.

## **2. Objective**

352 Polymerase Chain Reaction

Among the techniques of molecular biology that are currently used, as: ribotyping, the PCR amplification of repetitive extragenic palindromic sequences (REP-PCR) and the repetitive intragenic consensus sequences of *Enterobacteriaceae* (ERIC-PCR). These techniques can also be used in clinical studies to establish patterns of colonization and to identify sources of transmission of infectious microorganisms, which may contribute to a better understanding of the epidemiology and pathogenesis thereby helping to develop disease prevention

Ribotyping technique applied in the diagnostic of diseases has been used for differentiation of bacterial serotypes involved with the occurrence of outbreaks. Additionally, this technique has an extended use in the study of nosocomial fungus (Pavlic and Griffiths, 2009). Ribotyping is also used to study the ecology, the genotypic variation and the transmission of Streptococcus mutants from person to person (Alam et al., 1999). The patterns are simplified to ribotyping, making visible the DNA fragments containing parts or all of ribosomal genes, sometimes detected bacterial serotypes (Pavlic and Griffiths, 2009). Because of the epidemiological and clinical importance of some bacterial strains such as *M. tuberculosis*, it is interesting the application of related techniques like typing by PCR, in breaking through in a better understanding of the ecology and epidemiology of these bacteria. Some studies show this approach to evaluate the discriminatory power of different methods for genotyping of MTC isolates, they compared the performance of i) IS6110 DNA fingerprint, ii) spoligotyping and iii) 24-loci MIRU-VNTR (mycobacterial interspersed repetitive units - variable number of tandem repeats) typing in a long term study on the epidemiology of tuberculosis (TB) in Schleswig-Holstein, the most-northern federal state of Germany (Roetzer et al, 2011), other group studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variablenumber tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period (Pérez-Lago et al, 2011), another study was to compare polymerase chain reaction (PCR)-based methods- spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing--with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India (Varma-Basil et al 2011) and finally, a study where Forty three isoniazid (INH)-resistant *M. tuberculosis* isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and

Amplification of enterobacterial repetitive intergenic consensus by PCR (ERIC-PCR) has only been used sporadically to detect mycobacteria. ERIC sequences are repetitive elements

strategies (Struelens, MESGEM, 1996).

sequencing reaction (Soudani et al, 2011).

**1.6 ERIC-PCR** 

**1.5 Ribotyping** 

The aim of this study was to conduct a molecular characterization of mycobacteria strains by typing and drug resistance gene mutations from samples of potential TB patients in Northeast Mexico.

## **3. Methods**

Two strategies were conducted to study the samples isolated from patients with probable TB clinical diagnosis from the State Public Health Laboratory of Tamaulipas (LESPT) from The State of Tamaulipas, MX. The first one was the identification of strains as belonged or not to MTC. Second one was to detect mutations on the genes related to drug resistance to major antibiotics against *M. tuberculosis.*

## **3.1 Samples and cultures**

Specimens included in this study were collected over a period of 16 months (October 2008 to January 2010) from acid-fast bacilli AFB-positive sputum obtained from the State Public Health Laboratory (LESPT). Basically, LESPT concentrates most of the TB cases from Tamaulipas. All the samples were taken under the informed consent of the patients. . In addition, a structured test was used to obtain standard demographic and epidemiologic data of the patients. Two sputum consecutive specimens were collected from each individual. These samples were mixed with 1% cetylpyridinium chloride and immediately transported to the LESPT where they were stored at 4o C (Kent and Kubica, 1985). All strains cultured were identified to species level by standard microbiological procedures in the LESPT.

#### **3.2 DNA extraction**

Samples were first lysed (tissue samples were mechanically disrupted) and proteins simultaneously denatured in the appropriate lysis buffer. QIAGEN Proteinase K was then

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 355

A set of chosen primers, which amplified for desired sequences are shown below (Table 1).

Ribotyping by PCR was performed with two primers complementary to conserved regions. The sequences of the primers were described on Table 1. Amplifications were carried out in a final volume of 25 μl. Twenty five cycles of amplification were performed, with each cycle consisting of 2 min of denaturation at 94°C, 45 seconds of annealing at 62°C, and 1 min at 72°C. The last cycle consisted of a 7 min extension at 72°C. The amplification products were visualized after electrophoresis at 90 V for 90 min in a 2% agarose gel, and the gel was

For ERIC-PCR, a pair of primers (Sechi et al, 1998) used and their characteristics are

Amplification reactions were performed in a volume of 50 μl with final amounts of 1 U of Taq polymerase, 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 200 μM of deoxynucleoside triphosphate (Gibco, BRL, Life Technology, Paisley, United Kingdom). The reaction mixtures were then incubated for 5 min at 95°C, followed by 35 cycles of 94°C for 30 s, Touch-down (47-57°C), and 65°C for 4 min and a final extension at 70°C for 7 min. The amplification products were visualized after electrophoresis at 90 V for 90 min in a 2%

ERIC 1R 5'-ATGTAAGCT CCT GGGGATTCAC-3' 22 62.7 ERIC 2 5'-AAGTAAGTGACT GGGGTGAGCG-3' 22 64.5

Eight pairs of PCR primers (PR1 to PR16) were used to simultaneously amplify regions of eight genes associated with resistance to six antituberculosis drugs. In addition, eight pairs (PR17 to PR32) of internal PCR primers were then used to determine the DNA sequences of

PCR products obtained from only 36 out of 100 bacterial strains for ERIC-PCR and 15 bacteria drug resistant were purified with an EXO-SAP. Components were supplemented with gold buffer (Applied Biosystem) and sequenced on an Applied Biosystem 310 Genetic analyzer (ABI Prism 310 Genetic analyzer), using big dye terminator cycle sequencing

For Drug resistant, the purified samples were analyzed with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The DNA sequences are collected and edited with Data Collection software version 1.01 and Sequencing Analysis version 3.7 (Applied Biosystems)

(nt) Tm(°C)

agarose gel, and the gel was stained with SYBER Gold (Invitrogen).

Primer Nucleotide sequence Size

(Strom et al, 2002).

**3.5 ERIC-PCR** 

described below (Table 2).

Table 2. Primers for ERIC-PCR

these genes (Table 3 and 4)

Ready Kit (Applied Biosystem).

**3.7 Sequencing** 

**3.6 Gene drug resistant analysis** 

stained with SYBER Gold (Invitrogen).

added and after a suitable incubation period, lysates were loaded onto the QIAGEN Genomic-tip. DNA binds to the column while other cell constituents passed through. Following a wash step to remove any remaining contaminants, pure, high-molecular-weight DNA was eluted and precipitated with isopropanol. Hands-on time for the complete procedure was just 45 minutes for samples.

Bacterial strains obtained from patients with TB were preliminary analyzed by an antibiogram test to verify if these strains exhibit some class of antibiotic resistant. Approximately, One hundred consecutive strains were selected to further molecular characterization. Bacteria selected were growth in solid Lowenstein-Jensen and 7H9 Middlebrook broths supplemented with 10% (vol/vol) of oleic acid-albumin-dextrosecatalase. After that, the samples were incubated for at least 8 weeks. DNA from bacterial samples was obtained from those grew strains by used the QIAGEN kit (QIAGEN) of according to manufacturing instructions

## **3.3 Molecular detection of** *M. tuberculosis*

The following primers were used (Yeboah-Manu et al. 2001): spacer region-specific primers, spacer region 33 specific (5′ACACCGACATGACGGCGG3′) and spacer region 34 specific (5′CGACGGTGTGGGCGAGG3′); IS6110 (5′GGACAACGCCGAATTGCG′3 and 5′TAGGCGTCGGTGACAAAGGCCAC′3), and Mycobacterium genus-specific TB11 (sequence 5′ACCAACGATGGTGTGTCCAT3′) and TB12 (sequence 5′CTTGTCGAACCGCATACCCT3′). Expected PCR products are 550, 439, and 172 and 99 bp, respectively.

PCR mixtures contained 20 μl of 2× PCR mix, 10 μL of primer mix with each primer at 0.66 pmol/μL, 0.2 μL of Taq polymerase enzyme (Roche Diagnostics), and 10 μL of extracted DNA. The PCR conditions were 95°C for 3 min; 30 cycles of 95°C for 20 s, 65°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. After PCR, the products were analyzed by electrophoresis in agarose gel.

## **3.4 Typing methods**

For ribotyping, the standardization of PCR was done using three sets of primers to amplify the 16S region (Table 1). Note: The primers 16S and 16S R F were used for amplification of 16S Mycobacterium.


Table 1. Primers used for amplification of 16S Ribosomal DNA of Mycobacterium.

A set of chosen primers, which amplified for desired sequences are shown below (Table 1). (Strom et al, 2002).

Ribotyping by PCR was performed with two primers complementary to conserved regions. The sequences of the primers were described on Table 1. Amplifications were carried out in a final volume of 25 μl. Twenty five cycles of amplification were performed, with each cycle consisting of 2 min of denaturation at 94°C, 45 seconds of annealing at 62°C, and 1 min at 72°C. The last cycle consisted of a 7 min extension at 72°C. The amplification products were visualized after electrophoresis at 90 V for 90 min in a 2% agarose gel, and the gel was stained with SYBER Gold (Invitrogen).

## **3.5 ERIC-PCR**

354 Polymerase Chain Reaction

added and after a suitable incubation period, lysates were loaded onto the QIAGEN Genomic-tip. DNA binds to the column while other cell constituents passed through. Following a wash step to remove any remaining contaminants, pure, high-molecular-weight DNA was eluted and precipitated with isopropanol. Hands-on time for the complete

Bacterial strains obtained from patients with TB were preliminary analyzed by an antibiogram test to verify if these strains exhibit some class of antibiotic resistant. Approximately, One hundred consecutive strains were selected to further molecular characterization. Bacteria selected were growth in solid Lowenstein-Jensen and 7H9 Middlebrook broths supplemented with 10% (vol/vol) of oleic acid-albumin-dextrosecatalase. After that, the samples were incubated for at least 8 weeks. DNA from bacterial samples was obtained from those grew strains by used the QIAGEN kit (QIAGEN) of

The following primers were used (Yeboah-Manu et al. 2001): spacer region-specific primers, spacer region 33 specific (5′ACACCGACATGACGGCGG3′) and spacer region 34 specific (5′CGACGGTGTGGGCGAGG3′); IS6110 (5′GGACAACGCCGAATTGCG′3 and 5′TAGGCGTCGGTGACAAAGGCCAC′3), and Mycobacterium genus-specific TB11 (sequence 5′ACCAACGATGGTGTGTCCAT3′) and TB12 (sequence 5′CTTGTCGAACCGCATACCCT3′). Expected PCR products are 550, 439, and 172 and 99

PCR mixtures contained 20 μl of 2× PCR mix, 10 μL of primer mix with each primer at 0.66 pmol/μL, 0.2 μL of Taq polymerase enzyme (Roche Diagnostics), and 10 μL of extracted DNA. The PCR conditions were 95°C for 3 min; 30 cycles of 95°C for 20 s, 65°C for 30 s, and 72°C for 30 s; and 72°C for 7 min. After PCR, the products were analyzed by electrophoresis

For ribotyping, the standardization of PCR was done using three sets of primers to amplify the 16S region (Table 1). Note: The primers 16S and 16S R F were used for amplification of

(nt)

19 16

18 17

20 19 Tm (°C)

62.3 46.5

57.62 59.61

64 60 Reference

Sechi A, et al, 1998.

Strom et al,

Sorrell et al,

2002.

1996.

Primer Sequence Size

5'-TTGTACACACCGCCCGTCA-3' 5'-GAAACATCTAATACCT-3'

5'-GAGGAAGGTGGGGATGACGT-3' 5'-AGGCCCGGAACGTATTCAC-3'

Table 1. Primers used for amplification of 16S Ribosomal DNA of Mycobacterium.

5'AGAGTTTGATCCTGGCTC-3' 5'-CGGGAACGTATTCACCG-3'

procedure was just 45 minutes for samples.

according to manufacturing instructions

bp, respectively.

in agarose gel.

**3.4 Typing methods** 

16S Mycobacterium.

R1 R2

16S F 16S R

P13P F P11P R

**3.3 Molecular detection of** *M. tuberculosis*

For ERIC-PCR, a pair of primers (Sechi et al, 1998) used and their characteristics are described below (Table 2).

Amplification reactions were performed in a volume of 50 μl with final amounts of 1 U of Taq polymerase, 20 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, and 200 μM of deoxynucleoside triphosphate (Gibco, BRL, Life Technology, Paisley, United Kingdom). The reaction mixtures were then incubated for 5 min at 95°C, followed by 35 cycles of 94°C for 30 s, Touch-down (47-57°C), and 65°C for 4 min and a final extension at 70°C for 7 min. The amplification products were visualized after electrophoresis at 90 V for 90 min in a 2% agarose gel, and the gel was stained with SYBER Gold (Invitrogen).


Table 2. Primers for ERIC-PCR

## **3.6 Gene drug resistant analysis**

Eight pairs of PCR primers (PR1 to PR16) were used to simultaneously amplify regions of eight genes associated with resistance to six antituberculosis drugs. In addition, eight pairs (PR17 to PR32) of internal PCR primers were then used to determine the DNA sequences of these genes (Table 3 and 4)

#### **3.7 Sequencing**

PCR products obtained from only 36 out of 100 bacterial strains for ERIC-PCR and 15 bacteria drug resistant were purified with an EXO-SAP. Components were supplemented with gold buffer (Applied Biosystem) and sequenced on an Applied Biosystem 310 Genetic analyzer (ABI Prism 310 Genetic analyzer), using big dye terminator cycle sequencing Ready Kit (Applied Biosystem).

For Drug resistant, the purified samples were analyzed with the ABI PRISM 3100 Genetic Analyzer (Applied Biosystems). The DNA sequences are collected and edited with Data Collection software version 1.01 and Sequencing Analysis version 3.7 (Applied Biosystems)

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 357

segment of 250 nucleotides with an average size of 750 nucleotides for each sequence. Of these 12 sequences, only 3 isolates were grouped in NMTC, the 9 remaining isolated strains show polymorphism in their nucleotide sequences belong to the MTC. Two sequences of isolates tested showed 100% and 98% identity respectively with the species of *M. fortuitum* according to our analysis in the NCBI database (strain *M. fortuitum* 16S gene with accession number DQ973806.1 and strain *M. fortuitum* 16S gene with accession number AY457066.1,

Fig. 1. Comparison of genetic profiles from isolated mycobacteria by ERIC-PCR VS

analysis to identify their samples.

Ribotyping (a) In picture, letters A, B, C, and D show four different genetic profiles of ERIC-PCR grouped in 7 isolates of mycobacteria. Arrow on direction of figure (c) indicates 7 isolated strains are part of the MTC. (b) Image shows ERIC-PCR amplifications of 3 isolates (samples 2M, 10M, 19M) clustered in NMTC. (c) Phylogenetic tree based on comparison of 16S gene mycobacterium species from MTC and NMTC. *Mycobacterium spp* sample.

A third sequence of one isolated strain showed 100% identity with *M. chelonae* (*M. chelonae* strain T9 with AM884324.1 access number). In this sense, it is useful to mention that LESPT identified these strains as *M. tuberculosis* based only on their microbiological and biochemical results. It is important to mention that LESPT does not conducted molecular

respectively).

and compared with those of M. tuberculosis H37Rv (GenBank access no. NC\_000962) with the program Geneious version 4.5.4 (Software Development Biomatters Ltd).

Additionally, for ribotyping, sequence of 16S Ribosomal DNA of mycobacterial determined in an ABI Prism® 3130. Obtained sequences were analyzed in NCBI database using BlastN analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Alignment editing of 16S sequences was performed by Chromas Lite 2.0, BioEdit Sequence Alignment Editor Version 7.0.4.1 and CLC Sequence Viewer Version 6.1 software.

Finally, the comparison between isolated Mycobacteria and reported Mycobacterium tuberculosis strains were made by a microbial identification and phylogenetic analysis of obtained data using MEGA4 Software Version 4.0.2. Tree topologies were determined by methods of Minimum evolution criterion and Maximum Parsimony, with a value of reliability, "Bootstrap" of 100 replications for phylogenetic analysis.

## **4. Results and conclusion**

Male population in Tamaulipas is the most affected by TB with 61% of the isolates evaluated in this work. Geographical distribution of infected people represented a greater proportion in Central and South of the State with 52% and 45% of isolates evaluated, respectively.


Table 3. Primers for multiplex-PCR (Sekiguchi et al. 2007)

Thirty-seven out of 40 samples were analyzed by 16S gene sequences, 34 of them were grouped in the MTC, and the 3 remaining sequences were integrated into the NMTC (Figure 1). Moreover, from 37 sequences analyzed, only 12 of these showed polymorphisms on a

and compared with those of M. tuberculosis H37Rv (GenBank access no. NC\_000962) with

Additionally, for ribotyping, sequence of 16S Ribosomal DNA of mycobacterial determined in an ABI Prism® 3130. Obtained sequences were analyzed in NCBI database using BlastN analysis (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Alignment editing of 16S sequences was performed by Chromas Lite 2.0, BioEdit Sequence Alignment Editor Version

Finally, the comparison between isolated Mycobacteria and reported Mycobacterium tuberculosis strains were made by a microbial identification and phylogenetic analysis of obtained data using MEGA4 Software Version 4.0.2. Tree topologies were determined by methods of Minimum evolution criterion and Maximum Parsimony, with a value of

Male population in Tamaulipas is the most affected by TB with 61% of the isolates evaluated in this work. Geographical distribution of infected people represented a greater proportion in Central and South of the State with 52% and 45% of isolates evaluated, respectively.

Region Sequences Position Size

(bp)

315

2,223

1,362

2,748

670

572

1,329

398

1256–1275 1570–1551


640–665 3387–3303

> -80 a -61 590–572

428–447 1756–1737

4–23 575–556

1–19 397–379

1–32 223–2200

the program Geneious version 4.5.4 (Software Development Biomatters Ltd).

7.0.4.1 and CLC Sequence Viewer Version 6.1 software.

**4. Results and conclusion** 

katG PR3 (forward)

reliability, "Bootstrap" of 100 replications for phylogenetic analysis.

rpoB PR1 (forward) 5-CCGCGATCAAGGAGTTCTTC-3

mabA PR5 (forward) 5-ACATACCTGCTGCGCAATTC-3

pncA PR9 (forward) 5-GGCGTCATGGACCCTATATC-3

rpsL PR11 (forward) 5-CCAACCATCCAGCAGCTGGT-3

rrs PR13 (forward) 5-AAACCTCTTTCACCATCGAC-3

gyrA PR15 (forward) 5-GATGACAGACACGACGTTGC-3 PR16 (reverse) 5-GGGCTTCGGTGTACCTCAT-3

Table 3. Primers for multiplex-PCR (Sekiguchi et al. 2007)

PR2 (reverse) 5-ACACGATCTCGTCGCTAACC-3

PR6 (reverse) 5-GCATACGAATACGCCGAGAT-3

embB PR7 (forward) 5-CCGACCACGCTGAAACTGCTGGCGAT-3 PR8 (reverse) 5-GCCTGGTGCATACCGAGCAGCATAG-3

PR10 (reverse) 5-CAACAGTTCATCCCGGTTC-3

PR12 (reverse) 5-GTCGAGAGCCCGCTTGAGGG-3

PR14 (reverse) 5-GTATCCATTGATGCTCGCAA-3

Thirty-seven out of 40 samples were analyzed by 16S gene sequences, 34 of them were grouped in the MTC, and the 3 remaining sequences were integrated into the NMTC (Figure 1). Moreover, from 37 sequences analyzed, only 12 of these showed polymorphisms on a

5-GTGCCCGAGCAACACCCACCCATTACAGAAAC -3 PR4 (reverse) 5-TCAGCGCACGTCGAACCTGTCGAG-3

segment of 250 nucleotides with an average size of 750 nucleotides for each sequence. Of these 12 sequences, only 3 isolates were grouped in NMTC, the 9 remaining isolated strains show polymorphism in their nucleotide sequences belong to the MTC. Two sequences of isolates tested showed 100% and 98% identity respectively with the species of *M. fortuitum* according to our analysis in the NCBI database (strain *M. fortuitum* 16S gene with accession number DQ973806.1 and strain *M. fortuitum* 16S gene with accession number AY457066.1, respectively).

Fig. 1. Comparison of genetic profiles from isolated mycobacteria by ERIC-PCR VS Ribotyping (a) In picture, letters A, B, C, and D show four different genetic profiles of ERIC-PCR grouped in 7 isolates of mycobacteria. Arrow on direction of figure (c) indicates 7 isolated strains are part of the MTC. (b) Image shows ERIC-PCR amplifications of 3 isolates (samples 2M, 10M, 19M) clustered in NMTC. (c) Phylogenetic tree based on comparison of 16S gene mycobacterium species from MTC and NMTC. *Mycobacterium spp* sample.

A third sequence of one isolated strain showed 100% identity with *M. chelonae* (*M. chelonae* strain T9 with AM884324.1 access number). In this sense, it is useful to mention that LESPT identified these strains as *M. tuberculosis* based only on their microbiological and biochemical results. It is important to mention that LESPT does not conducted molecular analysis to identify their samples.

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 359

However, one aspect to consider in the results obtained in this work is that of obtaining the 4 different genetic profiles amplified by ERIC-PCR, they did not allow to discriminate among species of MTC and NMTC as it was expected, those profiles or genetic patterns of 3 isolated strains have been totally different from other profiles of 7 isolated strains (Figure 1),

Regarding to genetic relatedness of 40 isolates of mycobacteria studied, phylogenetic analysis of 16S gene sequences showed 37 sequences, which formed two groups. The first group of MTC consisting of 34 isolates and the second group resulted in NMTC consisting of 3 isolates. The percentages of identity were from 98% to 100% for isolates clustered in

The analysis on relationship between the isolates studied and their geographical origin revealed that the Mycobacterium tuberculosis complex is distributed both in the central and south of the state of Tamaulipas, MX. Meanwhile, species such as *M. fortuitum* and *M.* 

It should be noted that the isolates studied are only samples originating from the central and southern Tamaulipas. No isolated strains were obtained from northern part of Tamaulipas, which would have complemented the results of this research. For example, as mentioned before in those border states (US-Mexico border) there is a great number of people and cattle to move from different parts of country and abroad, which could suggest existence of different strains of *M. tuberculosis* in Tamaulipas, and even the presence of other species found in the central and southern Tamaulipas, MX, allowing us to know not only that isolated strains in each region are preferably, but also known how those isolated strains are

Note this work was limited to samples of LESPT; however the proposal will be to analyze samples from all health districts in Tamaulipas, and analyze samples of other states, such as Veracruz and/or Coahuila. Additionally, we will seek to refine the ERIC-PCR and implementing the MIRU-VNTR and spoligotyping for more complete diagnosis. In addition, arrangements are made between the County LESPT and McAllen, Texas, United States, to have samples (about 14,000 isolates) identified and stored in the United States from

Finally, the preliminary results were shown (Table 5), where mutations, insertions, transversions, and transitions were found. In general, the mutations obtained did not alter the chemical or structural composition of proteins that confer resistance to an antibiotic to the mycobacteria and their regions sequenced. In these particular cases, we selected to work with isolated strains were resistant to antibiotics commonly administered in Mexico, the results obtained for the case of pyrazinamide, a silent mutation was found, so that the resistance exhibited by the bacteria should be caused by mutations on the sequenced region. In the case of isoniazid *mab*A gene, we found an insertion within the gene that could be the

These results indicate that DNA sequencing-based method was effective for detection of MDR strains. However, when novel mutations in drug resistance-related genes are detected by the method, it is essential to also perform drug susceptibility testing, because novel

*chelonae* are only found circulating in the central region of Tamaulipas.

all of 10 isolated strains are in MTC.

circulating all around Tamaulipas.

cause of resistance exhibited.

mutation may not be associated with drug resistance.

both complexes.

Tamaulipas.

One out of the two identified strains was *M. fortuitum*, made by sequencing, but no for microbiology, since this was identified as *M. tuberculosis* by LESPT. The other isolated itself coincided with both techniques.This gives us a different result, although microbiology diagnostic and taken at this time, we do believe strongly that this is due to *M. fortuitum.*


Table 4. Primers for sequencing (Sekiguchi et al. 2007)

The use of the reference strain H37Rv of *M. tuberculosis* sequence served as a reference or guide for clustering of the isolates studied, since in the phylogenetic analysis, the type strain H37Rv was integrated with the 34 isolated MTC, thus reaffirming the phylogenetic relationship of isolates tested with the species *M. tuberculosis* (Figure 1). On the other hand, the reference sequence of *Nocardia arthritidis* (No. Access EU841600) showed no relation with the 37 evaluated strains in phylogenetic analysis, being totally separated from the two complexes formed, MTC and NMTC. This comparison is done, because of *Nocardia* is also acid-resistant and can be confused with *Mycobacteria* on microscopic analysis, hence the importance of making the comparison. Then, It should be mentioned that the identification of mycobacteria isolated from the 16S sequences was proved to be an appropriate strategy to establish the level of genetic relatedness among isolates studied and know how related isolates were isolated or if these could be separated into MTC and NMTC.

ERIC-PCR technique gave 4 different genetic profiles for mycobacteria (Figure 1). It should be emphasized that three of these genetic profiles are consistent with those reported in molecular epidemiology studies by amplifying sequences ERIC (Sechi et al, 1998). From 34 isolates clustered in the complex of M. tuberculosis, seven of these 4 make up the genetic profiles obtained by ERIC-PCR. Then they expect the rest of the isolates (27 isolates) that make up this complex terms grouped in the 4 genetic profiles (A, B, C and D) obtained by ERIC-PCR.

One out of the two identified strains was *M. fortuitum*, made by sequencing, but no for microbiology, since this was identified as *M. tuberculosis* by LESPT. The other isolated itself coincided with both techniques.This gives us a different result, although microbiology diagnostic and taken at this time, we do believe strongly that this is due to *M. fortuitum.*

Gene Sequences Position rpoB PR17 5-TACGGCGTTTCGATGAAC-3 (complementary strand) 1529–1512

mabA PR22 5-ACATACCTGCTGCGCAATTC-3 -217 a -198

pncA PR28 5-GGCGTCATGGACCCTATATC-3 -80 -61 rpsL PR29 5-CCAACCATCCAGCAGCTGGT-3 4–23

gyrA PR32 5-GATGACAGACACGACGTTGC-3 1–19

The use of the reference strain H37Rv of *M. tuberculosis* sequence served as a reference or guide for clustering of the isolates studied, since in the phylogenetic analysis, the type strain H37Rv was integrated with the 34 isolated MTC, thus reaffirming the phylogenetic relationship of isolates tested with the species *M. tuberculosis* (Figure 1). On the other hand, the reference sequence of *Nocardia arthritidis* (No. Access EU841600) showed no relation with the 37 evaluated strains in phylogenetic analysis, being totally separated from the two complexes formed, MTC and NMTC. This comparison is done, because of *Nocardia* is also acid-resistant and can be confused with *Mycobacteria* on microscopic analysis, hence the importance of making the comparison. Then, It should be mentioned that the identification of mycobacteria isolated from the 16S sequences was proved to be an appropriate strategy to establish the level of genetic relatedness among isolates studied and know how related

ERIC-PCR technique gave 4 different genetic profiles for mycobacteria (Figure 1). It should be emphasized that three of these genetic profiles are consistent with those reported in molecular epidemiology studies by amplifying sequences ERIC (Sechi et al, 1998). From 34 isolates clustered in the complex of M. tuberculosis, seven of these 4 make up the genetic profiles obtained by ERIC-PCR. Then they expect the rest of the isolates (27 isolates) that make up this complex terms grouped in the 4 genetic profiles (A, B, C and D) obtained by

PR25 5-GGTGGGCAGGATGAGGTAGT-3 (complementary strand)

689–670 574–593 1162–1181 1729–1748

646–665 1462–1481 1596–1577 2007–2026 2581–2601

979–959 1291–1310

katG PR18 5-ACGTAGATCAGCCCCATCTG-3 (complementary strand)

Rrs PR30 5-CAGGTAAGGTTCTTCGCGTTG-3 (complementary strand)

isolates were isolated or if these could be separated into MTC and NMTC.

PR19 5-GAGCCCGATGAGGTCTATTG-3 PR20 5-CCGATCTATGAGCGGATCAC-3 PR21 5-GAACAAACCGACGTGGAATC-3

PR24 5-GTCATCCTGACCGTGGTGTT-3

PR26 5-CACAATCTTTTTCGCCCTGT-3 PR27 5-GCGTGGTATCTCCTGCCTAAG-3

PR31 5-GTTCGGATCGGGGTCTGCAA-3

Table 4. Primers for sequencing (Sekiguchi et al. 2007)

ERIC-PCR.

embB PR23 5-ACGCTGAAACTGCTGGCGAT-3

However, one aspect to consider in the results obtained in this work is that of obtaining the 4 different genetic profiles amplified by ERIC-PCR, they did not allow to discriminate among species of MTC and NMTC as it was expected, those profiles or genetic patterns of 3 isolated strains have been totally different from other profiles of 7 isolated strains (Figure 1), all of 10 isolated strains are in MTC.

Regarding to genetic relatedness of 40 isolates of mycobacteria studied, phylogenetic analysis of 16S gene sequences showed 37 sequences, which formed two groups. The first group of MTC consisting of 34 isolates and the second group resulted in NMTC consisting of 3 isolates. The percentages of identity were from 98% to 100% for isolates clustered in both complexes.

The analysis on relationship between the isolates studied and their geographical origin revealed that the Mycobacterium tuberculosis complex is distributed both in the central and south of the state of Tamaulipas, MX. Meanwhile, species such as *M. fortuitum* and *M. chelonae* are only found circulating in the central region of Tamaulipas.

It should be noted that the isolates studied are only samples originating from the central and southern Tamaulipas. No isolated strains were obtained from northern part of Tamaulipas, which would have complemented the results of this research. For example, as mentioned before in those border states (US-Mexico border) there is a great number of people and cattle to move from different parts of country and abroad, which could suggest existence of different strains of *M. tuberculosis* in Tamaulipas, and even the presence of other species found in the central and southern Tamaulipas, MX, allowing us to know not only that isolated strains in each region are preferably, but also known how those isolated strains are circulating all around Tamaulipas.

Note this work was limited to samples of LESPT; however the proposal will be to analyze samples from all health districts in Tamaulipas, and analyze samples of other states, such as Veracruz and/or Coahuila. Additionally, we will seek to refine the ERIC-PCR and implementing the MIRU-VNTR and spoligotyping for more complete diagnosis. In addition, arrangements are made between the County LESPT and McAllen, Texas, United States, to have samples (about 14,000 isolates) identified and stored in the United States from Tamaulipas.

Finally, the preliminary results were shown (Table 5), where mutations, insertions, transversions, and transitions were found. In general, the mutations obtained did not alter the chemical or structural composition of proteins that confer resistance to an antibiotic to the mycobacteria and their regions sequenced. In these particular cases, we selected to work with isolated strains were resistant to antibiotics commonly administered in Mexico, the results obtained for the case of pyrazinamide, a silent mutation was found, so that the resistance exhibited by the bacteria should be caused by mutations on the sequenced region. In the case of isoniazid *mab*A gene, we found an insertion within the gene that could be the cause of resistance exhibited.

These results indicate that DNA sequencing-based method was effective for detection of MDR strains. However, when novel mutations in drug resistance-related genes are detected by the method, it is essential to also perform drug susceptibility testing, because novel mutation may not be associated with drug resistance.

Study of Mycobacterium Tuberculosis by Molecular Methods in Northeast Mexico 361

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Niemann S. 2011.Evaluation of Mycobacterium tuberculosis typing methods in a

Viridans Group Streptococci. Journal of Clinical Microbiology. Vol. 37, No. 9. p.

QFB. Norma Alicia Villareal Reyes from LESPT.

21st century. Eur Respir Rev. 20(120):71-84

virus infection. N. Engl. J. Med. 340, 367–373

Trop Med Hyg. 83(5):1056-8

Microbiology. No. 25. p. 17-21.

Clin Microbiol. 49(11):3771-3776.),

Infect Dis.;14 Suppl 3:e129-35

Control, Atlanta, Ga.

**8. References** 

2772-2776.


Table 5. Relationship of changes found in the sequences of the genes of interest.

## **5. Conclusion**

In conclusion, two strategies were carried out to study the samples isolated from patients with TB diagnostic of LESPT from Tamaulipas, MX. The first was the identification of isolates and determine if these isolates belonged or not to MTC. Second, to determinate if mutations in primary sequences of genes related to resistance to major antibiotics used to kill mycobacteria in Tamaulipas, could be detected.

For the first part of the study, there were used 3 strategies, a multiplex-PCR, ERIC-PCR, and ribotyping. For the second direct amplification of 16S DNA region was performed.

Multiplex-PCR for 99% of the samples coincided with the microbiological results, identifying *M. tuberculosis*, primary. In the case of ERIC-PCR, the samples could be grouped into 4 different groups; however it could differentiate between MTC and NMTC. Finally, ribotyping produced promising results by discriminating the isolated strains and identifying 99% as *M. tuberculosis*.

Finally, the results indicate that DNA sequencing-based method was effective for detection of MDR strains. However, when novel mutations in drug resistance-related genes are detected by the method, it is essential to also perform drug susceptibility testing, because novel mutations are not always associated with drug resistance.

### **6. Perspectives**

This kind of work will answer other questions: is it necessary ribotyping before or after ERIC-PCR or multiplex-PCR and it is important to recognize each species of Mycobacteria to understand if TB strains would circulate all around Tamaulipas and if those ones would be or get in USA too? In a few years we will understand this phenomenon; meanwhile this chapter makes the first approach to understand how TB strains are moving and if those strains are or not drug resistant on a border State between USA and Mexico. The present investigation continues, pending to sequence regions of resistance to pyrazinamide and ethambutol, which are largest genes.

#### **7. Acknowledgments**

Financing of this research was by Instituto Politécnico Nacional (projects SIP 20090679 and SIP 20100504). Narváez-Zapata J. A. and Reyes-López M. A. are fellows of CONACYT-SNI, COFAA, and EDI from Instituto Politécnico Nacional. Authors are also grateful for the support received from Network of Drug Development and Diagnostic Methods (RED FARMED) from CONACyT. Finally, authors really appreciated the samples submitted for QFB. Norma Alicia Villareal Reyes from LESPT.

## **8. References**

360 Polymerase Chain Reaction

*rpo*B 2 CGG-TGG R476W *rrs* 2 TGG-AGG W193R *rps*L 2 AAA-AAG K121K *pnc*A 2 GGT-GGC G75G


Table 5. Relationship of changes found in the sequences of the genes of interest.

In conclusion, two strategies were carried out to study the samples isolated from patients with TB diagnostic of LESPT from Tamaulipas, MX. The first was the identification of isolates and determine if these isolates belonged or not to MTC. Second, to determinate if mutations in primary sequences of genes related to resistance to major antibiotics used to

For the first part of the study, there were used 3 strategies, a multiplex-PCR, ERIC-PCR, and

Multiplex-PCR for 99% of the samples coincided with the microbiological results, identifying *M. tuberculosis*, primary. In the case of ERIC-PCR, the samples could be grouped into 4 different groups; however it could differentiate between MTC and NMTC. Finally, ribotyping produced promising results by discriminating the isolated strains and

Finally, the results indicate that DNA sequencing-based method was effective for detection of MDR strains. However, when novel mutations in drug resistance-related genes are detected by the method, it is essential to also perform drug susceptibility testing, because

This kind of work will answer other questions: is it necessary ribotyping before or after ERIC-PCR or multiplex-PCR and it is important to recognize each species of Mycobacteria to understand if TB strains would circulate all around Tamaulipas and if those ones would be or get in USA too? In a few years we will understand this phenomenon; meanwhile this chapter makes the first approach to understand how TB strains are moving and if those strains are or not drug resistant on a border State between USA and Mexico. The present investigation continues, pending to sequence regions of resistance to pyrazinamide and

Financing of this research was by Instituto Politécnico Nacional (projects SIP 20090679 and SIP 20100504). Narváez-Zapata J. A. and Reyes-López M. A. are fellows of CONACYT-SNI,

ribotyping. For the second direct amplification of 16S DNA region was performed.

Nucleotide Protein

Changes

Gene Number of

**5. Conclusion** 

sample

*mab*A 2 702 T insertion

kill mycobacteria in Tamaulipas, could be detected.

novel mutations are not always associated with drug resistance.

identifying 99% as *M. tuberculosis*.

ethambutol, which are largest genes.

**7. Acknowledgments** 

**6. Perspectives** 


**18** 

*1Belgium 2Italy* 

**Development of a Molecular Platform** 

Sylvia Broeders1, Nina Papazova1,

Marc Van den Bulcke2 and Nancy Roosens1

*Platform Biotechnology and Molecular Biology,* 

*Protection, Molecular Biology and Genomics Unit* 

 **for GMO Detection in Food and Feed on the** 

*1Wetenschappelijk Instituut Volksgezondheid, Institut Scientifique de Santé Publique,* 

Fifteen years after the first commercialisation of biotech crops, the global area of their cultivation comprises more than one billion hectares. The increase in the area between 1996 and 2010 is 87-fold which makes biotech crops the fastest adopted technology in modern

In 2010, 184 Genetically Modified (GM – see glossary) events, representing 24 crops have already received worldwide regulatory approval. To date, 29 countries have cultivated GM crops, whereas 59 countries have granted regulatory approvals for their import for food and feed use and release into the environment. The six main countries cultivating GM crops are USA, Brazil, Argentina, India, Canada and China. In the EU the cultivation area of biotech crops amounts only 0,1%of the cultivation area reaching 125 million hectares in 25 countries (Stein & Rodriguez-Cerezo, 2009). The most important biotech crop is soybean (50% of the biotech crops cultivation area), followed by maize (31%), cotton (14%) and oilseed rape (4%)

Herbicide tolerance and insect resistance are the main traits used in the first generation of GM crops. After 2009, many GM events conferring novel traits have entered the regulatory system. New traits were introduced in soybean, maize, cotton and oilseed rape. The second generation of traits comprises altered crop composition, new herbicide tolerances, virus and nematode resistance and abiotic stress tolerance. Furthermore, new crops such as potato and rice were approved in different countries (Stein & Rodriguez-Cerezo, 2009). Moreover, gene stacking is a trend that is likely to increase in the near future. There are new events containing up to four stacked traits in the regulatory pipeline. A maize stacked event containing up to eight traits is in an advanced research and development stage (Dow

**1. Introduction** 

(James, 2010).

agriculture (James, 2010).

AgroSciences SmartStax® platform; James, 2010).

*2European Commission, Joint Research Centre, Institute for Health and Consumer* 

**Basis of "Combinatory qPCR" Technology** 

four-year study in Schleswig-Holstein, Northern Germany. J Clin Microbiol. Oct 12 [Epub ahead of print])


## **Development of a Molecular Platform for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology**

Sylvia Broeders1, Nina Papazova1, Marc Van den Bulcke2 and Nancy Roosens1 *1Wetenschappelijk Instituut Volksgezondheid, Institut Scientifique de Santé Publique, Platform Biotechnology and Molecular Biology, 2European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Molecular Biology and Genomics Unit 1Belgium 2Italy* 

## **1. Introduction**

362 Polymerase Chain Reaction

Sala C, Hartkoorn RC. (2011). Tuberculosis drugs: new candidates and how to find more.

Sechi A., Zanetti S., Dupré I., Delogu G. and Fadda G. (1998). Enterobacterial Repetitive

Sonnenberg, P. et al. (2005) How soon after infection with HIV does the risk of tuberculosis

Sorrell TC, Chen SC, Ruma P, Meyer W, Pfeiffer TJ, Ellis DH, Brownlee AG. (1996).

Soudani A, Hadjfredj S, Zribi M, Messaadi F, Messaoud T, Masmoudi A, Zribi M, Fendri C.

Sougakoff W. (2011). Molecular epidemiology of multidrug-resistant strains of

Ström K, Sjögren J, Broberg A, Schnürer J. (2002). Lactobacillus plantarum MiLAB 393

tuberculosis in Delhi, North India. Mem Inst Oswaldo Cruz. 106(5):524-35) Venkatesh KK, Swaminathan S, Andrews JR, Mayer KH. (2011). Tuberculosis and HIV co-

Wagner KD, Pollini RA, Patterson TL, Lozada R, Ojeda VD, Brouwer KC, Vera A, Volkmann

Yew WW, Sotgiu G, Migliori GB. (2010). Update in tuberculosis and nontuberculous mycobacterial disease Am J Respir Crit Care Med. 2011 Jul 15;184(2):180-5

infection: screening and treatment strategies. Drugs. 71(9):1133-52

drug users in Tijuana, Mexico. Drug Alcohol Depend. 113(2-3):236-41 Yeboah-Manu D, Yates MD, Wilson SM. (2001). Application of a simple multiplex PCR to

Mycobacterium tuberculosis strains. 2011. J Microbiol. 49(3):413-7).

Mycobacterium tuberculosis. Clin Microbiol Infect. 17(6):800-5

[Epub ahead of print])

45(1):179-92.

39(11):4166-8.

Infect. Dis. 191, 150–158

fingerprinting. J Clin Microbiol. 34(5):1253-60.

SSA. (2009). Secretaría de Salud. Informe México 2009.

Future Microbiol. 6(6):617-33

four-year study in Schleswig-Holstein, Northern Germany. J Clin Microbiol. Oct 12

Consensus Sequences as Molecular Targets for Typing of Mycobacterium tuberculosis Strains. Journal of Clinical Microbiology. Vol. 3, No. 1. p. 128-132. Sekiguchi J, Miyoshi-Akiyama T, Augustynowicz-Kopeć E, Zwolska Z, Kirikae F, Toyota E,

Kobayashi I, Morita K, Kudo K, Kato S, Kuratsuji T, Mori T, Kirikae T. (2007). Detection of multidrug resistance in Mycobacterium tuberculosis. J Clin Microbiol.

start to increase? A retrospective cohort study in South African gold miners. J.

Concordance of clinical and environmental isolates of Cryptococcus neoformans var. gattii by random amplification of polymorphic DNA analysis and PCR

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant

Produces the Antifungal Cyclic Dipeptides Cyclo(L-Phe-L-Pro) and Cyclo(L-Phetrans-4-OH-L-Pro) and 3-Phenyllactic Acid. Appl Environ Microbiol. 68(9):4322-7. Struelens, J., and Members of the European Study Group on Epidemiological Markers

(MESGEM). (1996). Consensus guidelines for Appropriate use and evaluation of microbial epidemiologic typing systems. Clin Microbiology and Infect, 2 (1): 2-11. Varma-Basil M, Kumar S, Arora J, Angrup A, Zozio T, Banavaliker JN, Singh UB, Rastogi N,

Bose M. 2011. Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium

TA, Strathdee SA. (2011). Cross-border drug injection relationships among injection

aid in routine work of the mycobacterium reference laboratory. J Clin Microbiol.

Fifteen years after the first commercialisation of biotech crops, the global area of their cultivation comprises more than one billion hectares. The increase in the area between 1996 and 2010 is 87-fold which makes biotech crops the fastest adopted technology in modern agriculture (James, 2010).

In 2010, 184 Genetically Modified (GM – see glossary) events, representing 24 crops have already received worldwide regulatory approval. To date, 29 countries have cultivated GM crops, whereas 59 countries have granted regulatory approvals for their import for food and feed use and release into the environment. The six main countries cultivating GM crops are USA, Brazil, Argentina, India, Canada and China. In the EU the cultivation area of biotech crops amounts only 0,1%of the cultivation area reaching 125 million hectares in 25 countries (Stein & Rodriguez-Cerezo, 2009). The most important biotech crop is soybean (50% of the biotech crops cultivation area), followed by maize (31%), cotton (14%) and oilseed rape (4%) (James, 2010).

Herbicide tolerance and insect resistance are the main traits used in the first generation of GM crops. After 2009, many GM events conferring novel traits have entered the regulatory system. New traits were introduced in soybean, maize, cotton and oilseed rape. The second generation of traits comprises altered crop composition, new herbicide tolerances, virus and nematode resistance and abiotic stress tolerance. Furthermore, new crops such as potato and rice were approved in different countries (Stein & Rodriguez-Cerezo, 2009). Moreover, gene stacking is a trend that is likely to increase in the near future. There are new events containing up to four stacked traits in the regulatory pipeline. A maize stacked event containing up to eight traits is in an advanced research and development stage (Dow AgroSciences SmartStax® platform; James, 2010).

Development of a Molecular Platform

**Transformation event (Unique identifier)** 

**Bt11** 

**DAS1507 (DAS-Ø15Ø7-1)** 

**MON810** 

**T25** 

**(MON-ØØ81Ø-6)** 

**(ACS-ZMØØ3-2)** 

**MON88017 (MON-88Ø17-3)** 

**MON89034 (MON-89Ø34-3)** 

*3272 maize (SYN-E3272-5)* 

*(DP-098140-6)* 

**DAS1507xNK603 (DAS-Ø15Ø7-1xMON-**

**DAS59122xNK603 (DAS-59122-7xMON-**

**(SYN-BTØ11-1xMON-**

**ØØ6Ø3-6)** 

**ØØ6Ø3-6)** 

**Bt11xGA21** 

**ØØØ21-9)** 

*98140* 

**(SYN-BT Ø11-1)** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 365

**Maize single events** 

Insect resistance Herbicide tolerance (glufosinate)

Insect resistance (Lepidopteran insects)

Herbicide tolerance (glufosinate)

Herbicide tolerance (glufosinate)

Insect resistance (Coleopteran insects) Herbicide tolerance

Insect resistance (Lepidopteran insects)

*Altered composition (increased α-amilase* 

*Herbicide tolerance (ALS-inhibiting herbicides)* 

Insect resistance (Coleopteran insects) Double herbicide tolerance (glufosinate and glyfosate)

Insect resistance (Coleopteran insects) Double herbicide tolerance (glufosinate and glyfosate)

Insect resistance (Lepidopteran insects)

Double herbicide tolerance (glufosinate and glyfosate)

(glyfosate)

*content)* 

Insect resistance (Lepidopteran insects)

**Trait Transformation event** 

**DAS59122 (DAS-59122-7)** 

**(MON-ØØØ21-9)** 

**(MON-ØØ6Ø3-6)** 

**GA21** 

**MON863 (MON-ØØ863-5)** 

**NK603** 

**MIR604 (SYN-IR6Ø4-5)** 

*Bt176*

*MIR162 (SYN-IR162-4 )* 

**Maize stacked events** 

*(SYN-EV176-9)*

**NK603xMON810 (MON-ØØ6Ø3-6 x MON-ØØ81Ø-6)** 

**MON863xMON810 (MON-ØØ863-5 x MON-ØØ81Ø-6)** 

**MON863xNK603 (MON-ØØ863-5 x MON-ØØ6Ø3-6)** 

**(Unique identifier)** 

**Trait** 

Insect resistance (Coleopteran insects) Herbicide tolerance (glufosinate)

Herbicide tolerance

Insect resistance (Coleopteran insects)

Herbicide tolerance

Insect resistance (Coleopteran insects)

*Insect resistance (European corn borer) Herbicide tolerance (glufosinate)* 

*Insect resistance (Lepidopteran insects)* 

Insect resistance (Lepidopteran insects) Herbicide tolerance

(glyfosate)

Double insect resistance

(Lepidopteran and Coleopteran insects)

Insect resistance (Coleopteran insects) Herbicide tolerance

(glyfosate)

(glyfosate)

(glyfosate)

In the EU until 2010, 39 events were authorised for import and processing in food and feed and two for cultivation. This includes 23 maize events from which 12 containing double and triple stacked traits, seven cotton events from which two containing stacked traits, four oilseed rape events, three soybean events, one potato and one sugar beet event. A detailed list of the EU-authorised GM events per crop with their main traits is presented in table 1.

Another tendency is that new GM events are not solely developed and commercialised by international biotech companies anymore, but also by scientific governmental institutions. Many of these GM events are commercialised by Asian national research centres (e.g. China, India) and are intended for the local markets. However, as many food and feed materials are imported in the EU from third party countries, events that are not submitted for authorisation in the EU (unauthorised GMO or UGM) might accidentally end up into in the food and feed chain (Stein & Rodriguez-Cerezo, 2009).

In reaction to the public concern about the presence of Genetically Modified Organisms (GMO – see glossary) in the food chain, many countries have adopted a specific legislation with respect to the introduction of GMO on their market. The legislation requirements vary from country to country, but there are some common elements such as case by case safety assessment, distinction between contained use and release into the environment and a distinction between cultivation and use as raw products in processing. Commonly recognised is the concept of substantial equivalence (Shauzu, 2001). In many regulatory systems tolerances or labelling thresholds, varying between 0.9 and 5%, were introduced.

The EU legislation on GMO is complex and consists of several core elements: a preauthorisation safety assessment, use of a labelling threshold, strict requirements for traceability of the GM products along the food chain and post-market monitoring. Labelling and traceability of new GM products are regulated mainly under Commission Regulations 1829/2003 and 1830/2003. For all events submitted under EC/1829/2003 a safety assessment is performed by the European Food Safety Authority (EFSA- see glossary). Food, feed and environmental risks are evaluated based on the data provided by the company requesting authorisation of a GM product. The food and feed safety assessment includes several issues such as allergenicity, toxicology, nutritional characteristics and post-market monitoring of the GM food and feed. The environmental risk assessment includes evaluation of the potential of gene transfer, interaction of the GM plant with target and nontarget organisms and monitoring (EFSA, 2011).

A very important issue is the molecular characterisation of the GM event. The objective of this characterisation is to obtain information on the introduced trait or genetic modification and to assess if unintended effects due to the genetic modification have taken place (Organisation for Economic Co-operation and Development [OECD], 2010). The molecular characterisation is an evaluation of relevant scientific data on the transformation process and vector constructs used, inserted transgenic sequences, copy number of the inserts, presence of partial copies, expression of the transgenic protein, stability and the inheritance of the transgenic insert (EFSA, 2011). The information on the elements introduced in the GMO as well as the sequence information on the junction regions between the plant genome and the transgenic insert are an essential part as they are related to the development of detection methods.

In the EU until 2010, 39 events were authorised for import and processing in food and feed and two for cultivation. This includes 23 maize events from which 12 containing double and triple stacked traits, seven cotton events from which two containing stacked traits, four oilseed rape events, three soybean events, one potato and one sugar beet event. A detailed list of the EU-authorised GM events per crop with their main traits is presented in table 1. Another tendency is that new GM events are not solely developed and commercialised by international biotech companies anymore, but also by scientific governmental institutions. Many of these GM events are commercialised by Asian national research centres (e.g. China, India) and are intended for the local markets. However, as many food and feed materials are imported in the EU from third party countries, events that are not submitted for authorisation in the EU (unauthorised GMO or UGM) might accidentally end up into in the

In reaction to the public concern about the presence of Genetically Modified Organisms (GMO – see glossary) in the food chain, many countries have adopted a specific legislation with respect to the introduction of GMO on their market. The legislation requirements vary from country to country, but there are some common elements such as case by case safety assessment, distinction between contained use and release into the environment and a distinction between cultivation and use as raw products in processing. Commonly recognised is the concept of substantial equivalence (Shauzu, 2001). In many regulatory systems tolerances or labelling thresholds, varying between 0.9 and 5%, were

The EU legislation on GMO is complex and consists of several core elements: a preauthorisation safety assessment, use of a labelling threshold, strict requirements for traceability of the GM products along the food chain and post-market monitoring. Labelling and traceability of new GM products are regulated mainly under Commission Regulations 1829/2003 and 1830/2003. For all events submitted under EC/1829/2003 a safety assessment is performed by the European Food Safety Authority (EFSA- see glossary). Food, feed and environmental risks are evaluated based on the data provided by the company requesting authorisation of a GM product. The food and feed safety assessment includes several issues such as allergenicity, toxicology, nutritional characteristics and post-market monitoring of the GM food and feed. The environmental risk assessment includes evaluation of the potential of gene transfer, interaction of the GM plant with target and non-

A very important issue is the molecular characterisation of the GM event. The objective of this characterisation is to obtain information on the introduced trait or genetic modification and to assess if unintended effects due to the genetic modification have taken place (Organisation for Economic Co-operation and Development [OECD], 2010). The molecular characterisation is an evaluation of relevant scientific data on the transformation process and vector constructs used, inserted transgenic sequences, copy number of the inserts, presence of partial copies, expression of the transgenic protein, stability and the inheritance of the transgenic insert (EFSA, 2011). The information on the elements introduced in the GMO as well as the sequence information on the junction regions between the plant genome and the transgenic insert are an essential part as they are related to the development of

food and feed chain (Stein & Rodriguez-Cerezo, 2009).

target organisms and monitoring (EFSA, 2011).

introduced.

detection methods.


Development of a Molecular Platform

**Transformation event (Unique identifier)** 

*Ms1, Rf2, Ms1xRf2 (ACS-BNØØ4-7 ACS-BNØØ2-5 ACS-BNØØ4-7xACS-*

*BNØØ2-5)* 

**GTS40-3-2 (MON-Ø4Ø32-6)** 

**MON89788 (MON-89788-1)** 

*305423 (DP-305423-1)* 

*MON87701 (MON-877Ø1-2)* 

**EH92-527-1 (BPS-25271-9)** 

**(KM-ØØØ71-4)** 

**H7-1** 

*LLrice62 (ACS-OSØØ2-5)*

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 367

**Soybean single events** 

**Potato single events** 

**Sugar beet single events** 

**Rice single events** 

Table 1. GM events authorised in the EU and events under under EC/619/2011 (*in italic*).

A labelling threshold of 0,9% is set up for all authorised GM events in the EU. Food and feed products containing GM events above this threshold have to be labelled as 'containing GMO'. The existence of a labelling threshold requires development of a system for GMO detection and quantification. Several types of methods exist, primarily bioassays, both protein-based (immunological) and DNA-based (mainly based on the Polymerase Chain Reaction (PCR) technology). The protein assays are based on the immunological reaction between the target protein and the specific antibody coupled with colorimetric detection (Holst-Jensen, 2009). Practical applications are the ELISA test or flow strip tests, which are widely used in testing of seed or grain materials. For instance, the United States Department of Agriculture- Grain Inspection, Packers and Stockyards (USDA-GIPSA, 2011) has certified several protein-based rapid kits for detection of biotech-derived grain/oilseeds. However, sensitivity and reliable quantification are often a problem for the immunological assays, due to for example low protein expression. Additionally, proteins are instable and nearly impossible to be reliably detected in processed products. Therefore, the DNA-based methods provide a reliable alternative for detection. In the European Union (EU), the detection of GMO is based on DNA and the recommended technique is real-time PCR. Moreover, this technique also provides the possibility for quantification of the GM target. In this context it is recommended to express the GM percentage as a ratio between the GM

Herbicide tolerance (glufosinate) Fertility restoration

Herbicide tolerance

Herbicide tolerance

*High oleic acid content A5547-127*

(glyfosate)

(glyfosate)

*Insect resistance (Lepidopteran insects)* 

Low amylase content

Herbicide tolerance

*Herbicide tolerance (glufosinate)* 

(glyfosate)

**Trait Transformation event** 

*Topas 19/2 (ACS-BNØØ7-1)* 

**A2704-12** 

*356043 (DP-356043-5)* 

**(ACS-GMØØ5-3)** 

*(ACS-GM006-4)* 

**(Unique identifier)** 

**Trait** 

Herbicide tolerance (glufosinate)

Herbicide tolerance (glufosinate)

*Double herbicide tolerance (glyfosate and ALS-inhibiting herbicides)* 

*Herbicide tolerance (glufosinate)* 


Double insect resistance

(glyfosate)

Double insect resistance

Double insect resistance

(glyfosate)

*(glyfosate)*

(glyfosate)

(glyfosate)

(glyfosate)

(glyfosate)

Insect resistance (Lepidopteran insects) Herbicide tolerance

*Insect resistance (Lepidopteran insects) Herbicide tolerance (glufosinate)* 

Herbicide tolerance

Herbicide tolerance (glufosinate) Fertility restoration

*Insect resistance (Lepidopteran insects) Herbicide tolerance* 

Herbicide tolerance

Herbicide tolerance

(Lepidopteran and Coleopteran insects) Herbicide tolerance

(Lepidopteran and Coleopteran insects) Herbicide tolerance (glufosinate)

(Lepidopteran and Coleopteran insects) Herbicide tolerance

**Trait Transformation event** 

**(MON-89Ø34-3x MON-ØØ6Ø3-6)** 

**88Ø17-3)** 

**K603** 

**6)** 

**Cotton single events** 

**Cotton stacked events** 

**Oilseed rape single events** 

**T45** 

**(ACS-BNØØ8-2)** 

*Ms1, Rf1, Ms1xRf1 (ACS-BNØØ4-7 ACS-BNØØ1-4 ACS-BNØØ4-7xACS-*

*BNØØ1-4)* 

Insect resistance **LLcotton25** 

**MON15985 (MON-15985-7)** 

**(ACS-GHØØ1-3)** 

**MON531xMON1445 (MON-ØØ531-6 x MON-Ø1445-2)** 

**(Unique identifier)** 

**MON89034xMON88017 (MON-89Ø34-3x MON-**

**DAS59122xDAS1507xN**

**(DAS-59122-7xDAS-Ø15Ø7xMON-ØØ6Ø3-**

**MON89034xNK603** 

**Trait** 

Insect resistance (Lepidopteran) Herbicide tolerance

(glyfosate)

Double insect resistance

(glyfosate)

Double insect resistance

(Lepidopteran and Coleopteran insects) Herbicide tolerance

(Lepidopteran and Coleopteran insects) Double herbicide tolerance (glyfosate and glufosinate)

Insect resistance (Lepidopteran insects)

Insect resistance Herbicide tolerance

Herbicide tolerance (glufosinate)

Herbicide tolerance (glufosinate) Fertility restoration

(glyfosate)

Herbicide tolerance (glufosinate)

**Transformation event (Unique identifier)** 

**MON88017xMON810 (MON-88Ø17-3xMON-**

**DAS1507xDAS59122 (DAS-Ø15Ø7x DAS-**

**MON863xMON810XNK**

**(MON-ØØ863-5xMON-ØØ81Ø-6xMON-ØØ6Ø3-6)** 

*GA21xMON810 (MON-ØØØ21-9 x MON-ØØ81Ø-6)*

**MON1445 (MON-Ø1445-2)** 

**MON531 (MON-ØØ531-6)** 

**GHB614** 

**Ø1445-2)** 

*21Ø23-5)* 

**GT73** 

**(BCS-GHØØ2-5)** 

**MON15985xMON1445 (MON-15985-7 x MON-**

*281-24-236/3006-210-23 (DAS-24236-5 x DAS-*

**(MON-ØØØ73-7)** 

**Ms8, Rf3, MS8xRf3 (ACS-BNØØ5-8ACS-BNØØ3-6ACS-BNØØ5- 8 x ACS-BN003-6)** 

**ØØ81Ø-6)** 

**59122-7)** 

**603** 


Table 1. GM events authorised in the EU and events under under EC/619/2011 (*in italic*).

A labelling threshold of 0,9% is set up for all authorised GM events in the EU. Food and feed products containing GM events above this threshold have to be labelled as 'containing GMO'. The existence of a labelling threshold requires development of a system for GMO detection and quantification. Several types of methods exist, primarily bioassays, both protein-based (immunological) and DNA-based (mainly based on the Polymerase Chain Reaction (PCR) technology). The protein assays are based on the immunological reaction between the target protein and the specific antibody coupled with colorimetric detection (Holst-Jensen, 2009). Practical applications are the ELISA test or flow strip tests, which are widely used in testing of seed or grain materials. For instance, the United States Department of Agriculture- Grain Inspection, Packers and Stockyards (USDA-GIPSA, 2011) has certified several protein-based rapid kits for detection of biotech-derived grain/oilseeds. However, sensitivity and reliable quantification are often a problem for the immunological assays, due to for example low protein expression. Additionally, proteins are instable and nearly impossible to be reliably detected in processed products. Therefore, the DNA-based methods provide a reliable alternative for detection. In the European Union (EU), the detection of GMO is based on DNA and the recommended technique is real-time PCR. Moreover, this technique also provides the possibility for quantification of the GM target. In this context it is recommended to express the GM percentage as a ratio between the GM

Development of a Molecular Platform

efficient manner.

**2.1 Introduction** 

quantification.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 369

Given the fact that an increasing number of events have to be analysed in order to comply with the legislation requirements, the control laboratories need to develop analytical approaches (platforms) which allow them to perform the analyses in a fast, cost and time-

In view of the EU legislation on GMO commercialisation and the fact that GM events are being authorised, it is mandatory to have control on the products being used and brought onto the market in the EU. Hereto, detection of GM events in food and feed samples is necessary to decide on the conformity of a sample. To enable this detection, real-time PCR (qPCR) is to date the method of choice. For this purpose, DNA needs to be extracted from the sample under analysis. In this process it is important to obtain not only enough DNA to perform the necessary qPCR reaction(s) (part 3) but also DNA of high quality (i.e. purity and integrity). As PCR is an enzymatic reaction, it is kinetically sensitive and the presence of other substances in the reaction may affect the PCR efficiency by for example impairing the binding of the primers to the target sequence in the genomic DNA. Such interference can have an impact on the GMO analysis cascade, especially on the last step namely the GMO

It has indeed been shown (Corbisier et al., 2007) that the quality of the DNA used in the qPCR has an important influence on the GM% obtained. Depending on the DNA extraction method used and the degree of purity of the extracted genomic DNA (gDNA), a deviating GM% was recorded. An interlaboratory study designed for the maize event MON 810, further demonstrated a significant influence of the DNA extraction method on the measurement results when using the construct-specific qPCR method while this impact was not seen when the event-specific detection method was utilised (Charels et al., 2007). It must thus be noted that even using 'pure' materials such as reference materials, DNA extraction is not so straightforward and that attention should be paid to the choice of the applied extraction method. This becomes even more important for enforcement laboratories as they mainly have to deal with processed and mixed samples. In this respect, Peano et al. (2004) reported the effect of treatment (mechanical, technological, chemical) of a sample in combination with the applied extraction method on the quality of the gDNA. When the feed and food product showed extensive fragmentation, due to a certain treatment during the preparation, the detection of these DNA fragments was dependant on the kit used for DNA extraction. Furthermore, Bellocchi et al. (2010) demonstrated that the result of a quantification experiment may be affected by the DNA extraction method employed unless DNA extracts that do not comply with previously set criteria were removed from the GM% calculations.

This highlights the importance of taking into account different parameters when using a modular approach (Holst-Jensen & Berdal, 2004). It is necessary to set up criteria for DNA quantity, purity, integrity and inhibition prior to using the extracted DNA in the qPCR reactions and to choose an appropriate DNA extraction method. Furthermore, attention should be paid to the fact that different targets might not be affected in the same way by impurities or co-extracted substances. Both Corbisier et al. (2007) and Cankar et al. (2006) demonstrated that this would impair in a strong way the final result. If, in a GMO quantification the two targets (i.e. the transgene and the taxon-specific element) do not

**2. Plant DNA extraction and its impact on GMO detection** 

copy numbers and taxon-specific copy numbers (Commission Recommendation EC/787/2004).

The GMO detection policy in the EU is based on two important elements: availability of validated methods for detection and availability of Certified Reference Materials (CRM – see glossary). According to the EU legislation before a new GMO is approved to be released on the market a validated event-specific detection method should be available. The eventspecific methods are developed by the company submitting the GMO for authorisation. The company has to develop a method complying with the acceptance criteria described in the document "Definition of Minimum Performance Requirements for analytical methods of GM testing" (ENGL, 2008) developed by the European Network of GMO Laboratories (ENGL – see glossary). The ENGL is a consortium of National Reference Laboratories (NRL – see glossary) assisting the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF – see glossary) by providing scientific expertise. The EU-RL is responsible for testing and validation of the method submitted by the applicant. Upon validation the method is published on the EU-RL web site (http://gmocrl.jrc.ec.europa.eu/) and made available for further use in the control laboratories involved in GMO testing.

In addition to detection methods, the EU legislation requires availability of Certified Reference Materials for the authorised events (EC/641/2004; EC/1829/2003). The CRM for GM testing are produced by the EC-JRC Institute for Reference Materials and Measurements (IRMM, BE) and the American Oil Chemists' Society (AOCS, USA) and usually are powder or leaf DNA extract with a certified content of the GM event.

The GM testing laboratories have to verify that they are capable to achieve the method performance characteristics before using it for routine analyses by performing in house validation by testing the relevant validation parameters as described in the guidance document (ENGL, 2011). Additionally, the control laboratories must be accredited under ISO 17025 (2005) or another equivalent international standard (Commission Regulation EC/1981/2006).

Although the EU legislation regulates the availability of event-specific methods for GMO detection, other methods such as construct-specific (recognising the GM constructs with which several events are transformed) or element-specific (detecting the elements present in many GMO) methods are used in the control laboratories in order to perform the analysis. These methods are subject to development and introduction of the laboratories themselves: there are no official guidelines describing how to validate such methods and which parameters have to be assessed.

The increasing GM cultivation worldwide and the number of authorisations in the EU and elsewhere pose a significant challenge to the control laboratories. They have to be able to apply all official methods for GM detection of authorised events. A second problem, are the asynchronous approvals of GM events in the EU and third party countries which can lead to low level presence of non-authorised GMO in food and feed. The recently adopted Commission Regulation EC/619/2011 regulates the presence of events which are pending for authorisation or withdrawn from the market in feed and for which methods for detection and reference materials (RM – see glossary) are available (table 1).

Given the fact that an increasing number of events have to be analysed in order to comply with the legislation requirements, the control laboratories need to develop analytical approaches (platforms) which allow them to perform the analyses in a fast, cost and timeefficient manner.

## **2. Plant DNA extraction and its impact on GMO detection**

#### **2.1 Introduction**

368 Polymerase Chain Reaction

copy numbers and taxon-specific copy numbers (Commission Recommendation

The GMO detection policy in the EU is based on two important elements: availability of validated methods for detection and availability of Certified Reference Materials (CRM – see glossary). According to the EU legislation before a new GMO is approved to be released on the market a validated event-specific detection method should be available. The eventspecific methods are developed by the company submitting the GMO for authorisation. The company has to develop a method complying with the acceptance criteria described in the document "Definition of Minimum Performance Requirements for analytical methods of GM testing" (ENGL, 2008) developed by the European Network of GMO Laboratories (ENGL – see glossary). The ENGL is a consortium of National Reference Laboratories (NRL – see glossary) assisting the European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF – see glossary) by providing scientific expertise. The EU-RL is responsible for testing and validation of the method submitted by the applicant. Upon validation the method is published on the EU-RL web site (http://gmocrl.jrc.ec.europa.eu/) and made available for further use in the control laboratories

In addition to detection methods, the EU legislation requires availability of Certified Reference Materials for the authorised events (EC/641/2004; EC/1829/2003). The CRM for GM testing are produced by the EC-JRC Institute for Reference Materials and Measurements (IRMM, BE) and the American Oil Chemists' Society (AOCS, USA) and usually are powder

The GM testing laboratories have to verify that they are capable to achieve the method performance characteristics before using it for routine analyses by performing in house validation by testing the relevant validation parameters as described in the guidance document (ENGL, 2011). Additionally, the control laboratories must be accredited under ISO 17025 (2005) or another equivalent international standard (Commission Regulation

Although the EU legislation regulates the availability of event-specific methods for GMO detection, other methods such as construct-specific (recognising the GM constructs with which several events are transformed) or element-specific (detecting the elements present in many GMO) methods are used in the control laboratories in order to perform the analysis. These methods are subject to development and introduction of the laboratories themselves: there are no official guidelines describing how to validate such methods and which

The increasing GM cultivation worldwide and the number of authorisations in the EU and elsewhere pose a significant challenge to the control laboratories. They have to be able to apply all official methods for GM detection of authorised events. A second problem, are the asynchronous approvals of GM events in the EU and third party countries which can lead to low level presence of non-authorised GMO in food and feed. The recently adopted Commission Regulation EC/619/2011 regulates the presence of events which are pending for authorisation or withdrawn from the market in feed and for which methods for detection

or leaf DNA extract with a certified content of the GM event.

and reference materials (RM – see glossary) are available (table 1).

EC/787/2004).

involved in GMO testing.

EC/1981/2006).

parameters have to be assessed.

In view of the EU legislation on GMO commercialisation and the fact that GM events are being authorised, it is mandatory to have control on the products being used and brought onto the market in the EU. Hereto, detection of GM events in food and feed samples is necessary to decide on the conformity of a sample. To enable this detection, real-time PCR (qPCR) is to date the method of choice. For this purpose, DNA needs to be extracted from the sample under analysis. In this process it is important to obtain not only enough DNA to perform the necessary qPCR reaction(s) (part 3) but also DNA of high quality (i.e. purity and integrity). As PCR is an enzymatic reaction, it is kinetically sensitive and the presence of other substances in the reaction may affect the PCR efficiency by for example impairing the binding of the primers to the target sequence in the genomic DNA. Such interference can have an impact on the GMO analysis cascade, especially on the last step namely the GMO quantification.

It has indeed been shown (Corbisier et al., 2007) that the quality of the DNA used in the qPCR has an important influence on the GM% obtained. Depending on the DNA extraction method used and the degree of purity of the extracted genomic DNA (gDNA), a deviating GM% was recorded. An interlaboratory study designed for the maize event MON 810, further demonstrated a significant influence of the DNA extraction method on the measurement results when using the construct-specific qPCR method while this impact was not seen when the event-specific detection method was utilised (Charels et al., 2007). It must thus be noted that even using 'pure' materials such as reference materials, DNA extraction is not so straightforward and that attention should be paid to the choice of the applied extraction method. This becomes even more important for enforcement laboratories as they mainly have to deal with processed and mixed samples. In this respect, Peano et al. (2004) reported the effect of treatment (mechanical, technological, chemical) of a sample in combination with the applied extraction method on the quality of the gDNA. When the feed and food product showed extensive fragmentation, due to a certain treatment during the preparation, the detection of these DNA fragments was dependant on the kit used for DNA extraction. Furthermore, Bellocchi et al. (2010) demonstrated that the result of a quantification experiment may be affected by the DNA extraction method employed unless DNA extracts that do not comply with previously set criteria were removed from the GM% calculations.

This highlights the importance of taking into account different parameters when using a modular approach (Holst-Jensen & Berdal, 2004). It is necessary to set up criteria for DNA quantity, purity, integrity and inhibition prior to using the extracted DNA in the qPCR reactions and to choose an appropriate DNA extraction method. Furthermore, attention should be paid to the fact that different targets might not be affected in the same way by impurities or co-extracted substances. Both Corbisier et al. (2007) and Cankar et al. (2006) demonstrated that this would impair in a strong way the final result. If, in a GMO quantification the two targets (i.e. the transgene and the taxon-specific element) do not

Development of a Molecular Platform

overestimation of the concentration.

primers and thus affect the PCR efficiency.

performing a duplex reaction would thus be a good solution.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 371

fragments (Georgiou & Papapostolou 2006; Holden et al., 2009; Shokere et al., 2009). One of the possible sources of single-stranded DNA may be denaturation of DNA during the drying phase after ethanol precipitation, the final step in many extraction protocols (Svaren et al., 1996). Utilizing spectrophotometry to quantify the DNA in an extract may thus lead to

Although one should determine the concentration of an extract to ensure that the DNA amount in a quantification reaction is above the limit of quantification (LOQ –part 5), the exact DNA concentration is of less importance. As the determination of the GM content of a sample relies on a relative calculation (ratio transgene copies versus endogene copies – part 3), it is imperative that a same amount of DNA is engaged in both qPCR reactions necessary in quantification, i.e. the event-specific and taxon-specific qPCR methods, whereas the exact amount engaged is of lesser importance. Carrying out both reactions in a single well, i.e.

When using spectrophotometry, additional to measurements at 260 nm, also measurements at wavelengths of 230 and 280 nm may be done. The **purity** of the DNA can then be assessed using the absorbance ratios A260/280 and A260/230. The A260/280 ratio gives an idea of the occurrence of residual proteins. On the other hand, the A260/230 ratio gives an indication on the presence of carbohydrates. In an ideal situation, both ratios should tend to 2,0 (Glasel 1995; Manchester 1995). Any deviation could indicate the presence of coextracted materials that can impair the availability of the DNA for hybridisation with the

Another important aspect is the **integrity** or intactness of the gDNA (degradation). When the DNA becomes fragmented, the GM target which is less abundant (compared to the endogene) might fall below the quantification limit of the qPCR method. It is evident that this has a practical consequence on the correct quantification of the target. One must thus ensure that the average length of the extracted DNA molecules is longer than the size of the amplicon. To avoid that degradation of the DNA impairs the GMO quantification, the methods are generally designed to amplify sequences ranging in size between 70 and 100 bp. However, one should take into account the minimum length of an amplicon necessary to allow binding of the oligonucleotides (two primers in SYBR®Green chemistry, two primers and one probe used in TaqMan® chemistry). To this purpose for example MGB probes (Kutyavin et al., 2000) can be used to allow even shorter sequences that are stable and have an elevated melting temperature. Further, the amplicon sizes for the endogene and transgene target should not differ too much as shorter fragments are more efficiently amplified than longer ones. This difference in amplification efficiencies will have an impact on the correctness of the quantification reaction. The intactness of the extracted DNA can be assessed using agarose gel electrophoresis with ethidium bromide staining or an alternative.

Knowledge of the presence of co-extracted substances and RNA and the existence of fragmented DNA in the extract is however not sufficient. It is known that **PCR inhibitors** are one of the most important influencing factors of the reliability of quantification (Bickley & Hopkins, 1999). It is thus important to know the impact of these molecules, present in the solution, on the GM quantification. Hereto, a preliminary inhibition test should be performed to evaluate their possible effect on the PCR efficiency. In this view, it is important to check if both targets of the quantification reaction (i.e. endogene and transgene) are equally affected by

This technique also allows observing if any RNA has been co-extracted.

behave in the same way and the PCR efficiencies are deviating too much, the obtained GM% would be biased.

It should also be noted that the extraction method used has a double impact on GMO quantification as not only the sample needs to be extracted but also the CRM. As the DNA extracted from the CRM powder will be used to construct the calibration curve in the quantification experiment it should also be free of inhibitors as this otherwise will affect the PCR efficiency. DNA extracted from the CRM powder needs to be pure and free of inhibitors to obtain a curve falling within the ENGL criteria (ENGL, 2011). Additionally, the PCR efficiencies for the calibrant and the sample should be the same to obtain reliable quantification. As this is not always the case, controls such as dilutions of the sample to evaluate inhibition, should be included in the reaction (point 2.2).

Although many DNA extraction protocols are quite user friendly and many extraction kits exist, their downstream application in qPCR is not clear-cut and additional evaluation of the quality of the extracted gDNA is necessary as well as assessment of the presence of possible PCR inhibitors.

## **2.2 Assessment of DNA yield, purity, integrity and inhibition**

The determination of the DNA concentration in an extract is not straightforward and different techniques exist. The obtained **DNA yield** after extraction can, for example, be determined using spectrophotometry (UV). This determination is based on the absorbance of nucleic acids at a wavelength of 260 nm. It is a method that has been used commonly for the estimation of the concentration of nucleic acids in a range of applications (Sambrook & Russell, 2001). Although it is a fast and simple method, it allows only determination of the concentration in a range of 5 to 50 µg/ml. Another drawback of this method is the fact that it is not specific for double stranded DNA (dsDNA) but also detects RNA and single stranded DNA (ssDNA) molecules (Gallagher, 2011). Additionally, substances like proteins and phenolics also absorb between 220 and 340 nm and can thus interfere with the measurement.

Alternatively, fluorimetry can be used to determine the concentration of the extracted gDNA in the solution (Singer et al., 1997). This method uses a dye that fluoresces upon intercalating in the dsDNA such as the PicoGreen (Molecular Probes). This enables a more specific measurement of the dsDNA amount present in an extract as there is no binding with interfering proteins and only a limited interaction with RNA and ssDNA. This method is more sensitive than UV measurements permitting to work with samples with lower concentrations in a linear range of 0,05 to 1 µg/ml (Singer et al., 1997). The method is reliable and well introduced in GMO testing laboratories. It should however be noted that a standard curve using lambda DNA needs to be prepared which requests a little more time. Furthermore it has been observed that the presence of various compounds have an effect on the accuracy of PicoGreen-based measurements (Singer et al., 1997; Holden et al., 2009 – see below).

A deviation between the concentration obtained by UV measurement and fluorimetry is often seen (Holden et al., 2009), especially for highly processed products (Bellocchi et al., 2010). This may be due to the fact that short or single stranded nucleic acid fragments interfere more with UV than with the PicoGreen dye. It has been proven that the fluorescence signal decreases with increasing length of sonication time (and thus fragmentation) showing the inability of the PicoGreen dye to bind with single stranded

behave in the same way and the PCR efficiencies are deviating too much, the obtained GM%

It should also be noted that the extraction method used has a double impact on GMO quantification as not only the sample needs to be extracted but also the CRM. As the DNA extracted from the CRM powder will be used to construct the calibration curve in the quantification experiment it should also be free of inhibitors as this otherwise will affect the PCR efficiency. DNA extracted from the CRM powder needs to be pure and free of inhibitors to obtain a curve falling within the ENGL criteria (ENGL, 2011). Additionally, the PCR efficiencies for the calibrant and the sample should be the same to obtain reliable quantification. As this is not always the case, controls such as dilutions of the sample to

Although many DNA extraction protocols are quite user friendly and many extraction kits exist, their downstream application in qPCR is not clear-cut and additional evaluation of the quality of the extracted gDNA is necessary as well as assessment of the presence of possible

The determination of the DNA concentration in an extract is not straightforward and different techniques exist. The obtained **DNA yield** after extraction can, for example, be determined using spectrophotometry (UV). This determination is based on the absorbance of nucleic acids at a wavelength of 260 nm. It is a method that has been used commonly for the estimation of the concentration of nucleic acids in a range of applications (Sambrook & Russell, 2001). Although it is a fast and simple method, it allows only determination of the concentration in a range of 5 to 50 µg/ml. Another drawback of this method is the fact that it is not specific for double stranded DNA (dsDNA) but also detects RNA and single stranded DNA (ssDNA) molecules (Gallagher, 2011). Additionally, substances like proteins and phenolics also absorb between 220 and 340 nm and can thus interfere with the measurement. Alternatively, fluorimetry can be used to determine the concentration of the extracted gDNA in the solution (Singer et al., 1997). This method uses a dye that fluoresces upon intercalating in the dsDNA such as the PicoGreen (Molecular Probes). This enables a more specific measurement of the dsDNA amount present in an extract as there is no binding with interfering proteins and only a limited interaction with RNA and ssDNA. This method is more sensitive than UV measurements permitting to work with samples with lower concentrations in a linear range of 0,05 to 1 µg/ml (Singer et al., 1997). The method is reliable and well introduced in GMO testing laboratories. It should however be noted that a standard curve using lambda DNA needs to be prepared which requests a little more time. Furthermore it has been observed that the presence of various compounds have an effect on the accuracy of

PicoGreen-based measurements (Singer et al., 1997; Holden et al., 2009 – see below).

A deviation between the concentration obtained by UV measurement and fluorimetry is often seen (Holden et al., 2009), especially for highly processed products (Bellocchi et al., 2010). This may be due to the fact that short or single stranded nucleic acid fragments interfere more with UV than with the PicoGreen dye. It has been proven that the fluorescence signal decreases with increasing length of sonication time (and thus fragmentation) showing the inability of the PicoGreen dye to bind with single stranded

evaluate inhibition, should be included in the reaction (point 2.2).

**2.2 Assessment of DNA yield, purity, integrity and inhibition** 

would be biased.

PCR inhibitors.

fragments (Georgiou & Papapostolou 2006; Holden et al., 2009; Shokere et al., 2009). One of the possible sources of single-stranded DNA may be denaturation of DNA during the drying phase after ethanol precipitation, the final step in many extraction protocols (Svaren et al., 1996). Utilizing spectrophotometry to quantify the DNA in an extract may thus lead to overestimation of the concentration.

Although one should determine the concentration of an extract to ensure that the DNA amount in a quantification reaction is above the limit of quantification (LOQ –part 5), the exact DNA concentration is of less importance. As the determination of the GM content of a sample relies on a relative calculation (ratio transgene copies versus endogene copies – part 3), it is imperative that a same amount of DNA is engaged in both qPCR reactions necessary in quantification, i.e. the event-specific and taxon-specific qPCR methods, whereas the exact amount engaged is of lesser importance. Carrying out both reactions in a single well, i.e. performing a duplex reaction would thus be a good solution.

When using spectrophotometry, additional to measurements at 260 nm, also measurements at wavelengths of 230 and 280 nm may be done. The **purity** of the DNA can then be assessed using the absorbance ratios A260/280 and A260/230. The A260/280 ratio gives an idea of the occurrence of residual proteins. On the other hand, the A260/230 ratio gives an indication on the presence of carbohydrates. In an ideal situation, both ratios should tend to 2,0 (Glasel 1995; Manchester 1995). Any deviation could indicate the presence of coextracted materials that can impair the availability of the DNA for hybridisation with the primers and thus affect the PCR efficiency.

Another important aspect is the **integrity** or intactness of the gDNA (degradation). When the DNA becomes fragmented, the GM target which is less abundant (compared to the endogene) might fall below the quantification limit of the qPCR method. It is evident that this has a practical consequence on the correct quantification of the target. One must thus ensure that the average length of the extracted DNA molecules is longer than the size of the amplicon. To avoid that degradation of the DNA impairs the GMO quantification, the methods are generally designed to amplify sequences ranging in size between 70 and 100 bp. However, one should take into account the minimum length of an amplicon necessary to allow binding of the oligonucleotides (two primers in SYBR®Green chemistry, two primers and one probe used in TaqMan® chemistry). To this purpose for example MGB probes (Kutyavin et al., 2000) can be used to allow even shorter sequences that are stable and have an elevated melting temperature. Further, the amplicon sizes for the endogene and transgene target should not differ too much as shorter fragments are more efficiently amplified than longer ones. This difference in amplification efficiencies will have an impact on the correctness of the quantification reaction. The intactness of the extracted DNA can be assessed using agarose gel electrophoresis with ethidium bromide staining or an alternative. This technique also allows observing if any RNA has been co-extracted.

Knowledge of the presence of co-extracted substances and RNA and the existence of fragmented DNA in the extract is however not sufficient. It is known that **PCR inhibitors** are one of the most important influencing factors of the reliability of quantification (Bickley & Hopkins, 1999). It is thus important to know the impact of these molecules, present in the solution, on the GM quantification. Hereto, a preliminary inhibition test should be performed to evaluate their possible effect on the PCR efficiency. In this view, it is important to check if both targets of the quantification reaction (i.e. endogene and transgene) are equally affected by

Development of a Molecular Platform

to the amount of DNA in the assay (Holden et al., 2009).

investigation.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 373

(different plant species, processed versus raw material). The use of one universal DNA extraction method can thus difficultly be envisaged. The choice of an appropriate extraction procedure suitable for a particular sample matrix is thus a prerequisite for successful qPCR analysis. It must however be noted that this is not always straightforward as enforcement laboratories are not necessarily informed on the ingredients present in the sample under

The C-hexadecyl-Trimethyl-Ammonium-Bromide ('CTAB') extraction method is widely used in the enforcement laboratories for GMO detection (Pietsch et al., 1997). The method starts with lysis of the cells to release all contents. Addition of RNase and Proteinase K allows removal of respectively RNA and proteins. The ionic detergent CTAB forms an insoluble complex with the nucleic acids. The polyphenolic compounds, polysaccharides and other components remain in the supernatant and can be washed away. The DNA is released from the pellet by raising the salt content and is then concentrated by alcohol precipitation. It can be used for a variety of matrices such as maize, oilseed rape, potato and rice. The DNA yield is in most cases sufficient to conduct the necessary qPCR steps. However, the purity of the DNA solution is not always satisfactory. Yet, it is one of the more suitable methods for processed food and feed. In any case, an inhibition test is always advisable. In the GMOlab, inhibition is sometimes seen with very complex matrices such as processed feed products and liquid samples. The protocol is also less efficient for some rice containing materials. One of the drawbacks of the CTAB method is that the procedure is quite time-consuming as it contains different steps of incubation and centrifugation and also an overnight step necessary to ensure that the DNA pellet is completely dissolved. The method further requires some pre-extraction manipulations such as the preparation of specific buffers. It should also be noted that residues of the CTAB buffer can interfere with the PicoGreen dye and impair a correct measurement of the DNA concentration. It was observed that the magnitude of the effect of the CTAB detergent was in inverse proportion

The CTAB extraction method can alternatively be combined with an extra purification step. Hereto a Genomic-Tip 20 column can be used (QIAGEN). This is an anion-exchange chromatography column to which the DNA fragments will be bound by electrostatic interactions between the negatively charged phosphate groups of the DNA and the positively charged resin. Upon subsequent washing steps, the impurities are removed while the DNA remains bound to the column. Finally the DNA is eluted and precipitated with alcohol. The method is very efficient for DNA extraction from soybean and cotton matrices which are more difficult to extract using the classic CTAB extraction method. For cotton powders for example, this is also the method recommended by the EU-RL (http://gmocrl.jrc.ec.europa.eu/summaries/281-3006%20Cotton\_DNAExtr.pdf). Utilizing this alternative procedure, solutions of higher purity can be obtained although the DNA yields are lower. However, they are in most cases still sufficient to perform all necessary qPCR analyses. Due to the purification of the gDNA on the column, these extracts are most often free of inhibitors. As for the classic CTAB method, specific buffers need to be made and an overnight step has to be incorporated to allow the pellet to dissolve. Additionally, the Genomic-Tip 20 columns and buffers that need to be purchased tend to be rather expensive. A big advantage of the CTAB and CTAB-Tip20 methods is that there is no restriction on the sample intake. This allows the laboratories to easily scale up the extraction protocol. This is for example very convenient for the extraction of gDNA from CRM to ensure sufficient

the presence of the inhibitors. If this is not the case, it would influence the detection of the real number of targets and thus lead to a deviating result (Corbisier et al., 2007).

There are several ways to study the presence of inhibition in a qPCR reaction. It is for example possible to include Internal Amplification Controls (IAC; Nolan et al., 2006; Burggraf & Olgemoller, 2004) or to add a positive control nucleic acid to the sample (Cloud et al., 2003). Further, mathematical algorithms can provide a measure of PCR efficiency from analysis of the amplification curves (Tichopad et al., 2003; Ramakers et al., 2003; Liu and Saint, 2003; Lievens et al., 2011). A simple alternative is the use of dilution series to assess the impact of inhibitory substances on the PCR reaction.

Recently, the ENGL released a document wherein they describe an approach to evaluate inhibition of a PCR reaction (ENGL, 2011). To this purpose the gDNA is serially diluted and each dilution is measured in duplicate using the validated qPCR method that will be applied for quantification. According to the previously published ENGL document (2008), the difference between the measured and theoretical Ct value should not exceed 0,5 Ct to exclude inhibition. In practice, four four-fold dilutions (from 1/4 till 1/256) need to be prepared from a stock solution. Both the dilutions and the stock are subsequently analysed in qPCR. This yields five qPCR results: the undiluted sample and the four (four-fold) dilutions. Using the latter, a curve is constructed by regressing the Ct values against the log of the dilution factor. This relation then allows the calculation (extrapolation) of a theoretical Ct value for the undiluted sample. Subsequently, this 'extrapolated' Ct value is compared with the measured value: there should be no more than 0,5 difference. Additionally, the regression line should comply with the following criteria: the slope must be between -3,6 and -3,1 and the linearity (R2) must be equal or above 0,98.

A practical adaptation of this method is being used in the WIV-ISP-GMOlab. A series of dilutions is made from the gDNA under investigation and each dilution is analyzed using qPCR. Subsequently it is assumed that the last dilution contains the least inhibitors as the co-extracted substance will be diluted together with the DNA and will be below inhibitory concentration. The theoretical/expected Ct can be calculated for the other dilutions using knowledge of the dilution factors (e.g. a dilution of 2 corresponds to a Ct difference of 1). If the difference between the measured and theoretical Ct is equal or below 0,5, inhibition can be excluded. It must be noted that a difference of 0,5 for the highest concentration can be considered as an indication of inhibition. If this is observed for lower concentrations (more diluted samples) it is more probable that it comes from a dilution or pipeting mistake as it is unlikely that a low concentration would show inhibition that is not seen in the more concentrated solution.

These experiments and criteria should be set up by the laboratories prior to the quantification qPCR reaction to ensure correct quantification of a GM event in a sample. It should hereby be noted that also the DNA extracted from the CRM, used to construct the calibration curve, should be subjected to an inhibition test. Furthermore, these criteria should be evaluated for each DNA extraction method in combination with at least the most common matrices.

#### **2.3 Evaluation of DNA extraction methods**

Samples under investigation in GMO detection can vary to a great extend in the context of composition (single ingredient versus mixture), texture (solid versus liquid) and matrix

the presence of the inhibitors. If this is not the case, it would influence the detection of the real

There are several ways to study the presence of inhibition in a qPCR reaction. It is for example possible to include Internal Amplification Controls (IAC; Nolan et al., 2006; Burggraf & Olgemoller, 2004) or to add a positive control nucleic acid to the sample (Cloud et al., 2003). Further, mathematical algorithms can provide a measure of PCR efficiency from analysis of the amplification curves (Tichopad et al., 2003; Ramakers et al., 2003; Liu and Saint, 2003; Lievens et al., 2011). A simple alternative is the use of dilution series to assess

Recently, the ENGL released a document wherein they describe an approach to evaluate inhibition of a PCR reaction (ENGL, 2011). To this purpose the gDNA is serially diluted and each dilution is measured in duplicate using the validated qPCR method that will be applied for quantification. According to the previously published ENGL document (2008), the difference between the measured and theoretical Ct value should not exceed 0,5 Ct to exclude inhibition. In practice, four four-fold dilutions (from 1/4 till 1/256) need to be prepared from a stock solution. Both the dilutions and the stock are subsequently analysed in qPCR. This yields five qPCR results: the undiluted sample and the four (four-fold) dilutions. Using the latter, a curve is constructed by regressing the Ct values against the log of the dilution factor. This relation then allows the calculation (extrapolation) of a theoretical Ct value for the undiluted sample. Subsequently, this 'extrapolated' Ct value is compared with the measured value: there should be no more than 0,5 difference. Additionally, the regression line should comply with the following criteria: the slope must be between -3,6

A practical adaptation of this method is being used in the WIV-ISP-GMOlab. A series of dilutions is made from the gDNA under investigation and each dilution is analyzed using qPCR. Subsequently it is assumed that the last dilution contains the least inhibitors as the co-extracted substance will be diluted together with the DNA and will be below inhibitory concentration. The theoretical/expected Ct can be calculated for the other dilutions using knowledge of the dilution factors (e.g. a dilution of 2 corresponds to a Ct difference of 1). If the difference between the measured and theoretical Ct is equal or below 0,5, inhibition can be excluded. It must be noted that a difference of 0,5 for the highest concentration can be considered as an indication of inhibition. If this is observed for lower concentrations (more diluted samples) it is more probable that it comes from a dilution or pipeting mistake as it is unlikely that a low concentration would show inhibition that is not seen in the more

These experiments and criteria should be set up by the laboratories prior to the quantification qPCR reaction to ensure correct quantification of a GM event in a sample. It should hereby be noted that also the DNA extracted from the CRM, used to construct the calibration curve, should be subjected to an inhibition test. Furthermore, these criteria should be evaluated for

Samples under investigation in GMO detection can vary to a great extend in the context of composition (single ingredient versus mixture), texture (solid versus liquid) and matrix

each DNA extraction method in combination with at least the most common matrices.

number of targets and thus lead to a deviating result (Corbisier et al., 2007).

the impact of inhibitory substances on the PCR reaction.

and -3,1 and the linearity (R2) must be equal or above 0,98.

concentrated solution.

**2.3 Evaluation of DNA extraction methods** 

(different plant species, processed versus raw material). The use of one universal DNA extraction method can thus difficultly be envisaged. The choice of an appropriate extraction procedure suitable for a particular sample matrix is thus a prerequisite for successful qPCR analysis. It must however be noted that this is not always straightforward as enforcement laboratories are not necessarily informed on the ingredients present in the sample under investigation.

The C-hexadecyl-Trimethyl-Ammonium-Bromide ('CTAB') extraction method is widely used in the enforcement laboratories for GMO detection (Pietsch et al., 1997). The method starts with lysis of the cells to release all contents. Addition of RNase and Proteinase K allows removal of respectively RNA and proteins. The ionic detergent CTAB forms an insoluble complex with the nucleic acids. The polyphenolic compounds, polysaccharides and other components remain in the supernatant and can be washed away. The DNA is released from the pellet by raising the salt content and is then concentrated by alcohol precipitation. It can be used for a variety of matrices such as maize, oilseed rape, potato and rice. The DNA yield is in most cases sufficient to conduct the necessary qPCR steps. However, the purity of the DNA solution is not always satisfactory. Yet, it is one of the more suitable methods for processed food and feed. In any case, an inhibition test is always advisable. In the GMOlab, inhibition is sometimes seen with very complex matrices such as processed feed products and liquid samples. The protocol is also less efficient for some rice containing materials. One of the drawbacks of the CTAB method is that the procedure is quite time-consuming as it contains different steps of incubation and centrifugation and also an overnight step necessary to ensure that the DNA pellet is completely dissolved. The method further requires some pre-extraction manipulations such as the preparation of specific buffers. It should also be noted that residues of the CTAB buffer can interfere with the PicoGreen dye and impair a correct measurement of the DNA concentration. It was observed that the magnitude of the effect of the CTAB detergent was in inverse proportion to the amount of DNA in the assay (Holden et al., 2009).

The CTAB extraction method can alternatively be combined with an extra purification step. Hereto a Genomic-Tip 20 column can be used (QIAGEN). This is an anion-exchange chromatography column to which the DNA fragments will be bound by electrostatic interactions between the negatively charged phosphate groups of the DNA and the positively charged resin. Upon subsequent washing steps, the impurities are removed while the DNA remains bound to the column. Finally the DNA is eluted and precipitated with alcohol. The method is very efficient for DNA extraction from soybean and cotton matrices which are more difficult to extract using the classic CTAB extraction method. For cotton powders for example, this is also the method recommended by the EU-RL (http://gmocrl.jrc.ec.europa.eu/summaries/281-3006%20Cotton\_DNAExtr.pdf). Utilizing this alternative procedure, solutions of higher purity can be obtained although the DNA yields are lower. However, they are in most cases still sufficient to perform all necessary qPCR analyses. Due to the purification of the gDNA on the column, these extracts are most often free of inhibitors. As for the classic CTAB method, specific buffers need to be made and an overnight step has to be incorporated to allow the pellet to dissolve. Additionally, the Genomic-Tip 20 columns and buffers that need to be purchased tend to be rather expensive.

A big advantage of the CTAB and CTAB-Tip20 methods is that there is no restriction on the sample intake. This allows the laboratories to easily scale up the extraction protocol. This is for example very convenient for the extraction of gDNA from CRM to ensure sufficient

Development of a Molecular Platform

concentration of the extracted DNA.

**sequence used for qPCR analysis** 

**3.1 Introduction** 

**2.4 Conclusion** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 375

inhibition effect on the transgene compared to the endogene although that for other samples

It is thus advisable to validate an extraction method for different matrices. And although the extraction method is validated for a certain matrix, one should keep in mind that gDNA extracted from different samples is not necessarily equally suitable for quantitative analysis. Considering this, it is worthwhile for a GM detection laboratory to put some effort in the evaluation of the different existing extraction protocols in combination with the variety of samples that need to be analysed in GMO detection. And subsequently to chose the extraction method that is the most suitable to remove potential compounds such as lipids,

GM quantification is performed in different steps in which DNA extraction is the first one. This pre-PCR phase is of great importance for the trueness of the quantification result. The DNA extracted from different materials should be evaluated for yield, purity and integrity before performing the qPCR experiment. Furthermore, the DNA solution should be assessed for the presence of inhibitors and their impact on the two targets of the quantification i.e. the endogene and transgene. It is clear that these parameters not only have to be evaluated for the sample under investigation but also for the gDNA extracted from the Certified Reference Material used as a calibrant. Both the sample and CRM DNA need to meet the set criteria to ensure reliable quantification. Seen the diversity of products and matrices that need to be analysed by GM testing laboratories, several DNA extraction protocols exist including home-made buffers and kits. It is obvious, that the extraction protocol to be used needs to be evaluated and that the gDNA extracted has to pass the requirements set by the laboratories before it is used in subsequent PCR analysis. In addition to the choice of the DNA extraction method, thought should also be given to the method used to determine the

In general, the validated DNA extraction protocols used in routine such as the CTAB method are valid for different matrices. However, when dealing with a complex matrix it is important to verify the quality of the DNA. As the extraction method may in some cases have an influence on the GM content, optimalisation of the extraction procedure may be needed. Furthermore, the presence of inhibitors should be checked as they may impair the efficiency of the PCR reaction and thus influence the quantification of GM events in a sample. Hereto, the impact of co-extracted substances and products used in the extraction protocol should be evaluated on the sample, the CRM and the two targets under investigation. If a considerable

inhibitory effect is observed, further DNA purification should be performed.

**3. Description of the structure of a transgenic insert and the type of DNA** 

All the GM events currently on the EU market are plants in which a piece of foreign DNA has been introduced into the genome. This piece of DNA generally consists of a regulatory promoter region, a coding sequence and a terminator (Fig. 1) and is called the transgenic construct or insert. To introduce this construct into the plant genome, genetic engineering

such as the CRM, soybean milk and tortilla chips this was not observed.

polysaccharides and phenolics that could otherwise impair the PCR efficiency.

DNA for validation of methods. The production of large batches of CRM DNA allows the laboratory to have a tested material readily available for several subsequent experiments. Also for several samples such a scaling up is sometimes necessary as the DNA content of some samples may be very low (due to for instance processing).

To reduce the time of DNA extraction, several kits are commercially available. Different companies offer their own DNA extraction kit which is mostly based on isolation of the gDNA using a silica-based method. Usually these kits deliver very fast gDNA and are easy to handle. A drawback of these kits is that often the sample intake is limited which has an impact on the final DNA yield. In, for example, the Wizard Genomic DNA Purification Kit (Promega), a maximum intake of 20 mg is allowed. It is thus necessary to pool several extracts to obtain a sufficient DNA amount for the subsequent qPCR analysis. In addition, when using DNA extracted with this kit, fluctuations in PCR efficiencies upon repetitions were observed which could lead to over- or underestimation of the GMO content (Cankar et al., 2006). Moreover, when comparing the PCR efficiencies of different amplicons, the gDNA extracted with the Wizard kit showed a high dispersion of the data.

The GENESpin kit (Eurofins GeneScan) is one of the few kits where an indication for possible scaling up of the system is given. According to the manufacturers, the kit would be suitable for several food samples such as cakes, bread, sausages,… They also indicate adapted protocols for liquid and powdered hygroscopic samples.

Furthermore, it should be noted that the kits are not always suitable for the extraction of DNA from all matrices. The DNeasy plant kits (QIAGEN) for example, are very efficient kits for the purification of DNA from fresh material (leaves, roots,…) but are less suited for powder materials. Corbisier et al. (2007) showed in their pilot study that this kit yielded a DNA concentration that was twice as low in comparison to the CTAB method. However, using this protocol relatively pure extracts were obtained. In the same study, it was observed that the Nippon Gene GM Quicker protocol (Diagenode), although a low yield and purity was achieved, delivered DNA which was less contaminated by RNA in comparison to the other procedures used.

The situation is even more complicated when it comes to DNA extraction of real-life samples. These not only can contain different species but also additional substances that affect DNA extraction. One such example is the presence of lecithin. This substance is often used in bakery products and as emulgator, stabilisator or anti-oxidant. Additionally, some products such as soybeans contain natural lecithin. As soybean is widely used in food and feed materials and Roundup Ready Soybean is one of the most cultivated GM crops (James, 2010), GMO detection laboratories often have to deal with this product. Wurz et al. (1998) presented an efficient extraction protocol for the isolation of soybean DNA from soy lecithin and showed its application in downstream qPCR. This method can thus be used for extraction of DNA from products such as soymilk and soy sauce.

Last but not least, it should be taken into account that the same product (e.g. bread) can have a different composition when produced by different procedures and can thus contain different substances that could affect the efficiency of the PCR. Even when taking for example only soybean products into account, the PCR efficiency is very much dependant on the nature of the product (Cankar et al., 2006). It was reported that for example DNA extracted with the DNeasy kit (QIAGEN) from a soybean feed sample revealed a higher inhibition effect on the transgene compared to the endogene although that for other samples such as the CRM, soybean milk and tortilla chips this was not observed.

It is thus advisable to validate an extraction method for different matrices. And although the extraction method is validated for a certain matrix, one should keep in mind that gDNA extracted from different samples is not necessarily equally suitable for quantitative analysis. Considering this, it is worthwhile for a GM detection laboratory to put some effort in the evaluation of the different existing extraction protocols in combination with the variety of samples that need to be analysed in GMO detection. And subsequently to chose the extraction method that is the most suitable to remove potential compounds such as lipids, polysaccharides and phenolics that could otherwise impair the PCR efficiency.

## **2.4 Conclusion**

374 Polymerase Chain Reaction

DNA for validation of methods. The production of large batches of CRM DNA allows the laboratory to have a tested material readily available for several subsequent experiments. Also for several samples such a scaling up is sometimes necessary as the DNA content of

To reduce the time of DNA extraction, several kits are commercially available. Different companies offer their own DNA extraction kit which is mostly based on isolation of the gDNA using a silica-based method. Usually these kits deliver very fast gDNA and are easy to handle. A drawback of these kits is that often the sample intake is limited which has an impact on the final DNA yield. In, for example, the Wizard Genomic DNA Purification Kit (Promega), a maximum intake of 20 mg is allowed. It is thus necessary to pool several extracts to obtain a sufficient DNA amount for the subsequent qPCR analysis. In addition, when using DNA extracted with this kit, fluctuations in PCR efficiencies upon repetitions were observed which could lead to over- or underestimation of the GMO content (Cankar et al., 2006). Moreover, when comparing the PCR efficiencies of different amplicons, the gDNA

The GENESpin kit (Eurofins GeneScan) is one of the few kits where an indication for possible scaling up of the system is given. According to the manufacturers, the kit would be suitable for several food samples such as cakes, bread, sausages,… They also indicate

Furthermore, it should be noted that the kits are not always suitable for the extraction of DNA from all matrices. The DNeasy plant kits (QIAGEN) for example, are very efficient kits for the purification of DNA from fresh material (leaves, roots,…) but are less suited for powder materials. Corbisier et al. (2007) showed in their pilot study that this kit yielded a DNA concentration that was twice as low in comparison to the CTAB method. However, using this protocol relatively pure extracts were obtained. In the same study, it was observed that the Nippon Gene GM Quicker protocol (Diagenode), although a low yield and purity was achieved, delivered DNA which was less contaminated by RNA in comparison

The situation is even more complicated when it comes to DNA extraction of real-life samples. These not only can contain different species but also additional substances that affect DNA extraction. One such example is the presence of lecithin. This substance is often used in bakery products and as emulgator, stabilisator or anti-oxidant. Additionally, some products such as soybeans contain natural lecithin. As soybean is widely used in food and feed materials and Roundup Ready Soybean is one of the most cultivated GM crops (James, 2010), GMO detection laboratories often have to deal with this product. Wurz et al. (1998) presented an efficient extraction protocol for the isolation of soybean DNA from soy lecithin and showed its application in downstream qPCR. This method can thus be used for

Last but not least, it should be taken into account that the same product (e.g. bread) can have a different composition when produced by different procedures and can thus contain different substances that could affect the efficiency of the PCR. Even when taking for example only soybean products into account, the PCR efficiency is very much dependant on the nature of the product (Cankar et al., 2006). It was reported that for example DNA extracted with the DNeasy kit (QIAGEN) from a soybean feed sample revealed a higher

some samples may be very low (due to for instance processing).

extracted with the Wizard kit showed a high dispersion of the data.

adapted protocols for liquid and powdered hygroscopic samples.

extraction of DNA from products such as soymilk and soy sauce.

to the other procedures used.

GM quantification is performed in different steps in which DNA extraction is the first one. This pre-PCR phase is of great importance for the trueness of the quantification result. The DNA extracted from different materials should be evaluated for yield, purity and integrity before performing the qPCR experiment. Furthermore, the DNA solution should be assessed for the presence of inhibitors and their impact on the two targets of the quantification i.e. the endogene and transgene. It is clear that these parameters not only have to be evaluated for the sample under investigation but also for the gDNA extracted from the Certified Reference Material used as a calibrant. Both the sample and CRM DNA need to meet the set criteria to ensure reliable quantification. Seen the diversity of products and matrices that need to be analysed by GM testing laboratories, several DNA extraction protocols exist including home-made buffers and kits. It is obvious, that the extraction protocol to be used needs to be evaluated and that the gDNA extracted has to pass the requirements set by the laboratories before it is used in subsequent PCR analysis. In addition to the choice of the DNA extraction method, thought should also be given to the method used to determine the concentration of the extracted DNA.

In general, the validated DNA extraction protocols used in routine such as the CTAB method are valid for different matrices. However, when dealing with a complex matrix it is important to verify the quality of the DNA. As the extraction method may in some cases have an influence on the GM content, optimalisation of the extraction procedure may be needed. Furthermore, the presence of inhibitors should be checked as they may impair the efficiency of the PCR reaction and thus influence the quantification of GM events in a sample. Hereto, the impact of co-extracted substances and products used in the extraction protocol should be evaluated on the sample, the CRM and the two targets under investigation. If a considerable inhibitory effect is observed, further DNA purification should be performed.

## **3. Description of the structure of a transgenic insert and the type of DNA sequence used for qPCR analysis**

#### **3.1 Introduction**

All the GM events currently on the EU market are plants in which a piece of foreign DNA has been introduced into the genome. This piece of DNA generally consists of a regulatory promoter region, a coding sequence and a terminator (Fig. 1) and is called the transgenic construct or insert. To introduce this construct into the plant genome, genetic engineering

Development of a Molecular Platform

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 377

a preparative step namely DNA extraction (part 2) and three consequent qPCR steps namely screening, identification and quantification (Fig. 2). Hereto, in-house developed and validated SYBR®Green screening methods (part 4) are combined with EU-RL validated TaqMan® event-specific methods (part 5). In each step of the qPCR analysis, a different part of the transgenic construct is being targeted. The region in the construct targeted by the method is linked with the specificity of the method. By using a more specific method in each subsequent

In support of these analyses, a matrix-based approach called CoSYPS (Combinatory SYBR®Green qPCR Screening) has been developed (Van den Bulcke et al., 2010). This approach relies on the integration of the analytical results obtained for a sample in a mathematical Decision Support System and the application of a "prime-number"-based algorithm (part 6). Based on the outcome of the screening results of a set of markers in a

qPCR Quantification TaqMan event-spec methods

Transgene & Endogene

TaqMan event-spec method

DNA concentration, purity, integrity, inhibition test

Grinding, mixing, shaking

SYBR®Green element-spec method

CTAB, CTAB-Tip20, extraction kits

After DNA extraction, screening is the next crucial step in GMO detection. In view of the growing number of GM events introduced on the market and new upcoming traits, screening methods will become more and more important and necessary to enable the discrimination between the different GMO. Testing for each possible GM event separately

A screening method usually targets a sequence inside one of the elements of the transgenic construct (Fig. 1). Seen the fact that the elements that are used in transgenic constructs are

sample, the system will identify which GM events are possibly present in a sample.

step, it is possible to gradually narrow down the possibilities to a specific GM event.

Homogenisation

DNA extraction

DNA quantity and quality

qPCR Screening

qPCR Identification

Fig. 2. Flowchart of the analysis steps in GMO detection

would namely become too expensive and labour-intensive.

**3.2 GMO screening methods** 

techniques (Darbani et al., 2008), such as *Agrobacterium*-mediated transformation and particle bombardment, are being used. Hereto the transgene is cloned in a plasmid for example between two specific and unique sequences (T-DNA borders).

For *Agrobacterium*-mediated transformation, the plasmid carrying the transgene is introduced into this bacterium. Further, the intrinsic properties of this soil bacterium are used to incorporate the transgenic construct into the plant genome: the bacterium namely infects the plant and transfers the T-DNA part of the plasmid to the plant genome. In this way the transgene is stably inherited in the subsequent generations (Chilton et al., 1977). Different explants such as leaves (Horsch et al., 1985), roots (Valvekens et al., 1988), embryos (Hensel et al., 2009), ovules (Holme et al., 2006) and microspores (Kumlehn et al., 2006) can be used for transformation. In particle bombardment, gold or tungsten particles are coated with the plasmid containing the transgene (Kikkert et al., 2004). Subsequently, these particles are fired onto the explants with high voltage allowing the incorporation of the transgene into the plant genome. Compared to *Agrobacterium*-mediated transformation, particle bombardment more often leads to multiple inserts of the transgenic construct into the genome.

The detection of this transgenic insert forms the basis of the EU legislation concerning the introduction of GMO onto the market and thus requests the development of GMO detection methods. This detection is carried out by enforcement laboratories and the method of choice is real-time PCR (qPCR). At WIV-ISP, a GMO detection platform, allowing the verification of the presence of GM material in food and feed samples was developed. The platform consists of

Fig. 1. Plant transformation and type of sequence targeted by the different steps in qPCR analysis.

In screening, a sequence inside one of the elements of the transgenic construct is targeted. A construct-specific method used for the identification of the GMO targets the junction between two elements within the transgenic construct. An event-specific method, used in identification and quantification of a GM event, targets the junction between the transgenic insert and the plant genome DNA.

a preparative step namely DNA extraction (part 2) and three consequent qPCR steps namely screening, identification and quantification (Fig. 2). Hereto, in-house developed and validated SYBR®Green screening methods (part 4) are combined with EU-RL validated TaqMan® event-specific methods (part 5). In each step of the qPCR analysis, a different part of the transgenic construct is being targeted. The region in the construct targeted by the method is linked with the specificity of the method. By using a more specific method in each subsequent step, it is possible to gradually narrow down the possibilities to a specific GM event.

Fig. 2. Flowchart of the analysis steps in GMO detection

In support of these analyses, a matrix-based approach called CoSYPS (Combinatory SYBR®Green qPCR Screening) has been developed (Van den Bulcke et al., 2010). This approach relies on the integration of the analytical results obtained for a sample in a mathematical Decision Support System and the application of a "prime-number"-based algorithm (part 6). Based on the outcome of the screening results of a set of markers in a sample, the system will identify which GM events are possibly present in a sample.

## **3.2 GMO screening methods**

376 Polymerase Chain Reaction

techniques (Darbani et al., 2008), such as *Agrobacterium*-mediated transformation and particle bombardment, are being used. Hereto the transgene is cloned in a plasmid for

For *Agrobacterium*-mediated transformation, the plasmid carrying the transgene is introduced into this bacterium. Further, the intrinsic properties of this soil bacterium are used to incorporate the transgenic construct into the plant genome: the bacterium namely infects the plant and transfers the T-DNA part of the plasmid to the plant genome. In this way the transgene is stably inherited in the subsequent generations (Chilton et al., 1977). Different explants such as leaves (Horsch et al., 1985), roots (Valvekens et al., 1988), embryos (Hensel et al., 2009), ovules (Holme et al., 2006) and microspores (Kumlehn et al., 2006) can be used for transformation. In particle bombardment, gold or tungsten particles are coated with the plasmid containing the transgene (Kikkert et al., 2004). Subsequently, these particles are fired onto the explants with high voltage allowing the incorporation of the transgene into the plant genome. Compared to *Agrobacterium*-mediated transformation, particle bombardment more often leads to multiple inserts of the transgenic construct into the genome. The detection of this transgenic insert forms the basis of the EU legislation concerning the introduction of GMO onto the market and thus requests the development of GMO detection methods. This detection is carried out by enforcement laboratories and the method of choice is real-time PCR (qPCR). At WIV-ISP, a GMO detection platform, allowing the verification of the presence of GM material in food and feed samples was developed. The platform consists of

Fig. 1. Plant transformation and type of sequence targeted by the different steps in qPCR

In screening, a sequence inside one of the elements of the transgenic construct is targeted. A construct-specific method used for the identification of the GMO targets the junction between two elements within the transgenic construct. An event-specific method, used in identification and quantification of a GM event, targets the junction between the transgenic

analysis.

insert and the plant genome DNA.

example between two specific and unique sequences (T-DNA borders).

After DNA extraction, screening is the next crucial step in GMO detection. In view of the growing number of GM events introduced on the market and new upcoming traits, screening methods will become more and more important and necessary to enable the discrimination between the different GMO. Testing for each possible GM event separately would namely become too expensive and labour-intensive.

A screening method usually targets a sequence inside one of the elements of the transgenic construct (Fig. 1). Seen the fact that the elements that are used in transgenic constructs are

Development of a Molecular Platform

oxygenase

(*Zea mays* L.)

*napus*)

*sativa*)

Virus

Virus

Virus

EPSPS-CP4

Rbcl Ribulose-1,5-biphosphate carboxylase

Adh Alcohol dehydrogenase I gene from maize

Cru Cruciferin gene from oilseed rape (*Brassica* 

PLD Phospholipase D gene from rice (*Oryza* 

Sad 1 Stearoyl-acyl carrier protein desaturase gene of cotton (Gossypium genus)

Glu3 Glutamine synthetase gene from sugar

p35S Promoter of the 35S Cauliflower Mosaic

pFMV Promoter of the 34S Figworth Mosaic

t35S Terminator of the Cauliflower Mosaic

CryIAb Gene encoding the *Bacillus thuringiensis* δendotoxin (insect resistance)

Cry3Bb Gene encoding the *Bacillus thuringiensis* δendotoxin (insect resistance)

Pat Phosphinotricin-*N*-acetyltransferases gene from *Streptomyces* v*iridochromogenes*

Bar Phosphinotricin-*N*-acetyltransferases gene from *Streptomyces hygroscopicus*

*tumefasciens* strain CP4

CRT Reverse transcriptase gene from the Cauliflower Mosaic Virus

5-enolpyruvylshikimate-3-phosphate synthase gene from *Agrobacterium* 

beet (*Beta vulgaris*)

**Method name** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 379

**Plant kingdom marker** 

**Plant taxon-specific methods** Lectin Lectin gene of soybean (*Glycine max* L.) 81 Mbongolo Mbella

**Methods specific for generic element** 

tNOS Terminator of the nopaline synthase gene 69 Barbau-Piednoir

pNOS Promoter of the nopaline synthase gene 75 Broeders et al., (in

**Methods specific for GM elements** 

**P35S discriminating method** 

Table 2. List of SYBR®Green screening methods developed and validated by the GMOlab.

**size (bp)** 

**Reference** 

95 Mbongolo Mbella et al., 2011

et al., 2011

83 Mbongolo Mbella et al., 2011

85 Mbongolo Mbella et al., 2011

80 Mbongolo Mbella et al., 2011

107 Mbongolo Mbella et al., 2011

118 Mbongolo Mbella et al., 2011

75 Barbau-Piednoir et al., 2010

et al., 2010

preparation)

79 Broeders et al., (in preparation)

107 Broeders et al., (in preparation)

73 Barbau-Piednoir et al., 2011

105 Broeders et al., (personal communication)

109 Barbau-Piednoir et al., 2011

69 Barbau-Piednoir et al., 2011

108 Barbau-Piednoir et al., 2011

94 Papazova et al.,

(in preparation)

**Target Fragment** 

recurrent, detection of a single element often does not confer high specificity and, as a consequence, does not allow deciding on which GM event might be present. A combination of different screening markers is therefore necessary to get a better idea of the possible GM events occurring in a sample. This allows the reduction of the number of identifications to be performed.

To date several screening methods for the detection of GM materials in food and feed samples have already been published. These methods often target the Cauliflower Mosaic Virus 35S promoter (p35S) and/or the *Agrobacterium tumefaciens* nopaline synthase terminator (tNOS) seen the fact that these elements are the most represented in the EU authorised GM events. From the twenty four authorised events, nineteen events contain the p35S target, fifteen the tNOS element and eleven combine both markers (GMO Compass website; Agbios website). Additionally, methods for the detection of herbicide tolerance (HT) genes used in transgenic constructs have been reported. These mainly target two classes of HT sequences: the bacterial phosphinotricin-*N*-acetyltransferases from *Streptomyces* v*iridochromogenes* (*pat*) and from *Streptomyces hygroscopicus* (*bar*) (Wehrmann et al., 1996), and the 5-enolpyruvylshikimate-3-phosphate synthase (*epsps*) from *Agrobacterium tumefaciens* strain CP4 or from plant origin (*in casu* petunia) (Kishore et al., 1988; Padgette et al., 1996). Apart from herbicide tolerance, the GM events currently on the market are transformed with insect resistance traits. Hereto the *Bacillus thuringiensis* endotoxin encoding genes (e.g. the *cryIAb/Ac*) are being used and detection methods have been developed (Bravo et al., 2007). It should however be noted that the above-mentioned methods are mostly either end-point detection on agarose gel or real-time qPCR using TaqMan® chemistry (Hamels et al., 2009; Raymond et al., 2010; Nadal et al., 2009; Prins et al., 2008). Development of screening methods using the SYBR®Green qPCR technology only started recently (Barbau-Piednoir et al., 2010; Barbau-Piednoir et al., 2011; Mbongolo Mbella et al., 2011) although this approach offers a number of advantages over the TaqMan chemistry. The use of melting temperature analysis for instance allows detection of the expected target but also allows distinction between closely-related elements, which is important in the evaluation of the specificity of the method. But more important for enforcement laboratories is the fact that SYBR®Green methods do not require the use of fluorescent labelled oligonucleotides which is much more cost effective.

In view of the growing amount of GM events and the lack of cost-effective screening methods, the WIV-ISP platform puts a major effort in the development of an extensive number of qPCR SYBR®Green screening methods. They form a unique combination targeting different elements within the transgenic construct in addition to plant sequences and are gathered in the patented CoSYPS matrix (Combinatory SYBR®Green qPCR Screening; Van den Bulcke et al., 2010). The methods used to build the CoSYPS were inhouse developed and validated (part 4). They are used together with the CoSYPS matrix in the routine analysis of food and feed samples in the GMOlab under ISO 17025 accreditation. To cover the increasing number of GM events and to add discriminative power to the CoSYPS system, new screening methods are being developed on a regular basis and are subsequently being introduced in the CoSYPS (part 6) after in-house validation.

The in-house developed methods target different types of DNA elements (table 2). Firstly, a screening method aiming to target the chloroplastic *rbcl* gene (plant kingdom marker) was developed. This element will permit to decide on the presence of vegetative DNA in an unknown sample. Secondly, methods that detect plant taxon-specific sequences (Mbongolo

recurrent, detection of a single element often does not confer high specificity and, as a consequence, does not allow deciding on which GM event might be present. A combination of different screening markers is therefore necessary to get a better idea of the possible GM events occurring in a sample. This allows the reduction of the number of identifications to

To date several screening methods for the detection of GM materials in food and feed samples have already been published. These methods often target the Cauliflower Mosaic Virus 35S promoter (p35S) and/or the *Agrobacterium tumefaciens* nopaline synthase terminator (tNOS) seen the fact that these elements are the most represented in the EU authorised GM events. From the twenty four authorised events, nineteen events contain the p35S target, fifteen the tNOS element and eleven combine both markers (GMO Compass website; Agbios website). Additionally, methods for the detection of herbicide tolerance (HT) genes used in transgenic constructs have been reported. These mainly target two classes of HT sequences: the bacterial phosphinotricin-*N*-acetyltransferases from *Streptomyces* v*iridochromogenes* (*pat*) and from *Streptomyces hygroscopicus* (*bar*) (Wehrmann et al., 1996), and the 5-enolpyruvylshikimate-3-phosphate synthase (*epsps*) from *Agrobacterium tumefaciens* strain CP4 or from plant origin (*in casu* petunia) (Kishore et al., 1988; Padgette et al., 1996). Apart from herbicide tolerance, the GM events currently on the market are transformed with insect resistance traits. Hereto the *Bacillus thuringiensis* endotoxin encoding genes (e.g. the *cryIAb/Ac*) are being used and detection methods have been developed (Bravo et al., 2007). It should however be noted that the above-mentioned methods are mostly either end-point detection on agarose gel or real-time qPCR using TaqMan® chemistry (Hamels et al., 2009; Raymond et al., 2010; Nadal et al., 2009; Prins et al., 2008). Development of screening methods using the SYBR®Green qPCR technology only started recently (Barbau-Piednoir et al., 2010; Barbau-Piednoir et al., 2011; Mbongolo Mbella et al., 2011) although this approach offers a number of advantages over the TaqMan chemistry. The use of melting temperature analysis for instance allows detection of the expected target but also allows distinction between closely-related elements, which is important in the evaluation of the specificity of the method. But more important for enforcement laboratories is the fact that SYBR®Green methods do not require the use of

fluorescent labelled oligonucleotides which is much more cost effective.

subsequently being introduced in the CoSYPS (part 6) after in-house validation.

In view of the growing amount of GM events and the lack of cost-effective screening methods, the WIV-ISP platform puts a major effort in the development of an extensive number of qPCR SYBR®Green screening methods. They form a unique combination targeting different elements within the transgenic construct in addition to plant sequences and are gathered in the patented CoSYPS matrix (Combinatory SYBR®Green qPCR Screening; Van den Bulcke et al., 2010). The methods used to build the CoSYPS were inhouse developed and validated (part 4). They are used together with the CoSYPS matrix in the routine analysis of food and feed samples in the GMOlab under ISO 17025 accreditation. To cover the increasing number of GM events and to add discriminative power to the CoSYPS system, new screening methods are being developed on a regular basis and are

The in-house developed methods target different types of DNA elements (table 2). Firstly, a screening method aiming to target the chloroplastic *rbcl* gene (plant kingdom marker) was developed. This element will permit to decide on the presence of vegetative DNA in an unknown sample. Secondly, methods that detect plant taxon-specific sequences (Mbongolo

be performed.


Table 2. List of SYBR®Green screening methods developed and validated by the GMOlab.

Development of a Molecular Platform

**3.4 GMO quantification methods** 

(LLP) legislation (EC/619/2011).

the legislations or not.

become a major task of enforcement laboratories.

**3.5 Conclusion** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 381

As the location of the transgenic insert into the plant genome is unique, the event-specific methods are specific to a sole GM event. Indeed, one and the same construct can be inserted into the genome of different plant species and will not be discriminated by using a construct-specific method alone whereas the plant-insert junction, targeted by the eventspecific method, will be unique. This makes the event-specific methods the technique of choice in GMO identification. These methods are in fact part of the GM quantification methods available. They are laid down by the GM Company together with the request for GM authorisation. Subsequently the EU-RL validates them in a ring trial in which the NRL for GMO detection participate. Once the validated method is published and a CRM is available, the enforcement laboratories need to be able to implement the method in their laboratory (part 5). The construct-specific methods, on the other hand, can be in-house developed methods, methods developed by research groups or the qPCR methods that are published by the EU-RL for quantification of GM events. As they are less specific than the event-specific methods, they have a less discriminative power and are thus not recommended.

However, for some GM events (e.g. rice GM events) no other methods exist to date.

quantification), a third step will be involved namely quantification of the GM event.

At the GMOlab, the coming out of the different identifications are gathered in a Decision Support System (part 6) which will further indicate at which level a specific GM event is present. Only if the GMO is found at quantifiable levels (i.e. above the limit of

In this last step in the process of GMO detection, the amount of the present GM event will be determined. This quantification is necessary to assess the compliance of a sample with the 0,9% labelling threshold (EC/1829/2003) and the recently voted 'Low Level Presence'

Quantification of a GM event in a sample relies on the relative determination of the number of copies of the transgene in relation to the number of copies of the endogene (i.e. the taxonspecific sequence). Hereto a combination of a GM event-specific method and a taxon-specific method will be used. Both methods need to be provided by the GM plant developing companies when requesting EU authorisation and are subsequently validated by the EU-RL. Each laboratory involved in GMO detection needs then to verify in-house if the method complies with the set acceptance criteria before to use it in routine analysis of samples (part 5). The result of GMO quantification is expressed as a GM mass percentage in relation to the ingredient for authorised events and in relation to the GM material for the LLP events. This result is reported to the competent authorities who will decide if the sample is conform to

As the number of GM events being introduced on the market is rapidly increasing, screening will become a necessary first step in GMO detection. Additionally, an intensive screening provides an indication on the presence of GM material originating from unauthorised and unapproved GMO. Indeed, countries that produce GM plants only for local consumption will not request for EU authorisation but these crops might still "escape" and end up in the EU food chain. As a consequence also the detection of these UGM will

Mbella et al., 2011) have been developed. These methods target the main GM commodity crops such as soybean, maize, oilseed rape, cotton, sugar beet and rice. They make it possible determining the species composition of the sample and allow a first discrimination of GM events (e.g. the presence of a soybean GM event can be excluded if the soybean taxon-specific marker is negative). Thirdly, methods specific for GM generic elements were developed (Barbau-Piednoir et al., 2010). These are elements that are included in many transgenic constructs used in commercial GM plants. Such elements are represented by promoter and terminator sequences such as the Cauliflower Mosaic Virus promoter (p35S) and the *Agrobacterium tumefaciens* nopaline synthase terminator (tNOS). Adding the information from the qPCR experiments targeting these generic elements gives a first idea of the putative presence of a GM event in the sample. However, seen these elements are widespread in the transgenic constructs currently used, they do not contain enough discriminative power to sufficiently reduce the number of possible GM events present. These elements need thus, in a fourth step, to be combined with methods targeting other GM specific elements such as herbicide tolerance and insect resistance genes (e.g. Cry genes, bar, pat). Such methods have also been developed and were recently published (Barbau-Piednoir et al., 2011). Last but not least, a marker was developed to be able to discriminate between the p35S present in a GM event and the one due to possible natural presence of the Cauliflower Mosaic Virus from which the transgenic sequence was originally taken (the socalled donor organism). The combination of the results of the eighteen markers, currently used in routine, will allow defining the putative GM events present in a sample. Utilizing the CoSYPS to this purpose, a list of possible events to be identified will be obtained. Additionally, the use of the various markers in combination with the CoSYPS is a powerful tool in the detection of unauthorised GMO (UGM) events. In principle, the elements that are positive in the screening qPCR should be covered by the EU authorised events (EC/1829/2003) or the GM events included in the 'Low Level Presence' legislation (EC/619/2011). If this is not the case, one might suspect the presence of an unauthorised event in the sample.

For each of the screening methods developed and validated at the WIV-ISP-GMOlab, the corresponding amplicon is cloned in a pUC18 background. These plasmids, called Sybricons, are submitted under "Safe Deposit" at the BCCM (Ghent, BE). They can be used to determine the nominal Tm value of the target and further utilized as positive controls in routine analysis.

In addition to the 18 SYBR®Green screening markers, the GMOlab applies two markers in TaqMan® chemistry for the detection of potato (UGPase) and linseed (SAD).

#### **3.3 GMO identification methods**

Based on the outcome of the screening step, a second phase will be necessary namely identification of the GM event.

Identification methods are directed to the detection of a specific GM event. These qPCR methods, contrary to the screening methods, use TaqMan® chemistry. They can be either construct-specific or event-specific qPCR methods. A construct-specific method targets the junction between two elements within the transgenic construct. They are thus directed to the sequence covering a part of the promoter and coding sequence or of the coding sequence and the terminator (Fig. 1). Event-specific methods, in contrast, target the junction between the transgenic insert and the plant genome DNA. They are thus designed to cover part of the sequence of the plant and the promoter or of the terminator and the plant DNA (Fig. 1).

As the location of the transgenic insert into the plant genome is unique, the event-specific methods are specific to a sole GM event. Indeed, one and the same construct can be inserted into the genome of different plant species and will not be discriminated by using a construct-specific method alone whereas the plant-insert junction, targeted by the eventspecific method, will be unique. This makes the event-specific methods the technique of choice in GMO identification. These methods are in fact part of the GM quantification methods available. They are laid down by the GM Company together with the request for GM authorisation. Subsequently the EU-RL validates them in a ring trial in which the NRL for GMO detection participate. Once the validated method is published and a CRM is available, the enforcement laboratories need to be able to implement the method in their laboratory (part 5). The construct-specific methods, on the other hand, can be in-house developed methods, methods developed by research groups or the qPCR methods that are published by the EU-RL for quantification of GM events. As they are less specific than the event-specific methods, they have a less discriminative power and are thus not recommended. However, for some GM events (e.g. rice GM events) no other methods exist to date.

At the GMOlab, the coming out of the different identifications are gathered in a Decision Support System (part 6) which will further indicate at which level a specific GM event is present. Only if the GMO is found at quantifiable levels (i.e. above the limit of quantification), a third step will be involved namely quantification of the GM event.

## **3.4 GMO quantification methods**

380 Polymerase Chain Reaction

Mbella et al., 2011) have been developed. These methods target the main GM commodity crops such as soybean, maize, oilseed rape, cotton, sugar beet and rice. They make it possible determining the species composition of the sample and allow a first discrimination of GM events (e.g. the presence of a soybean GM event can be excluded if the soybean taxon-specific marker is negative). Thirdly, methods specific for GM generic elements were developed (Barbau-Piednoir et al., 2010). These are elements that are included in many transgenic constructs used in commercial GM plants. Such elements are represented by promoter and terminator sequences such as the Cauliflower Mosaic Virus promoter (p35S) and the *Agrobacterium tumefaciens* nopaline synthase terminator (tNOS). Adding the information from the qPCR experiments targeting these generic elements gives a first idea of the putative presence of a GM event in the sample. However, seen these elements are widespread in the transgenic constructs currently used, they do not contain enough discriminative power to sufficiently reduce the number of possible GM events present. These elements need thus, in a fourth step, to be combined with methods targeting other GM specific elements such as herbicide tolerance and insect resistance genes (e.g. Cry genes, bar, pat). Such methods have also been developed and were recently published (Barbau-Piednoir et al., 2011). Last but not least, a marker was developed to be able to discriminate between the p35S present in a GM event and the one due to possible natural presence of the Cauliflower Mosaic Virus from which the transgenic sequence was originally taken (the socalled donor organism). The combination of the results of the eighteen markers, currently used in routine, will allow defining the putative GM events present in a sample. Utilizing the CoSYPS to this purpose, a list of possible events to be identified will be obtained. Additionally, the use of the various markers in combination with the CoSYPS is a powerful tool in the detection of unauthorised GMO (UGM) events. In principle, the elements that are positive in the screening qPCR should be covered by the EU authorised events (EC/1829/2003) or the GM events included in the 'Low Level Presence' legislation (EC/619/2011). If this is not the

case, one might suspect the presence of an unauthorised event in the sample.

TaqMan® chemistry for the detection of potato (UGPase) and linseed (SAD).

routine analysis.

**3.3 GMO identification methods** 

identification of the GM event.

For each of the screening methods developed and validated at the WIV-ISP-GMOlab, the corresponding amplicon is cloned in a pUC18 background. These plasmids, called Sybricons, are submitted under "Safe Deposit" at the BCCM (Ghent, BE). They can be used to determine the nominal Tm value of the target and further utilized as positive controls in

In addition to the 18 SYBR®Green screening markers, the GMOlab applies two markers in

Based on the outcome of the screening step, a second phase will be necessary namely

Identification methods are directed to the detection of a specific GM event. These qPCR methods, contrary to the screening methods, use TaqMan® chemistry. They can be either construct-specific or event-specific qPCR methods. A construct-specific method targets the junction between two elements within the transgenic construct. They are thus directed to the sequence covering a part of the promoter and coding sequence or of the coding sequence and the terminator (Fig. 1). Event-specific methods, in contrast, target the junction between the transgenic insert and the plant genome DNA. They are thus designed to cover part of the sequence of the plant and the promoter or of the terminator and the plant DNA (Fig. 1).

In this last step in the process of GMO detection, the amount of the present GM event will be determined. This quantification is necessary to assess the compliance of a sample with the 0,9% labelling threshold (EC/1829/2003) and the recently voted 'Low Level Presence' (LLP) legislation (EC/619/2011).

Quantification of a GM event in a sample relies on the relative determination of the number of copies of the transgene in relation to the number of copies of the endogene (i.e. the taxonspecific sequence). Hereto a combination of a GM event-specific method and a taxon-specific method will be used. Both methods need to be provided by the GM plant developing companies when requesting EU authorisation and are subsequently validated by the EU-RL. Each laboratory involved in GMO detection needs then to verify in-house if the method complies with the set acceptance criteria before to use it in routine analysis of samples (part 5).

The result of GMO quantification is expressed as a GM mass percentage in relation to the ingredient for authorised events and in relation to the GM material for the LLP events. This result is reported to the competent authorities who will decide if the sample is conform to the legislations or not.

#### **3.5 Conclusion**

As the number of GM events being introduced on the market is rapidly increasing, screening will become a necessary first step in GMO detection. Additionally, an intensive screening provides an indication on the presence of GM material originating from unauthorised and unapproved GMO. Indeed, countries that produce GM plants only for local consumption will not request for EU authorisation but these crops might still "escape" and end up in the EU food chain. As a consequence also the detection of these UGM will become a major task of enforcement laboratories.

Development of a Molecular Platform

et al., 2010).

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 383

important transgenic elements occurring in unauthorised GM events which might be

The development of a new screening method depends on several prerequisites: information on the elements inserted in a GM event, their copy number and the nucleotide sequence of the inserted element. Information on the elements of the transgenic construct inserted in a GMO can be obtained from publicly available dossiers submitted by the applicant for authorisation or patent databases. This information is usually available after the authorisation is granted or after the competent authorities have given a positive advice. Important information sources are the GMO crop database of the Centre for Environmental Risk Assessment (CERA) (http://www.cera-gmc.org/?action=gm\_crop\_database) and the GMO database on authorisations and approval of GMO in the EU (http://www.gmocompass.org/eng/gmo/db/). The nucleotide sequences are available in public databases such as the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), patent databases and scientific publications. One must however take care when using the information present in these databases as for example Single Nucleotide Polymorphisms (SNP) may exist in the sequence of the elements inserted in different GM events (Morisset et al., 2009). Therefore, the information in the public databases is not always completely reliable and more than one source should be consulted. Additionally, variations in the sequences used to design taxon-specific assays exists as for instance SNP can occur between the varieties of one plant species (Broothaerts et al., 2008; Papazova et al., 2010). The difficulty here is that information on the nucleotide sequence in different plant varieties is not available. This problem can be partially solved by designing the SYBR®Green primers on basis of existing TaqMan® taxon-specific assays for which experimental tests have been performed. Presence of SNP in the primer annealing sites can lead to a false negative result and to the conclusion that an event containing this target is not present when the assay is applied to an unknown sample (Broothaerts et al., 2008; Papazova

Upon selection of the suitable sequence different primer pairs are designed by using appropriate bioinformatic tools. One of the most widely used programs is Primer3 (Rozen & Scaletzky, 2001). These primer pairs are further assessed *in silico* for their specificity. This can be done by means of bioinformatic tools such as the primer search module in the EMBOSS bioinformatic platform, BLAST searches etc. For transgenic elements, this theoretical specificity test is performed using sequences from authorised GM events. If the primers target a reference taxon-specific sequence, it should be tested if they are specific for the target taxon and do not amplify closely related species. Here, the criteria for specificity

As the goal is to use all the methods simultaneously under uniform conditions, particular attention is paid on the amplicon size and the primer annealing temperature (Tm) when developing the primers. Amplicons with a size lower than 100 bp are preferred although the size for real-time PCR amplicons can be as large as 250 bp. For qPCR detection smaller amplicons are favoured in order to avoid lack of amplification due to the possible fragmented status of the DNA in the sample (part 2). In addition, the melting temperature of the primers should be around 60°C according to the general requirements for qPCR primers (www.appliedbiosystems.com). The formation of primer dimers and hairpins should be checked and primer pairs showing this feature should be excluded for further analysis.

for reference assays of the event-specific quantification methods also apply (part 5).

necessary to test for by the enforcement laboratories should be targeted.

The GMO platform developed by the WIV-ISP-GMOlab allows detection of authorised GM events as well as UGM in a cost- and time effective manner. It consists of a preparative DNA extraction step and three consecutive qPCR steps. The CoSYPS system, including in-house developed SYBR®Green screening methods, forms an innovative tool in GMO detection allowing reducing the number of identifications to be carried out. The TaqMan® identification further allows a narrowing down of the GM events present to a specific GMO and quantification permits the determination of the GM content.

## **4. Development and validation of a qualitative qPCR method in view of its application for screening purposes in the WIV-ISP GMO detection platform**

## **4.1 Introduction**

As described previously, in order to face the rapidly increasing number of GMO in food and feed products, new methods facilitating an initial screening of analytical samples is needed. Therefore, one of the major objectives of the molecular platform at WIV-ISP is to develop qualitative screening methods targeting either new genetic elements commonly found in transgenic constructs or species frequently used in food and feed in view of rationalizing GMO detection.

The methods developed are singleplex qPCR, based on SYBR®Green chemistry. Additionally, the methods are designed to work under uniform conditions (primer concentrations, PCR program) in order to facilitate their simultaneous application in a 96 well plate format. These SYBR®Green methods were in-house validated in order to be applied under ISO 17025 accreditation. As there is no 'golden standard' for the validation of qualitative methods related to GMO detection, enforcement laboratories need to decide which parameters need to be evaluated in the validation. In addition, the laboratories have to set their own criteria based on the guidance document for quantitative qPCR methods.

Part 4.3 of this chapter focuses on the method validation criteria and proposes a pragmatic approach for the in-house validation of singleplex real-time PCR qualitative methods. This proposal is mainly based on the recently adopted Codex Alimentarius guidelines on performance criteria and validation of methods for GMO analysis (Codex, 2010), and on the minimum performance requirements for methods for GMO testing set forward by the ENGL (ENGL, 2008). During the in-house validation critical values are determined for the screening methods to be introduced in the Decision Support System currently used in the routine analyses, namely the CoSYPS (part 6).

#### **4.2 Development of SYBR® Green methods for screening purposes**

The first step of method development is to determine the screening qPCR target. Targets for screening can be any element present in the transgenic construct inserted in authorised or unauthorised GMO and taxon-specific sequences. Application of the screening approach requires development of many targets in order to cover the growing range of GM events. Selection of the methods to be developed is based on a number of priorities. Firstly, methods targeting the main commodity crops used in transformation events are of high importance. Secondly, priority is given to transgenic elements frequently occurring in EU authorised GM events in addition to targets that provide an extra discriminative power. Thirdly, other

The GMO platform developed by the WIV-ISP-GMOlab allows detection of authorised GM events as well as UGM in a cost- and time effective manner. It consists of a preparative DNA extraction step and three consecutive qPCR steps. The CoSYPS system, including in-house developed SYBR®Green screening methods, forms an innovative tool in GMO detection allowing reducing the number of identifications to be carried out. The TaqMan® identification further allows a narrowing down of the GM events present to a specific GMO

**4. Development and validation of a qualitative qPCR method in view of its application for screening purposes in the WIV-ISP GMO detection platform** 

As described previously, in order to face the rapidly increasing number of GMO in food and feed products, new methods facilitating an initial screening of analytical samples is needed. Therefore, one of the major objectives of the molecular platform at WIV-ISP is to develop qualitative screening methods targeting either new genetic elements commonly found in transgenic constructs or species frequently used in food and feed in view of rationalizing

The methods developed are singleplex qPCR, based on SYBR®Green chemistry. Additionally, the methods are designed to work under uniform conditions (primer concentrations, PCR program) in order to facilitate their simultaneous application in a 96 well plate format. These SYBR®Green methods were in-house validated in order to be applied under ISO 17025 accreditation. As there is no 'golden standard' for the validation of qualitative methods related to GMO detection, enforcement laboratories need to decide which parameters need to be evaluated in the validation. In addition, the laboratories have to set their own criteria based on the guidance document for quantitative qPCR

Part 4.3 of this chapter focuses on the method validation criteria and proposes a pragmatic approach for the in-house validation of singleplex real-time PCR qualitative methods. This proposal is mainly based on the recently adopted Codex Alimentarius guidelines on performance criteria and validation of methods for GMO analysis (Codex, 2010), and on the minimum performance requirements for methods for GMO testing set forward by the ENGL (ENGL, 2008). During the in-house validation critical values are determined for the screening methods to be introduced in the Decision Support System currently used in the

**Green methods for screening purposes** 

The first step of method development is to determine the screening qPCR target. Targets for screening can be any element present in the transgenic construct inserted in authorised or unauthorised GMO and taxon-specific sequences. Application of the screening approach requires development of many targets in order to cover the growing range of GM events. Selection of the methods to be developed is based on a number of priorities. Firstly, methods targeting the main commodity crops used in transformation events are of high importance. Secondly, priority is given to transgenic elements frequently occurring in EU authorised GM events in addition to targets that provide an extra discriminative power. Thirdly, other

and quantification permits the determination of the GM content.

**4.1 Introduction** 

GMO detection.

methods.

routine analyses, namely the CoSYPS (part 6).

**4.2 Development of SYBR®**

important transgenic elements occurring in unauthorised GM events which might be necessary to test for by the enforcement laboratories should be targeted.

The development of a new screening method depends on several prerequisites: information on the elements inserted in a GM event, their copy number and the nucleotide sequence of the inserted element. Information on the elements of the transgenic construct inserted in a GMO can be obtained from publicly available dossiers submitted by the applicant for authorisation or patent databases. This information is usually available after the authorisation is granted or after the competent authorities have given a positive advice. Important information sources are the GMO crop database of the Centre for Environmental Risk Assessment (CERA) (http://www.cera-gmc.org/?action=gm\_crop\_database) and the GMO database on authorisations and approval of GMO in the EU (http://www.gmocompass.org/eng/gmo/db/). The nucleotide sequences are available in public databases such as the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/), patent databases and scientific publications. One must however take care when using the information present in these databases as for example Single Nucleotide Polymorphisms (SNP) may exist in the sequence of the elements inserted in different GM events (Morisset et al., 2009). Therefore, the information in the public databases is not always completely reliable and more than one source should be consulted.

Additionally, variations in the sequences used to design taxon-specific assays exists as for instance SNP can occur between the varieties of one plant species (Broothaerts et al., 2008; Papazova et al., 2010). The difficulty here is that information on the nucleotide sequence in different plant varieties is not available. This problem can be partially solved by designing the SYBR®Green primers on basis of existing TaqMan® taxon-specific assays for which experimental tests have been performed. Presence of SNP in the primer annealing sites can lead to a false negative result and to the conclusion that an event containing this target is not present when the assay is applied to an unknown sample (Broothaerts et al., 2008; Papazova et al., 2010).

Upon selection of the suitable sequence different primer pairs are designed by using appropriate bioinformatic tools. One of the most widely used programs is Primer3 (Rozen & Scaletzky, 2001). These primer pairs are further assessed *in silico* for their specificity. This can be done by means of bioinformatic tools such as the primer search module in the EMBOSS bioinformatic platform, BLAST searches etc. For transgenic elements, this theoretical specificity test is performed using sequences from authorised GM events. If the primers target a reference taxon-specific sequence, it should be tested if they are specific for the target taxon and do not amplify closely related species. Here, the criteria for specificity for reference assays of the event-specific quantification methods also apply (part 5).

As the goal is to use all the methods simultaneously under uniform conditions, particular attention is paid on the amplicon size and the primer annealing temperature (Tm) when developing the primers. Amplicons with a size lower than 100 bp are preferred although the size for real-time PCR amplicons can be as large as 250 bp. For qPCR detection smaller amplicons are favoured in order to avoid lack of amplification due to the possible fragmented status of the DNA in the sample (part 2). In addition, the melting temperature of the primers should be around 60°C according to the general requirements for qPCR primers (www.appliedbiosystems.com). The formation of primer dimers and hairpins should be checked and primer pairs showing this feature should be excluded for further analysis.

Development of a Molecular Platform

samples to the existing dataset.

unknown sample.

**4.4 Conclusion** 

AFNOR XP V 03-020-2).

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 385

confidence interval can be updated regularly by adding data from analysis of routine

Using the data from the *in silico* and experimental specificity tests, mostly only one primer pair is selected for determination of the method sensitivity (LOD and LOQ) and repeatability. To assess the **sensitivity** of the developed method, a GM event containing the target is used (usually a CRM with a known GM%). It should however be noted that the GM-specific CRM are certified for the content of a specific GM event and not for the content of the screening target (promoter, coding sequence, terminator). This demonstrates that the preliminary information on the elements inserted in a GM event and their copy number is crucial in order to estimate the correct copy number of the target. For taxon-specific markers, this assessment can be done using a wild type (non-GM) material. The LOD and the LOQ are determined on basis of serial dilutions starting from at least 2000 target copies until the

The **LOD** is set up at the level where less than 5% false negatives are observed (Codex Alimentarius, 2009). As it is not feasible to perform the analysis on a large number of PCR replicates, six repeats are run per dilution point. If all six repeats are positive, this means that 95% of the time a positive sample will indeed be detected. Therefore the LOD of the screening method is set at the haploid genome copy level at which all six replicates provide

The **LOQ** is defined as the target copy number with a similar positive PCR result (expressed as Ct value) upon six-fold measurement of the target sequence in the same DNA sample with a minor standard deviation (SDCt<0,5) (AFNOR XP V 03-020-2). A screening target is in principle not quantified, but the LOQ can give an idea about the content of the target in an

Additionally, the **precision** (inter-run repeatability) of the method is determined. In practice this is done by calculating the relative repeatability standard deviation (RSDr%) on each of the dilutions used to determine the LOD and LOQ. Hereto, the experiment is performed under repeatability conditions (in a short period of time, on the same qPCR instrument by the same operator) in four independent runs. The RSDr% is calculated according to the

As, to date, no instructions on the development and validation of screening methods are available, the laboratories need to set up their own experimental plan and criteria. At the WIV-ISP-GMOlab, development and validation of SYBR®Green methods for screening purposes is done in a harmonized way to allow applying the methods in a single qPCR run. The parameters evaluated, the way to perform this assessment and the acceptance criteria are based on previously published documents (ENGL, 2008; Codex Alimentarius, 2009;

Upon evaluation of all the necessary parameters and their accordance with the set criteria, a validation dossier is established. The LOD, LOQ (expressed as a Ct value) and the Tm interval are introduced into the CoSYPS Decision Support System and serve as decision values to conclude if a sample is positive for the target or not (part 6). Subsequently the

theoretical zero copy numbers. Each of the dilutions is run in six replicates.

a specific positive signal (n = 6; 6/6 specific signals) (AFNOR XP V 03-020-2).

ISO 5725-2. The method is accepted as valid when the RSDr% is below 25%.

method is implemented in routine GMO detection under ISO 17025.

#### **4.3 Validation of a SYBR® Green screening method**

The in-house validation of a SYBR®Green screening method is based on the determination of several method characteristics that are required for the validation of event-specific quantitative methods (ENGL, 2008 - part 5), namely applicability, practicability, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ) and precision (RSDr%). The definitions of these parameters can be found in the glossary. The GMOlab has developed its own experimental set up in order to assess these parameters. Upon validation the results are evaluated and if they meet the acceptance criteria the method can be used under accreditation. Additionally, the critical values which are introduced in the CoSYPS (part 6) are determined during the in-house validation.

The method is **applicable** when it detects the target in the respective GMO for which it was designed. To test this aspect of a method a list of GM events containing the target (positive samples) and events not containing the target (negative samples) is made. Usually, this list is limited to GM events which are authorised and for which (certified) reference materials are available. If possible, different matrices (e.g. gDNA, pDNA, raw material, processed material,…) are included and different GM concentrations are used. Further the applicability of the methods is assessed by screening certified reference materials which are used in the GMOlab for validation and calibration purposes.

The **practicability** of the SYBR®Green screening methods follows directly from the fact that all methods have been developed in-house. During the development, the use of the same conditions (qPCR program, reaction volume, …) and qPCR instruments have been taken into account. This will thus allow using all methods in a same run during routine analysis of a sample.

The **specificity** of the method is first assessed *in silico* (part 4.2) and further experimentally. The screening method should be specific for the target for which it is developed and should not be homologous and give an amplification product with other sequences. The specificity is experimentally tested on all materials to which the analysis can be applied. The GM events or taxa containing the target should give a positive amplification signal, while the ones which do not contain it should give no amplification signal. An amplification signal is considered positive when a Ct value and a melting curve analysis are recorded. Absence of amplification is considered when either no Ct is recorded or when a Ct value at least 10 Ct higher than the one of the positive samples is measured. To assess the nominal Tm value, a plasmid containing the construct under analysis may be used.

As the screening methods developed and validated at the GMOlab are based on the SYBR®Green detection chemistry, the melting temperature of the amplicon is an important parameter related to the specificity of the method. The melting temperature (Tm) of a DNA sequence is dependent on a large number of factors, among which the ionic conditions in the sample solution, the DNA nature (sequence, secondary structure, etc.) and the starting concentration of the DNA molecule (Hillen et al., 1981; Rouzina & Bloomfield, 2001). Moreover different qPCR instruments tend to measure slightly different values for a given amplicon (due to differences in heating block control, mathematical integration, extrapolation, etc.). The variation of the Tm follows a normal distribution and the Tm of the method is calculated as the average Tm from all the data obtained during validation. Additionally, a Tm confidence interval is calculated (Tm ± 3 standard deviations) which is used further to decide whether the correct target has been amplified (part 6). The Tm and its confidence interval can be updated regularly by adding data from analysis of routine samples to the existing dataset.

Using the data from the *in silico* and experimental specificity tests, mostly only one primer pair is selected for determination of the method sensitivity (LOD and LOQ) and repeatability.

To assess the **sensitivity** of the developed method, a GM event containing the target is used (usually a CRM with a known GM%). It should however be noted that the GM-specific CRM are certified for the content of a specific GM event and not for the content of the screening target (promoter, coding sequence, terminator). This demonstrates that the preliminary information on the elements inserted in a GM event and their copy number is crucial in order to estimate the correct copy number of the target. For taxon-specific markers, this assessment can be done using a wild type (non-GM) material. The LOD and the LOQ are determined on basis of serial dilutions starting from at least 2000 target copies until the theoretical zero copy numbers. Each of the dilutions is run in six replicates.

The **LOD** is set up at the level where less than 5% false negatives are observed (Codex Alimentarius, 2009). As it is not feasible to perform the analysis on a large number of PCR replicates, six repeats are run per dilution point. If all six repeats are positive, this means that 95% of the time a positive sample will indeed be detected. Therefore the LOD of the screening method is set at the haploid genome copy level at which all six replicates provide a specific positive signal (n = 6; 6/6 specific signals) (AFNOR XP V 03-020-2).

The **LOQ** is defined as the target copy number with a similar positive PCR result (expressed as Ct value) upon six-fold measurement of the target sequence in the same DNA sample with a minor standard deviation (SDCt<0,5) (AFNOR XP V 03-020-2). A screening target is in principle not quantified, but the LOQ can give an idea about the content of the target in an unknown sample.

Additionally, the **precision** (inter-run repeatability) of the method is determined. In practice this is done by calculating the relative repeatability standard deviation (RSDr%) on each of the dilutions used to determine the LOD and LOQ. Hereto, the experiment is performed under repeatability conditions (in a short period of time, on the same qPCR instrument by the same operator) in four independent runs. The RSDr% is calculated according to the ISO 5725-2. The method is accepted as valid when the RSDr% is below 25%.

## **4.4 Conclusion**

384 Polymerase Chain Reaction

The in-house validation of a SYBR®Green screening method is based on the determination of several method characteristics that are required for the validation of event-specific quantitative methods (ENGL, 2008 - part 5), namely applicability, practicability, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ) and precision (RSDr%). The definitions of these parameters can be found in the glossary. The GMOlab has developed its own experimental set up in order to assess these parameters. Upon validation the results are evaluated and if they meet the acceptance criteria the method can be used under accreditation. Additionally, the critical values which are introduced in the CoSYPS (part 6)

The method is **applicable** when it detects the target in the respective GMO for which it was designed. To test this aspect of a method a list of GM events containing the target (positive samples) and events not containing the target (negative samples) is made. Usually, this list is limited to GM events which are authorised and for which (certified) reference materials are available. If possible, different matrices (e.g. gDNA, pDNA, raw material, processed material,…) are included and different GM concentrations are used. Further the applicability of the methods is assessed by screening certified reference materials which are

The **practicability** of the SYBR®Green screening methods follows directly from the fact that all methods have been developed in-house. During the development, the use of the same conditions (qPCR program, reaction volume, …) and qPCR instruments have been taken into account. This will thus allow using all methods in a same run during routine analysis of a

The **specificity** of the method is first assessed *in silico* (part 4.2) and further experimentally. The screening method should be specific for the target for which it is developed and should not be homologous and give an amplification product with other sequences. The specificity is experimentally tested on all materials to which the analysis can be applied. The GM events or taxa containing the target should give a positive amplification signal, while the ones which do not contain it should give no amplification signal. An amplification signal is considered positive when a Ct value and a melting curve analysis are recorded. Absence of amplification is considered when either no Ct is recorded or when a Ct value at least 10 Ct higher than the one of the positive samples is measured. To assess the nominal Tm value, a

As the screening methods developed and validated at the GMOlab are based on the SYBR®Green detection chemistry, the melting temperature of the amplicon is an important parameter related to the specificity of the method. The melting temperature (Tm) of a DNA sequence is dependent on a large number of factors, among which the ionic conditions in the sample solution, the DNA nature (sequence, secondary structure, etc.) and the starting concentration of the DNA molecule (Hillen et al., 1981; Rouzina & Bloomfield, 2001). Moreover different qPCR instruments tend to measure slightly different values for a given amplicon (due to differences in heating block control, mathematical integration, extrapolation, etc.). The variation of the Tm follows a normal distribution and the Tm of the method is calculated as the average Tm from all the data obtained during validation. Additionally, a Tm confidence interval is calculated (Tm ± 3 standard deviations) which is used further to decide whether the correct target has been amplified (part 6). The Tm and its

**Green screening method** 

**4.3 Validation of a SYBR®**

sample.

are determined during the in-house validation.

used in the GMOlab for validation and calibration purposes.

plasmid containing the construct under analysis may be used.

As, to date, no instructions on the development and validation of screening methods are available, the laboratories need to set up their own experimental plan and criteria. At the WIV-ISP-GMOlab, development and validation of SYBR®Green methods for screening purposes is done in a harmonized way to allow applying the methods in a single qPCR run. The parameters evaluated, the way to perform this assessment and the acceptance criteria are based on previously published documents (ENGL, 2008; Codex Alimentarius, 2009; AFNOR XP V 03-020-2).

Upon evaluation of all the necessary parameters and their accordance with the set criteria, a validation dossier is established. The LOD, LOQ (expressed as a Ct value) and the Tm interval are introduced into the CoSYPS Decision Support System and serve as decision values to conclude if a sample is positive for the target or not (part 6). Subsequently the method is implemented in routine GMO detection under ISO 17025.

Development of a Molecular Platform

**quantification** 

harmonised methods applicable in official GMO detection.

the world and against those still in development.

used for different GM events is needed.

applied in a routine laboratory.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 387

with the results of the study and the validated protocol. These are submitted to the European Food Safety Authority (EFSA) and are subsequently published on the EU-RL GMFF official website. Upon publication the validated methods become official methods. The method validation thus provides the enforcement laboratories with standardised and

The requirements for method **specificity** are laid down in the legislation. The method submitted has to be event-specific (based on the specific sequence of the plant-transgenic construct junction, part 3) and should detect only the specific GMO submitted for authorisation to be useful for unequivocal detection/identification/quantification of the GM event (EC/641/2004). To demonstrate that the method is event-specific, it has to be tested against all GM events from the applicant which are currently authorised in different parts of

As the submitted methods are quantitative, they also include a reference taxon-specific assay. The specificity of this assay should also be tested. For taxon-specific assays the target should be preferably a unique sequence present in a single copy in the target plant genome. The copy number and the specificity have to be assessed *in silico* by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches against known databases. In addition, the taxon-specific target should not show amplification signals with close relatives or taxa of the most important food crops. Usually, the different biotech companies develop their own taxon-specific method and test it on a range of taxa selected by them. This can pose several problems for the laboratories applying the methods. Firstly, there is no standard list of taxa and varieties to be included in the test. Ideally, the reference assay should be tested on a large range of varieties covering the existing natural variation within the taxon in order to assure that it will amplify any material from the plant species targeted by the method. Secondly, the existence of more than one reference system for events of the same plant taxon requires the use of several reference assays in quantification, which increases the costs of the analysis by the laboratory. In this context the requirements for the specificity of the taxonspecific reference assays should be made more precise and harmonisation in the methods

Information on the **applicability** of the method should be provided. This includes information on the scope of the method. In addition, information on known interferences

The **practicability** of the method should be demonstrated. For instance, methods where the reference and the event-specific assays are run on different PCR plates or under different PCR cycling conditions are less practicable and would be time and cost consuming when

Besides these criteria, other parameters related to the method performance are assessed namely the **dynamic range, linearity, amplification efficiency, LOD and LOQ, trueness, precision and robustness**. The definitions of all parameters can be found in the glossary.

with other analytes and the applicability to certain matrices should be supplied.

**5.2 Evaluated parameters for newly developed event-specific methods for GMO** 

**5.2.1 Evaluation of method performance characteristics by the EURL-GMFF** 

## **5. Validation of a qPCR method for GMO quantification and its implementation in a routine laboratory under ISO 17025 accreditation**

## **5.1 Introduction into the legal context**

Regulation (EC) 1829/2003 on genetically modified food and feed defines that food and feed products containing or derived from GMO must be labelled. The labelling requirements do not apply to food and feed containing GMO in a proportion not higher than 0,9% of the ingredients, provided that this presence is adventitious or technically unavoidable. Moreover, the recently adopted "Low Level Presence" Commission Regulation (EC/619/2011) requires a reliable quantification at a level of 0.1%. Member States are responsible for monitoring the GMO content of products and compliance with GMO labelling requirements. In this context, the enforcement of the EU legislation on GMO labelling requires GMO detection methods that are sound, precise and robust. It is, therefore, an essential requirement to use validated methods for GMO detection and quantification. Only in this manner it can be assured that independent control laboratories achieve comparable analysis results and are able to fulfil regulatory tasks (JRC, 2010).

The submission and validation of a GMO detection method is an integral part of the regulatory and approval process for GM food and feed to be placed on the market (EC/1829/2003). This Commission Regulation states that the application for authorisation should include, amongst others, "methods for detection, sampling and identification of the transformation event". As a consequence, the biotech companies have to provide detection protocols and control samples to validate the event-specific method to the EU-RL GMFF. These methods should be based on the real-time PCR technology (EC/787/2004). In view of the European harmonisation and standardisation of methods for sampling, detection, identification and quantification of GMO, the EU-RL has published a list of parameters to be tested and their acceptance criteria in the a document "Definition of minimum performance requirements for analytical methods of GMO testing" (ENGL, 2008).

A GM event cannot be authorised in the EU before a relevant detection method has been validated. The method validation process is conducted by the European Commission's Joint Research Centre (JRC) in its capacity as European Union Reference Laboratory for GM Food and Feed, and is assisted in its task by the European Network of GMO Laboratories. Commission Regulation EC/882/2004 establishes that analytical methods used for food and feed control must be verified by control laboratories before their use (JRC, 2010). In practice, after testing of the material and protocol, the JRC distributes the sample material and corresponding reagents to the participating laboratories in a ring trial. The validation ring trials are organised according to the requirements set up in ISO 5725 and following the IUPAC protocol (IUPAC, 1995). In such a collaborative validation trial, the EU-RL is assisted by the National Reference Laboratories (NRL) which are assigned as official control laboratories at national level (EC/882/2004). The NRL have to be accredited under ISO 17025 standard. Usually there are 12-13 participating laboratories, randomly selected from all available NRL. The validation ring trial aims at determining the method performance characteristics.

In this way the submitted method is evaluated with regard to the validation criteria. Failure to meet these criteria leads to rejection of the method and consequently to a delay in the authorisation of the GMO. Upon acceptance, the EU-RL GMFF prepares a validation report

Regulation (EC) 1829/2003 on genetically modified food and feed defines that food and feed products containing or derived from GMO must be labelled. The labelling requirements do not apply to food and feed containing GMO in a proportion not higher than 0,9% of the ingredients, provided that this presence is adventitious or technically unavoidable. Moreover, the recently adopted "Low Level Presence" Commission Regulation (EC/619/2011) requires a reliable quantification at a level of 0.1%. Member States are responsible for monitoring the GMO content of products and compliance with GMO labelling requirements. In this context, the enforcement of the EU legislation on GMO labelling requires GMO detection methods that are sound, precise and robust. It is, therefore, an essential requirement to use validated methods for GMO detection and quantification. Only in this manner it can be assured that independent control laboratories achieve comparable analysis results and are able to fulfil regulatory tasks (JRC, 2010).

The submission and validation of a GMO detection method is an integral part of the regulatory and approval process for GM food and feed to be placed on the market (EC/1829/2003). This Commission Regulation states that the application for authorisation should include, amongst others, "methods for detection, sampling and identification of the transformation event". As a consequence, the biotech companies have to provide detection protocols and control samples to validate the event-specific method to the EU-RL GMFF. These methods should be based on the real-time PCR technology (EC/787/2004). In view of the European harmonisation and standardisation of methods for sampling, detection, identification and quantification of GMO, the EU-RL has published a list of parameters to be tested and their acceptance criteria in the a document "Definition of minimum performance

A GM event cannot be authorised in the EU before a relevant detection method has been validated. The method validation process is conducted by the European Commission's Joint Research Centre (JRC) in its capacity as European Union Reference Laboratory for GM Food and Feed, and is assisted in its task by the European Network of GMO Laboratories. Commission Regulation EC/882/2004 establishes that analytical methods used for food and feed control must be verified by control laboratories before their use (JRC, 2010). In practice, after testing of the material and protocol, the JRC distributes the sample material and corresponding reagents to the participating laboratories in a ring trial. The validation ring trials are organised according to the requirements set up in ISO 5725 and following the IUPAC protocol (IUPAC, 1995). In such a collaborative validation trial, the EU-RL is assisted by the National Reference Laboratories (NRL) which are assigned as official control laboratories at national level (EC/882/2004). The NRL have to be accredited under ISO 17025 standard. Usually there are 12-13 participating laboratories, randomly selected from all available NRL. The validation ring trial aims at determining the method

In this way the submitted method is evaluated with regard to the validation criteria. Failure to meet these criteria leads to rejection of the method and consequently to a delay in the authorisation of the GMO. Upon acceptance, the EU-RL GMFF prepares a validation report

requirements for analytical methods of GMO testing" (ENGL, 2008).

**5. Validation of a qPCR method for GMO quantification and its** 

**5.1 Introduction into the legal context** 

performance characteristics.

**implementation in a routine laboratory under ISO 17025 accreditation** 

with the results of the study and the validated protocol. These are submitted to the European Food Safety Authority (EFSA) and are subsequently published on the EU-RL GMFF official website. Upon publication the validated methods become official methods. The method validation thus provides the enforcement laboratories with standardised and harmonised methods applicable in official GMO detection.

#### **5.2 Evaluated parameters for newly developed event-specific methods for GMO quantification**

## **5.2.1 Evaluation of method performance characteristics by the EURL-GMFF**

The requirements for method **specificity** are laid down in the legislation. The method submitted has to be event-specific (based on the specific sequence of the plant-transgenic construct junction, part 3) and should detect only the specific GMO submitted for authorisation to be useful for unequivocal detection/identification/quantification of the GM event (EC/641/2004). To demonstrate that the method is event-specific, it has to be tested against all GM events from the applicant which are currently authorised in different parts of the world and against those still in development.

As the submitted methods are quantitative, they also include a reference taxon-specific assay. The specificity of this assay should also be tested. For taxon-specific assays the target should be preferably a unique sequence present in a single copy in the target plant genome. The copy number and the specificity have to be assessed *in silico* by using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) searches against known databases. In addition, the taxon-specific target should not show amplification signals with close relatives or taxa of the most important food crops. Usually, the different biotech companies develop their own taxon-specific method and test it on a range of taxa selected by them. This can pose several problems for the laboratories applying the methods. Firstly, there is no standard list of taxa and varieties to be included in the test. Ideally, the reference assay should be tested on a large range of varieties covering the existing natural variation within the taxon in order to assure that it will amplify any material from the plant species targeted by the method. Secondly, the existence of more than one reference system for events of the same plant taxon requires the use of several reference assays in quantification, which increases the costs of the analysis by the laboratory. In this context the requirements for the specificity of the taxonspecific reference assays should be made more precise and harmonisation in the methods used for different GM events is needed.

Information on the **applicability** of the method should be provided. This includes information on the scope of the method. In addition, information on known interferences with other analytes and the applicability to certain matrices should be supplied.

The **practicability** of the method should be demonstrated. For instance, methods where the reference and the event-specific assays are run on different PCR plates or under different PCR cycling conditions are less practicable and would be time and cost consuming when applied in a routine laboratory.

Besides these criteria, other parameters related to the method performance are assessed namely the **dynamic range, linearity, amplification efficiency, LOD and LOQ, trueness, precision and robustness**. The definitions of all parameters can be found in the glossary.

Development of a Molecular Platform

**5.4 Conclusion** 

quantifiable.

**6.1 General strategy** 

range of (-2;2) should have been obtained.

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 389

not deviate more than 25% from the true value. In the case of a PT sample a z-score in the

The **Relative Repeatability Standard Deviation (RSDr)** should be calculated on at least 16 single test results obtained under repeatability conditions. Repeatability should be available

Furthermore, the enforcement laboratory should estimate the **sensitivity** of the method. Hereto, four parameters can be calculated. The *Relative LOQ (LOQrel)* is estimated at low concentration(s) of positive material e.g. 0,1%. The LOQrel is set at this level if the RSDr is below 25%. The *Absolute LOQ (LOQabs)* is estimated by measuring dilution series of low copy numbers of the target. The LOQabs is set as the last dilution where the RSDr is lower than 25%. The *Relative LOD (LODrel)* is estimated using ten replicates of a positive control material with a low GM level. The LODrel is set at this level if the ten replicates show a positive amplification. The *Absolute LOD (LODabs)* is estimated as the copy number at which not more than 5% false negatives are obtained. In practice this is performed by evaluating ten PCR replicates of low copy number of the target. The LODabs is set at this level if the ten replicates score positive.

A GMO quantification method filed by the biotech companies together with the application for authorisation follows different steps. Firstly, the developer needs to provide information on the performance of the method. Hereto, he needs to evaluate different parameters as laid down in the ENGL document (ENGL, 2008). Secondly, the EU-RL GMFF evaluates the submitted information and decides whether the dossier is in compliance with the set criteria. Thirdly, the EU-RL organises a ring trial to validate the method. Hereto it gets the support of the different NRL that participate in the validation. Fourthly, the enforcement laboratories need to assess a number of parameters before to implement the method in their

At WIV-ISP-GMOlab, the assessed parameters and the data obtained during the in-house verification are gathered in a validation dossier. The event-specific method is in a first time used as a qualitative identification method in the second step of GMO analysis. The critical parameters determined during the in-house validation for these methods are the LODabs and LOQabs. These parameters, expressed as Ct values, are introduced into the DSS and serve as a threshold to decide if the GM event is present in the sample and in case of presence if it is

For quantification methods, no real DSS exists but different parameters are evaluated at each use in routine analysis and have to be in compliance with the set criteria. In a first step, the parameters of the calibration curves of the event-specific and the taxon-specific method (linearity, slope, PCR efficiency) are evaluated. Additionally, control samples (0,1% and 1%) are quantified and the result has to fulfil the acceptance criterion for trueness. In this way

**6. Introduction of the qPCR methods in the Decision Support System (DSS)** 

As described before (part 3), to cover the broadest GMO spectra, SYBR®Green qPCR methods have been developed and validated in the GMO detection platform. In this context,

laboratory for routine analysis under ISO 17025 accreditation.

the obtained quantitative results for unknown samples are validated.

for all tested GM levels. The RSDr needs to be equal or below 25% to be acceptable.

#### **5.2.2 Evaluation of method performance characteristics, performed by the analysis of the results of the inter-laboratory collaborative trial**

Once the EU-RL GMFF has made a scientific evaluation of the method based on the performance of the above-mentioned parameters (as provided by the method developer), it organizes a validation ring trial (concerning dynamic range, precision, relative reproducibility standard deviation and trueness). The participating laboratories receive the necessary samples and reagents and a detailed experimental protocol. It should be noted that the purpose of the ring trial is to assess the performance of the method and not of the laboratory. Therefore each participant has to follow the experimental procedure strictly. The results obtained by the laboratories are expressed as GM% for each tested level. These results are further scrutinised for outliers by the EU-RL GMFF using statistical methods recommended by ISO 5725. In addition, the mean value is calculated for each GM level analysed. Based on the parameters assessed during the ring trial, a conclusion is made on the compliance of the method with the ENGL method acceptance criteria and if it can be considered applicable in regard to the requirements of EC/641/2004.

### **5.3 Implementation of a validated event-specific method in a testing laboratory**

When the interlaboratory validation study is completed and the method is considered as applicable, the method is ready to be implemented in routine testing laboratories like the GMOlab.

On the one hand, Commission Regulation EC/882/2004 states that official laboratories shall be accredited according to the ISO 17025 standard. An ISO 17025 accreditation, under a fixed or flexible scope, implies that "the laboratory shall confirm that it can properly operate standard methods before introducing the tests for calibrations". On the other hand, according to the same regulation, it is the task of the EU-RL GMFF to provide the NRL with details of analytical methods, including reference methods. In this context, guidelines for implementation of the validated methods in the routine laboratory are set up by the ENGL in the document "Verification of analytical methods for GMO testing when implementing interlaboratory validated methods" (ENGL, 2011). These guidelines reflect the requirements set up in the document "Definition of the Minimum Performance Requirements for analytical methods of GMO testing" (ENGL, 2008), but also give additional guidance on how to design the experimental set up and to calculate the required values. In practice the laboratories have to design the quantification experiment in which two or three GM levels are quantified and the parameters described hereunder have to be assessed.

**Dynamic range, R2 coefficient and amplification efficiency:** these parameters can be calculated simultaneously from calibration curves when testing other parameters (trueness and precision). For each target, the average values of at least two calibration curves should be taken. The dynamic range should be tested between 1/10th of the threshold value and 5 times this value i.e. between 0,09% and 4,5% for the 0,9% labelling threshold. The PCR efficiency should be between 90 and 100% and the R2 coefficient needs to be equal or above 0,98 to have a linear curve.

**Trueness** should be determined at a level close to the level set in the legislation (0,9%) or according to the intended use of the method and additionally at a level close to the LOQ. The trueness can be measured using a CRM or if not available on a sample from a proficiency test (PT). To comply with the acceptance criterion, the measured value should not deviate more than 25% from the true value. In the case of a PT sample a z-score in the range of (-2;2) should have been obtained.

The **Relative Repeatability Standard Deviation (RSDr)** should be calculated on at least 16 single test results obtained under repeatability conditions. Repeatability should be available for all tested GM levels. The RSDr needs to be equal or below 25% to be acceptable.

Furthermore, the enforcement laboratory should estimate the **sensitivity** of the method. Hereto, four parameters can be calculated. The *Relative LOQ (LOQrel)* is estimated at low concentration(s) of positive material e.g. 0,1%. The LOQrel is set at this level if the RSDr is below 25%. The *Absolute LOQ (LOQabs)* is estimated by measuring dilution series of low copy numbers of the target. The LOQabs is set as the last dilution where the RSDr is lower than 25%.

The *Relative LOD (LODrel)* is estimated using ten replicates of a positive control material with a low GM level. The LODrel is set at this level if the ten replicates show a positive amplification. The *Absolute LOD (LODabs)* is estimated as the copy number at which not more than 5% false negatives are obtained. In practice this is performed by evaluating ten PCR replicates of low copy number of the target. The LODabs is set at this level if the ten replicates score positive.

## **5.4 Conclusion**

388 Polymerase Chain Reaction

**5.2.2 Evaluation of method performance characteristics, performed by the analysis of** 

Once the EU-RL GMFF has made a scientific evaluation of the method based on the performance of the above-mentioned parameters (as provided by the method developer), it organizes a validation ring trial (concerning dynamic range, precision, relative reproducibility standard deviation and trueness). The participating laboratories receive the necessary samples and reagents and a detailed experimental protocol. It should be noted that the purpose of the ring trial is to assess the performance of the method and not of the laboratory. Therefore each participant has to follow the experimental procedure strictly. The results obtained by the laboratories are expressed as GM% for each tested level. These results are further scrutinised for outliers by the EU-RL GMFF using statistical methods recommended by ISO 5725. In addition, the mean value is calculated for each GM level analysed. Based on the parameters assessed during the ring trial, a conclusion is made on the compliance of the method with the ENGL method acceptance criteria and if it can be

**the results of the inter-laboratory collaborative trial** 

considered applicable in regard to the requirements of EC/641/2004.

GMOlab.

0,98 to have a linear curve.

**5.3 Implementation of a validated event-specific method in a testing laboratory** 

are quantified and the parameters described hereunder have to be assessed.

When the interlaboratory validation study is completed and the method is considered as applicable, the method is ready to be implemented in routine testing laboratories like the

On the one hand, Commission Regulation EC/882/2004 states that official laboratories shall be accredited according to the ISO 17025 standard. An ISO 17025 accreditation, under a fixed or flexible scope, implies that "the laboratory shall confirm that it can properly operate standard methods before introducing the tests for calibrations". On the other hand, according to the same regulation, it is the task of the EU-RL GMFF to provide the NRL with details of analytical methods, including reference methods. In this context, guidelines for implementation of the validated methods in the routine laboratory are set up by the ENGL in the document "Verification of analytical methods for GMO testing when implementing interlaboratory validated methods" (ENGL, 2011). These guidelines reflect the requirements set up in the document "Definition of the Minimum Performance Requirements for analytical methods of GMO testing" (ENGL, 2008), but also give additional guidance on how to design the experimental set up and to calculate the required values. In practice the laboratories have to design the quantification experiment in which two or three GM levels

**Dynamic range, R2 coefficient and amplification efficiency:** these parameters can be calculated simultaneously from calibration curves when testing other parameters (trueness and precision). For each target, the average values of at least two calibration curves should be taken. The dynamic range should be tested between 1/10th of the threshold value and 5 times this value i.e. between 0,09% and 4,5% for the 0,9% labelling threshold. The PCR efficiency should be between 90 and 100% and the R2 coefficient needs to be equal or above

**Trueness** should be determined at a level close to the level set in the legislation (0,9%) or according to the intended use of the method and additionally at a level close to the LOQ. The trueness can be measured using a CRM or if not available on a sample from a proficiency test (PT). To comply with the acceptance criterion, the measured value should A GMO quantification method filed by the biotech companies together with the application for authorisation follows different steps. Firstly, the developer needs to provide information on the performance of the method. Hereto, he needs to evaluate different parameters as laid down in the ENGL document (ENGL, 2008). Secondly, the EU-RL GMFF evaluates the submitted information and decides whether the dossier is in compliance with the set criteria. Thirdly, the EU-RL organises a ring trial to validate the method. Hereto it gets the support of the different NRL that participate in the validation. Fourthly, the enforcement laboratories need to assess a number of parameters before to implement the method in their laboratory for routine analysis under ISO 17025 accreditation.

At WIV-ISP-GMOlab, the assessed parameters and the data obtained during the in-house verification are gathered in a validation dossier. The event-specific method is in a first time used as a qualitative identification method in the second step of GMO analysis. The critical parameters determined during the in-house validation for these methods are the LODabs and LOQabs. These parameters, expressed as Ct values, are introduced into the DSS and serve as a threshold to decide if the GM event is present in the sample and in case of presence if it is quantifiable.

For quantification methods, no real DSS exists but different parameters are evaluated at each use in routine analysis and have to be in compliance with the set criteria. In a first step, the parameters of the calibration curves of the event-specific and the taxon-specific method (linearity, slope, PCR efficiency) are evaluated. Additionally, control samples (0,1% and 1%) are quantified and the result has to fulfil the acceptance criterion for trueness. In this way the obtained quantitative results for unknown samples are validated.

## **6. Introduction of the qPCR methods in the Decision Support System (DSS)**

#### **6.1 General strategy**

As described before (part 3), to cover the broadest GMO spectra, SYBR®Green qPCR methods have been developed and validated in the GMO detection platform. In this context,

Development of a Molecular Platform

present.

an integer).

**interpretation** 

crl.jrc.ec.europa.eu).

conditions (part 4).

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 391

multiple targets are present in a GMO and the CoSYPS contains methods for each of these targets, all targets present in that GMO must be positive to conclude that this GMO might be

The second step in the CoSYPS algorithm is based on a mathematical model. A unique prime number (a prime number is a natural number that has exactly two distinct natural number divisors: 1 and itself) is associated with each particular screening method. When the sample is considered positive for a certain screening element, this specific prime number is assigned to the sample. When it is considered negative, the number 1 (neutral element in multiplication) is assigned. By multiplying all assigned values, the algorithm calculates the "Gödel prime product" (GPPsample) of the sample (the product of the prime numbers corresponding to the positive scoring screening methods). In a similar way each GMO can be represented by a product of the different prime numbers corresponding to the elements belonging to the GMO. This product is designed as the "Gödel prime product" (GPPGMO) of the GMO and represents a "mathematical tag" for this GMO. Note that several GMO can be

associated with a same GPP product as they comprise the same genetic elements.

potential GMO present in the sample by a series of simple divisions.

previously introduced in the Decision Support System.

The third step of the CoSYPS is based on the fact that, as a consequence of the nature of prime numbers, the division of the GPP by any of the prime numbers used in the generation of the GPP is an integer. Therefore the presence of a target in a GMO can be mathematically traced by generating this fraction: the program makes the ratio between the GPPsample and the GPPGMO to identify which GMO could be present in the sample (the division generates

Consequently, on the basis of the positive signal(s) obtained during the screening for each specific SYBR®Green qPCR method, the specific prime number assigned to each method is scored by the CoSYPS. The multiplication of these prime numbers allows the CoSYPS to calculate the GPP for the analysed sample. From this number, the CoSYPS can select all the

On the basis of outcome of the CoSYPS analysis a set of candidate GMO which could possibly reside within the product can be identified. In order to confirm the presence of a certain GM event in this product, event-specific Taqman® qPCR analysis is performed in a next step by applying methods validated and published by the EU-RL (http://gmo-

During the sample analysis, the Ct value obtained as outcome of the event-specific qPCR is recorded. This Ct value is compared to the LOD and LOQ (as determined during the verification of the identification method in the laboratory - part 5). These values were

A GM event is considered detectable by the DSS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained. The LOD was obtained under repeatability

To conclude which GM events are effectively present and identified in the sample, the DSS retains all prime numbers of the GM event with a Ct value below the Ct value of the LOD (+

**6.3 Integration of an event-specific method in the Decision Support System and** 

it rapidly becomes tedious in routine analyses to manually combine all the screening results in order to decide which GMO are potentially present in a sample. Therefore, in support to the qPCR data, a simple mathematical model has been developed to automatically calculate the possible presences in a product based on the outcome of the qPCR screening analysis (Van den Bulcke et al., 2010). The CoSYPS, standing for Combinatory SYBR®Green qPCR screening, represents a novel tool for GMO analysis based on the SYBR®Green qPCR technology. Using this decision support system alone is not sufficient. The suspected GM events need to be specifically identified in a second step, using e.g. the EU-RL Taqman® event-specific qPCR method(s). In a third step, the positively identified GM events are quantified to asses if their content complies or not with the 0,9% labelling threshold (EC1830/2003).

This newly developed tool is a versatile, cost-effective and time-efficient approach in assessing the GMO presence in analytical samples and can be applied in routine analysis for enforcement purposes. The full system has been patent protected (Van den Bulcke et al., 2008).

Here the construction, functioning and the theoretical basis of the CoSYPS will be described. Further explanation on the mathematical functioning of the CoSYPS may be found in the recently published paper "A theoretical introduction to "Combinatory SYBR®Green qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants" (Van den Bulcke et al., 2010).

## **6.2 Screening for GMO candidates by CoSYPS analysis**

The CoSYPS is based on the determination of the presence of certain element(s) originating from GMO and plant taxa frequently occurring in food and feed products. Hereto, SYBR®Green qPCR analysis of gDNA extracted from the product is performed, using primer pairs targeting different (multiple) discriminatory marker amplicons (part 3 and table 2).

During the SYBR®Green qPCR analysis of the sample, two critical qPCR parameters are recorded for each method used: the Ct and Tm values. Within the Decision Support System the obtained values are then compared to the LOD (expressed as a Ct value – see glossary) determined in the validation of the qPCR screening method and the nominal Tm value of the amplicon (see glossary). Both parameters are used as decision criteria for the analysis and are incorporated as such in the CoSYPS Decision Support System.

In a first step, the CoSYPS algorithm compares the measured Ct and the Tm values for each screening element with the corresponding "decision values" in the DSS. The latter values are determined during the in-house validation of the method (part 4). A signal generated in SYBR®Green qPCR analysis for a sample is considered as positive by the CoSYPS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained and the amplicon has a Tm value that falls within the determined Tm confidence interval (part 4). In agreement with the decision principles of the ISO norm 24276 (twice positive, twice negative), all decisions within the CoSYPS are based on the extraction and analysis of two distinct representative sub-extracts and eventually confirmed by a third analysis in case of ambiguous results (one positive, one negative). Therefore, a sample is positive for a specific screening element when the Ct and Tm results are unambiguously for both sub-extracts. Any positive signal obtained with a SYBR®Green qPCR method targeting a particular GM element indicates that a GMO comprising this target could be present in the sample. When several GMO contain the same target, a positive result generated by this screening method indicates that potentially all these GMO may be present in the sample. However, when

it rapidly becomes tedious in routine analyses to manually combine all the screening results in order to decide which GMO are potentially present in a sample. Therefore, in support to the qPCR data, a simple mathematical model has been developed to automatically calculate the possible presences in a product based on the outcome of the qPCR screening analysis (Van den Bulcke et al., 2010). The CoSYPS, standing for Combinatory SYBR®Green qPCR screening, represents a novel tool for GMO analysis based on the SYBR®Green qPCR technology. Using this decision support system alone is not sufficient. The suspected GM events need to be specifically identified in a second step, using e.g. the EU-RL Taqman® event-specific qPCR method(s). In a third step, the positively identified GM events are quantified to asses if their

This newly developed tool is a versatile, cost-effective and time-efficient approach in assessing the GMO presence in analytical samples and can be applied in routine analysis for enforcement purposes. The full system has been patent protected (Van den Bulcke et al., 2008). Here the construction, functioning and the theoretical basis of the CoSYPS will be described. Further explanation on the mathematical functioning of the CoSYPS may be found in the recently published paper "A theoretical introduction to "Combinatory SYBR®Green qPCR screening", a matrix-based approach for the detection of materials derived from genetically

The CoSYPS is based on the determination of the presence of certain element(s) originating from GMO and plant taxa frequently occurring in food and feed products. Hereto, SYBR®Green qPCR analysis of gDNA extracted from the product is performed, using primer pairs targeting different (multiple) discriminatory marker amplicons (part 3 and table 2).

During the SYBR®Green qPCR analysis of the sample, two critical qPCR parameters are recorded for each method used: the Ct and Tm values. Within the Decision Support System the obtained values are then compared to the LOD (expressed as a Ct value – see glossary) determined in the validation of the qPCR screening method and the nominal Tm value of the amplicon (see glossary). Both parameters are used as decision criteria for the analysis and

In a first step, the CoSYPS algorithm compares the measured Ct and the Tm values for each screening element with the corresponding "decision values" in the DSS. The latter values are determined during the in-house validation of the method (part 4). A signal generated in SYBR®Green qPCR analysis for a sample is considered as positive by the CoSYPS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained and the amplicon has a Tm value that falls within the determined Tm confidence interval (part 4). In agreement with the decision principles of the ISO norm 24276 (twice positive, twice negative), all decisions within the CoSYPS are based on the extraction and analysis of two distinct representative sub-extracts and eventually confirmed by a third analysis in case of ambiguous results (one positive, one negative). Therefore, a sample is positive for a specific screening element when the Ct and Tm results are unambiguously for both sub-extracts. Any positive signal obtained with a SYBR®Green qPCR method targeting a particular GM element indicates that a GMO comprising this target could be present in the sample. When several GMO contain the same target, a positive result generated by this screening method indicates that potentially all these GMO may be present in the sample. However, when

content complies or not with the 0,9% labelling threshold (EC1830/2003).

modified plants" (Van den Bulcke et al., 2010).

**6.2 Screening for GMO candidates by CoSYPS analysis** 

are incorporated as such in the CoSYPS Decision Support System.

multiple targets are present in a GMO and the CoSYPS contains methods for each of these targets, all targets present in that GMO must be positive to conclude that this GMO might be present.

The second step in the CoSYPS algorithm is based on a mathematical model. A unique prime number (a prime number is a natural number that has exactly two distinct natural number divisors: 1 and itself) is associated with each particular screening method. When the sample is considered positive for a certain screening element, this specific prime number is assigned to the sample. When it is considered negative, the number 1 (neutral element in multiplication) is assigned. By multiplying all assigned values, the algorithm calculates the "Gödel prime product" (GPPsample) of the sample (the product of the prime numbers corresponding to the positive scoring screening methods). In a similar way each GMO can be represented by a product of the different prime numbers corresponding to the elements belonging to the GMO. This product is designed as the "Gödel prime product" (GPPGMO) of the GMO and represents a "mathematical tag" for this GMO. Note that several GMO can be associated with a same GPP product as they comprise the same genetic elements.

The third step of the CoSYPS is based on the fact that, as a consequence of the nature of prime numbers, the division of the GPP by any of the prime numbers used in the generation of the GPP is an integer. Therefore the presence of a target in a GMO can be mathematically traced by generating this fraction: the program makes the ratio between the GPPsample and the GPPGMO to identify which GMO could be present in the sample (the division generates an integer).

Consequently, on the basis of the positive signal(s) obtained during the screening for each specific SYBR®Green qPCR method, the specific prime number assigned to each method is scored by the CoSYPS. The multiplication of these prime numbers allows the CoSYPS to calculate the GPP for the analysed sample. From this number, the CoSYPS can select all the potential GMO present in the sample by a series of simple divisions.

#### **6.3 Integration of an event-specific method in the Decision Support System and interpretation**

On the basis of outcome of the CoSYPS analysis a set of candidate GMO which could possibly reside within the product can be identified. In order to confirm the presence of a certain GM event in this product, event-specific Taqman® qPCR analysis is performed in a next step by applying methods validated and published by the EU-RL (http://gmocrl.jrc.ec.europa.eu).

During the sample analysis, the Ct value obtained as outcome of the event-specific qPCR is recorded. This Ct value is compared to the LOD and LOQ (as determined during the verification of the identification method in the laboratory - part 5). These values were previously introduced in the Decision Support System.

A GM event is considered detectable by the DSS when an exponential amplification below the Ct value of the LOD (+ 1 Ct) is obtained. The LOD was obtained under repeatability conditions (part 4).

To conclude which GM events are effectively present and identified in the sample, the DSS retains all prime numbers of the GM event with a Ct value below the Ct value of the LOD (+

Development of a Molecular Platform

Screening methods

Prime numbers

*+ 1 Ct* Subsample

Subsample

*Taqman* 

*Results Present* 

**6.5 Conclusion** 

*Quantifiable* 

cost-saving tool in GMO detection.

1

2

a. Accredited SYBR®Green qPCR available

b. CoSYPS algorithm and screening methods

GPP 1057485

*CoSYPS step 2 - Calculation of the Gödel prime product of the sample* 

*CoSYPS step 3 - Assessment of potential GMO present in the sample*  GMO GPP of GMO GPPsample/ GPPGMO Decision

c. DSS and confirmation by event-specific Taqman method

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 393

*CoSYPs step1 - Sample analysis "+" is assigned when value < LOD+1 Ct; " –" is assigned when value > LOD* 

Product of 3 1 11 5 13 17 1 1 29 1 1

MON810 5655 187 Confirmation by event-specific Taqman method T25 16269 65 Confirmation by event-specific Taqman method GA21 145 7293 Confirmation by event-specific Taqman method GTS 40-3-2 8835 119,69 No confirmation by event-specific Taqman

MON 810 T25 GTS 40-3-2 GA21 Subsample 1 LOD > Ct < LOQ LOD > Ct > LOQ LOD < Ct > LOQ

Subsample 2 LOD > Ct < LOQ LOD > Ct > LOQ LOD < Ct > LOQ

Table 3. Mathematical functioning of the CoSYPS, allowing demonstrating the possible presence of a set of GMO in a product based on the outcome of a qPCR screening analysis.

By combining the results of the screening analysis, the CoSYPS allows to decide in a fast way which GM events are possibly present in the sample under analysis. The use of the mathematical algorithm, which compares the GPPsample and GPPGMO, excludes the need for manual calculations and comparisons. The only thing that needs to be done by the operator is the preliminary introduction of the critical values (Ct corresponding to the LOD and LOQ, Tm) obtained during method validation in the system. Further, in identification, the obtained results for a sample are compared with the LOD and LOQ values determined during inhouse validation of the event-specific methods. From this comparison, the Decision Support System will indicate which GM events are present and at which level and thus allow deciding which GMO needs to be quantified in the sample. This Decision Support System, developed and patented by the WIV-ISP-GMOlab is thus a very efficient, user friendly and

p35S pNOS t35S tNOS CryIAb PAT/pat PAT/bar CP4 ADH1 LEC CRU

3 7 11 5 13 17 23 19 29 31 37

+ - + + + + - - + - -

+ - + + + + - - + - -

method

*Present Not detectable* 

1 Ct) threshold level. The Ct value is also compared with the LOQ + 1 Ct to decide if the GM event is present at a quantifiable level.

If no authorised GMO can explain the presence of a set of screening targets, it can be concluded that the sample contains one or more unassigned targets. The unassigned signals are mostly due to unauthorised GMO or donor organisms (bacterial, viral and plant sources of transgenic elements). In such cases more complex analysis like DNA walking, DNA sequencing has to be performed outside of the routine to elucidate their origin.

## **6.4 Practical case**

As an example, the accredited SYBR®Green qPCR methods available in a qPCR platform for GMO detection and their associated prime numbers are p35S, tNOS, pNOS, t35S, CryIAb, PAT/pat, CP4, PAT/bar for the transgenic elements and ADH1, LEC and CRU for the taxon-specific markers (table 3a). The elements targeted by these methods can be found in part 3 table 2.

During the screening analysis a positive signal (correct Tm and Ct < Ct of LOD + 1 Ct) is found for the p35S, tNOS, CryIAb, t35S , PAT/pat and ADH1 elements while no positive signal was obtained for pNOS, CP4, PAT/bar and the other species-specific targets (table 3b–step1). For each positive screening marker (p35S, tNOS, CryIAb, t35S and PAT/pat and ADH1) the specific prime number is assigned to each of the corresponding methods. As the pNOS, CP4, PAT/bar targets and the other taxon-specific markers are considered as negative the assigned number for all of these methods is 1. The "Gödel prime product" of the sample (= 1057485) is calculated by multiplying all the assigned prime numbers (table 3b-step2). The CoSYPS will compare this GPPsample with the GPP of all GM events that have previously been introduced in the system. The example is given here for four GM events.

The transgenic MON 810 and T25 events are described as a function of three transgenic elements (p35S, tNOS, CryIAb) and (p35S, t35S, PAT/pat) respectively and one maizespecific (ADH1). The GA21 maize is covered by the tNOS and maize–specific element. The GTS40-3-2 event is defined by three transgenic elements (p35S, tNOS, CP4) and the soybean endogen (LEC). Consequently, the "Gödel prime product" of the MON 810, T25, GA21 and GTS40-3-2 are 5655 (= 3 X 5 X 13 X 29), 16269 (= 3 X 11 X17 X 29), 145 (= 5 X 29) and 8835 (= 3 X 5 X 19 X 31) respectively (table 3b-step 3).

To assess which GMO are potentially present in the sample, the "Gödel prime product" of the sample is divided by the GPP of each GMO (table 3b-step 3). The result is an integer only for MON 810, T25 and GA21. From the screening analysis, the CoSYPS thus predicts that MON 810, T25 and GA21 are potentially present while GTS40-3-2 is not. As a consequence MON 810, GA21 and T25 have to be further analysed with the event-specific method to confirm their presence.

In order to confirm the presence of MON 810, T25 and GA21 in the sample product, the event-specific qPCR analyses are performed. The results (expressed as Ct values) confirm the presence of MON 810 and T25 while GA21 is not detectable (table 3c). The Ct values obtained are compared with the LOQ + 1 Ct of each method and show that only MON 810 can be quantified. Finally the GM% of this event will be compared to the labelling threshold (0,9% mass per ingredient) in order to conclude on the conformity of the sample.

#### Development of a Molecular Platform for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 393


#### a. Accredited SYBR®Green qPCR available

392 Polymerase Chain Reaction

1 Ct) threshold level. The Ct value is also compared with the LOQ + 1 Ct to decide if the GM

If no authorised GMO can explain the presence of a set of screening targets, it can be concluded that the sample contains one or more unassigned targets. The unassigned signals are mostly due to unauthorised GMO or donor organisms (bacterial, viral and plant sources of transgenic elements). In such cases more complex analysis like DNA walking, DNA

As an example, the accredited SYBR®Green qPCR methods available in a qPCR platform for GMO detection and their associated prime numbers are p35S, tNOS, pNOS, t35S, CryIAb, PAT/pat, CP4, PAT/bar for the transgenic elements and ADH1, LEC and CRU for the taxon-specific markers (table 3a). The elements targeted by these methods can be found in

During the screening analysis a positive signal (correct Tm and Ct < Ct of LOD + 1 Ct) is found for the p35S, tNOS, CryIAb, t35S , PAT/pat and ADH1 elements while no positive signal was obtained for pNOS, CP4, PAT/bar and the other species-specific targets (table 3b–step1). For each positive screening marker (p35S, tNOS, CryIAb, t35S and PAT/pat and ADH1) the specific prime number is assigned to each of the corresponding methods. As the pNOS, CP4, PAT/bar targets and the other taxon-specific markers are considered as negative the assigned number for all of these methods is 1. The "Gödel prime product" of the sample (= 1057485) is calculated by multiplying all the assigned prime numbers (table 3b-step2). The CoSYPS will compare this GPPsample with the GPP of all GM events that have previously been introduced in the system. The example is given here for four GM events.

The transgenic MON 810 and T25 events are described as a function of three transgenic elements (p35S, tNOS, CryIAb) and (p35S, t35S, PAT/pat) respectively and one maizespecific (ADH1). The GA21 maize is covered by the tNOS and maize–specific element. The GTS40-3-2 event is defined by three transgenic elements (p35S, tNOS, CP4) and the soybean endogen (LEC). Consequently, the "Gödel prime product" of the MON 810, T25, GA21 and GTS40-3-2 are 5655 (= 3 X 5 X 13 X 29), 16269 (= 3 X 11 X17 X 29), 145 (= 5 X 29) and 8835 (= 3

To assess which GMO are potentially present in the sample, the "Gödel prime product" of the sample is divided by the GPP of each GMO (table 3b-step 3). The result is an integer only for MON 810, T25 and GA21. From the screening analysis, the CoSYPS thus predicts that MON 810, T25 and GA21 are potentially present while GTS40-3-2 is not. As a consequence MON 810, GA21 and T25 have to be further analysed with the event-specific method to

In order to confirm the presence of MON 810, T25 and GA21 in the sample product, the event-specific qPCR analyses are performed. The results (expressed as Ct values) confirm the presence of MON 810 and T25 while GA21 is not detectable (table 3c). The Ct values obtained are compared with the LOQ + 1 Ct of each method and show that only MON 810 can be quantified. Finally the GM% of this event will be compared to the labelling threshold

(0,9% mass per ingredient) in order to conclude on the conformity of the sample.

sequencing has to be performed outside of the routine to elucidate their origin.

event is present at a quantifiable level.

X 5 X 19 X 31) respectively (table 3b-step 3).

confirm their presence.

**6.4 Practical case** 

part 3 table 2.

#### b. CoSYPS algorithm and screening methods


c. DSS and confirmation by event-specific Taqman method


Table 3. Mathematical functioning of the CoSYPS, allowing demonstrating the possible presence of a set of GMO in a product based on the outcome of a qPCR screening analysis.

#### **6.5 Conclusion**

By combining the results of the screening analysis, the CoSYPS allows to decide in a fast way which GM events are possibly present in the sample under analysis. The use of the mathematical algorithm, which compares the GPPsample and GPPGMO, excludes the need for manual calculations and comparisons. The only thing that needs to be done by the operator is the preliminary introduction of the critical values (Ct corresponding to the LOD and LOQ, Tm) obtained during method validation in the system. Further, in identification, the obtained results for a sample are compared with the LOD and LOQ values determined during inhouse validation of the event-specific methods. From this comparison, the Decision Support System will indicate which GM events are present and at which level and thus allow deciding which GMO needs to be quantified in the sample. This Decision Support System, developed and patented by the WIV-ISP-GMOlab is thus a very efficient, user friendly and cost-saving tool in GMO detection.

Development of a Molecular Platform

our team.

**8. Glossary** 

**Applicability** 

**Amplification Efficiency** 

by the following equation:

method can be applied.

metrological traceability.

regression analysis. **Dynamic Range** 

**Correlation Coefficient (R2)** 

**Certified Reference Material (CRM)** 

**European Food Safety Authority (EFSA)** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 395

broad range of GMO and UGM and thus to improve the resolution of the system. Particular importance will be given to their use in a modular approach associated with a decision tree cascade. Moreover, our strategy aiming at developing harmonised SYBR®Green qPCR screening methods incorporated in the Combinatory SYBR®Green qPCR Screening (CoSYPS) system has a potential to be applied in other scientific fields than GMO detection. The application of this strategy for food borne pathogenic bacteria is now under development in

The amplification efficiency is the rate of amplification that leads to a theoretical slope of – 3,32 with an efficiency of 100% in each cycle. The efficiency of the reaction can be calculated

10 1 *slope Efficiency*

Applicability is the description of analytes, matrices and concentrations to which the

A Certified Reference Material is a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of

The R2 coefficient is the correlation coefficient of a (calibration) curve obtained by linear

The dynamic range is the range of concentrations over which the method performs in a

EFSA is an agency of the EU that provides independent scientific advice and

The European Network of GMO Laboratories is a platform of EU experts that plays an eminent role in the development, harmonisation and standardisation of means and methods for sampling, detection, identification and quantification of Genetically Modified Organisms (GMO) or derived products in a wide variety of matrices, covering seeds, grains, food, feed and environmental samples. The network was inaugurated in Brussels on December

linear manner with an acceptable level of trueness and precision.

communication on all matters concerning food and feed safety.

**European Network of GMO Laboratories (ENGL)** 

1

<sup>−</sup> = − (1)

## **7. Conclusion**

In the near future, the number and the diversity of GM crops will continue to increase, as well as the requests for authorisation for their import for food and feed in the EU. Beside the notifications of GM events produced by multinational biotech companies, many GM events will be developed by universities, national research centres and small private companies. Thus, the chance for accidental occurrence of unapproved GMO in the EU food and feed chain trough importation will be higher. As the EU's general policy supports strong commitment to consumer protection and freedom of choice, and therefore mandatory product labelling, the development of sensitive, reliable but also cost-effective and flexible strategies for the detection of GMO in products through establishment of molecular platforms will become more and more crucial.

The GMO detection platform developed at WIV-ISP consists of a pre-PCR step namely DNA extraction and three consecutive qPCR phases. In this view, the choice of efficient methods to extract good quality DNA, in particular for processed food and feed, is a critical factor. A pre-PCR evaluation of the extracted gDNA is necessary as well as setting criteria for the purity and integrity of the DNA. Furthermore, the presence of PCR inhibitors is a major obstacle for efficient amplification in qPCR. This step may even become more important as the number of GM plant taxa becomes larger. Developing simple standard methods for genomic DNA extraction minimizing inhibition will therefore be the key for providing concordant results when using qPCR techniques.

Due to the broad range of GMO that my occur in the EU food and feed chains, the use of screening strategies only based on the 35S promoter of the Cauliflower Mosaic Virus (p35S) and the nopaline synthase terminator of *Agrobacterium tumefaciens* (tNOS) followed by the analysis of the sample with event-specific EU validated methods by the enforcement laboratories will become insufficient. As a consequence, new methods focusing on an intensive screening analysis need to be developed.

At the present time several high-tech strategies like multiplex PCR and consecutive detection and identification of the amplification products using micro-arrays (Chaouachi et al., 2008, Morisset et al., 2008, Hamels et al., 2009) or PCR combined with capillary electrophoresis (Nadal et al., 2009) have been proposed to deal with this discriminative problem and the broad diversity of GMO. However, at the present time, these technologies require additional costly equipment and investments in technical support. Furthermore, they need technological optimalisation as they show a high background at low target level. These difficulties make them less suitable for routine or enforcement purposes.

Contrary to the above-mentioned technically complex strategies, our approach based on numerous singleplex qPCR-based methods developed to function under the same reaction conditions combined with the informatics decision support tool CoSYPS may in the future represent a very effective alternative. This newly developed tool is considered as a versatile, cost-effective and time-efficient platform assessing the GMO presence in analytical samples. In addition, it functions in routine analysis for enforcement purposes in a commonly applied 96-well plate qPCR format.

In the future, the research of the molecular platform of the WIV-ISP will focus on the development of more discriminative SYBR®Green qPCR screening methods to cover the broad range of GMO and UGM and thus to improve the resolution of the system. Particular importance will be given to their use in a modular approach associated with a decision tree cascade. Moreover, our strategy aiming at developing harmonised SYBR®Green qPCR screening methods incorporated in the Combinatory SYBR®Green qPCR Screening (CoSYPS) system has a potential to be applied in other scientific fields than GMO detection. The application of this strategy for food borne pathogenic bacteria is now under development in our team.

## **8. Glossary**

394 Polymerase Chain Reaction

In the near future, the number and the diversity of GM crops will continue to increase, as well as the requests for authorisation for their import for food and feed in the EU. Beside the notifications of GM events produced by multinational biotech companies, many GM events will be developed by universities, national research centres and small private companies. Thus, the chance for accidental occurrence of unapproved GMO in the EU food and feed chain trough importation will be higher. As the EU's general policy supports strong commitment to consumer protection and freedom of choice, and therefore mandatory product labelling, the development of sensitive, reliable but also cost-effective and flexible strategies for the detection of GMO in products through establishment of molecular

The GMO detection platform developed at WIV-ISP consists of a pre-PCR step namely DNA extraction and three consecutive qPCR phases. In this view, the choice of efficient methods to extract good quality DNA, in particular for processed food and feed, is a critical factor. A pre-PCR evaluation of the extracted gDNA is necessary as well as setting criteria for the purity and integrity of the DNA. Furthermore, the presence of PCR inhibitors is a major obstacle for efficient amplification in qPCR. This step may even become more important as the number of GM plant taxa becomes larger. Developing simple standard methods for genomic DNA extraction minimizing inhibition will therefore be the key for providing

Due to the broad range of GMO that my occur in the EU food and feed chains, the use of screening strategies only based on the 35S promoter of the Cauliflower Mosaic Virus (p35S) and the nopaline synthase terminator of *Agrobacterium tumefaciens* (tNOS) followed by the analysis of the sample with event-specific EU validated methods by the enforcement laboratories will become insufficient. As a consequence, new methods focusing on an

At the present time several high-tech strategies like multiplex PCR and consecutive detection and identification of the amplification products using micro-arrays (Chaouachi et al., 2008, Morisset et al., 2008, Hamels et al., 2009) or PCR combined with capillary electrophoresis (Nadal et al., 2009) have been proposed to deal with this discriminative problem and the broad diversity of GMO. However, at the present time, these technologies require additional costly equipment and investments in technical support. Furthermore, they need technological optimalisation as they show a high background at low target level.

Contrary to the above-mentioned technically complex strategies, our approach based on numerous singleplex qPCR-based methods developed to function under the same reaction conditions combined with the informatics decision support tool CoSYPS may in the future represent a very effective alternative. This newly developed tool is considered as a versatile, cost-effective and time-efficient platform assessing the GMO presence in analytical samples. In addition, it functions in routine analysis for enforcement purposes in a commonly applied

In the future, the research of the molecular platform of the WIV-ISP will focus on the development of more discriminative SYBR®Green qPCR screening methods to cover the

These difficulties make them less suitable for routine or enforcement purposes.

**7. Conclusion** 

platforms will become more and more crucial.

concordant results when using qPCR techniques.

intensive screening analysis need to be developed.

96-well plate qPCR format.

#### **Amplification Efficiency**

The amplification efficiency is the rate of amplification that leads to a theoretical slope of – 3,32 with an efficiency of 100% in each cycle. The efficiency of the reaction can be calculated by the following equation:

$$\text{Efficiency} = 10^{\left(\frac{-1}{\text{slope}}\right)} - 1 \tag{1}$$

### **Applicability**

Applicability is the description of analytes, matrices and concentrations to which the method can be applied.

#### **Certified Reference Material (CRM)**

A Certified Reference Material is a reference material characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability.

#### **Correlation Coefficient (R2)**

The R2 coefficient is the correlation coefficient of a (calibration) curve obtained by linear regression analysis.

#### **Dynamic Range**

The dynamic range is the range of concentrations over which the method performs in a linear manner with an acceptable level of trueness and precision.

#### **European Food Safety Authority (EFSA)**

EFSA is an agency of the EU that provides independent scientific advice and communication on all matters concerning food and feed safety.

#### **European Network of GMO Laboratories (ENGL)**

The European Network of GMO Laboratories is a platform of EU experts that plays an eminent role in the development, harmonisation and standardisation of means and methods for sampling, detection, identification and quantification of Genetically Modified Organisms (GMO) or derived products in a wide variety of matrices, covering seeds, grains, food, feed and environmental samples. The network was inaugurated in Brussels on December

Development of a Molecular Platform

**Reference material (RM)** 

in a measurement process.

**Robustness** 

**Specificity** 

**Threshold cycle (Ct)** 

usually expressed in terms of bias.

**9. Acknowledgement** 

Science Policy.

interest.

**Trueness** 

for GMO Detection in Food and Feed on the Basis of "Combinatory qPCR" Technology 397

results are obtained with the same method, on identical test items, in the same laboratory,

The relative reproducibility standard deviation is the relative standard deviation of test results obtained under reproducibility conditions. Reproducibility conditions are conditions where the test results are obtained with the same method, on identical test items, in different laboratories, with different operators, using different equipment. Reproducibility standard

A Reference Material is a material that is sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use

The robustness of a method is a measure of its capacity to remain unaffected by small, but

Specificity is a property of a method to respond exclusively to the characteristic or analyte of

The threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The Ct value assigned to a particular well thus reflects the point during the reaction

The trueness is defined as the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. The measure of trueness is

The authors like to acknowledge Lievens Antoon for his critical review of the manuscript and De Keersmaecker Sigrid for her help with figures and layout. This work of the GMOlab was financially supported by four projects: the British Food Standard Agency (FSA, contract G03032) and the German Federal Office of Consumer Protection and Food Safety (BVL) through the Project GMOseek, under the European ERA-NET consortium SAFEFOODERA; the GMODETEC project (RT-06/6) of the Belgian Federal Ministry ''Health, Food Chain Safety and Environment'; the European Commission through the Integrated Project Co-Extra (Contract No. 007158), under the 6th Framework Program and by the Belgian Science Policy projects SPSD I & II of the Belgian Federal Ministry of

deliberate deviations from the experimental conditions described in the procedure.

at which a sufficient number of amplicons has been accumulated.

by the same operator, using the same equipment within short intervals of time.

**Precision – Relative Reproducibility Standard Deviation (RSDR%)** 

deviation describes the inter-laboratory variation.

4th 2002 and it currently consists of more than 100 national enforcement laboratories, representing all 27 EU Member States plus Norway and Switzerland. Its plenary meetings are open to particular observers, such as to representatives from Acceding and Candidate Countries.

## **European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF)**

The core task of the EU-RL GMFF is the scientific assessment and validation of detection methods for GM Food and Feed as part of the EU authorisation procedure. The Joint Research Centre (JRC) of the European Commission and, more precisely, the Molecular Biology and Genomics Unit of the Institute for Health and Consumer Protection (IHCP), has been given the mandate for the operation of the EU-RL GMFF. Activities are carried out in close collaboration with European Network of GMO Laboratories (ENGL).

## **Genetically Modified (GM) event**

A GM event refers to the unique DNA recombination event that took place in one plant cell, which was then used to generate entire transgenic plants

## **Genetically Modified Organism (GMO)**

A Genetically Modified Organism is officially defined in the EU legislation as "organisms, not from human origin, in which the genetic material (DNA) has been altered in a way that does not occur naturally by mating and/or natural recombination"

#### **Limit of Detection (LOD)**

The limit of detection is the lowest amount or concentration of analyte in a sample, which can be reliably detected but not necessarily quantified, as demonstrated by single-laboratory validation.

#### **Limit of Quantification (LOQ)**

The limit of quantification is the lowest amount or concentration of analyte in a sample that can be reliably quantified with an acceptable level of precision and accuracy.

### **Melting temperature (Tm)**

The melting temperature is the temperature at which 50% of the DNA is single stranded.

#### **National Reference Laboratory (NRL)**

A National Reference Laboratory on GMO operates in the frame of Commission Regulation EC/1829/2003 on GM Food and Feed and Commission regulation EC/1830/2003 on labelling and traceability of GMO. It assists the EU-RL and the NRL from the different member states are gathered in the ENGL.

#### **Practicability**

Practicability is the ease of operations, the feasibility and efficiency of implementation, the associated unitary costs (e.g. cost/sample) of the method.

#### **Precision - Relative Repeatability Standard Deviation (RSDr%)**

The relative repeatability standard deviation is the relative standard deviation of test results obtained under repeatability conditions. Repeatability conditions are conditions where test results are obtained with the same method, on identical test items, in the same laboratory, by the same operator, using the same equipment within short intervals of time.

## **Precision – Relative Reproducibility Standard Deviation (RSDR%)**

The relative reproducibility standard deviation is the relative standard deviation of test results obtained under reproducibility conditions. Reproducibility conditions are conditions where the test results are obtained with the same method, on identical test items, in different laboratories, with different operators, using different equipment. Reproducibility standard deviation describes the inter-laboratory variation.

#### **Reference material (RM)**

A Reference Material is a material that is sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurement process.

#### **Robustness**

396 Polymerase Chain Reaction

4th 2002 and it currently consists of more than 100 national enforcement laboratories, representing all 27 EU Member States plus Norway and Switzerland. Its plenary meetings are open to particular observers, such as to representatives from Acceding and Candidate

The core task of the EU-RL GMFF is the scientific assessment and validation of detection methods for GM Food and Feed as part of the EU authorisation procedure. The Joint Research Centre (JRC) of the European Commission and, more precisely, the Molecular Biology and Genomics Unit of the Institute for Health and Consumer Protection (IHCP), has been given the mandate for the operation of the EU-RL GMFF. Activities are carried out in

A GM event refers to the unique DNA recombination event that took place in one plant cell,

A Genetically Modified Organism is officially defined in the EU legislation as "organisms, not from human origin, in which the genetic material (DNA) has been altered in a way that

The limit of detection is the lowest amount or concentration of analyte in a sample, which can be reliably detected but not necessarily quantified, as demonstrated by single-laboratory

The limit of quantification is the lowest amount or concentration of analyte in a sample that

The melting temperature is the temperature at which 50% of the DNA is single stranded.

A National Reference Laboratory on GMO operates in the frame of Commission Regulation EC/1829/2003 on GM Food and Feed and Commission regulation EC/1830/2003 on labelling and traceability of GMO. It assists the EU-RL and the NRL from the different

Practicability is the ease of operations, the feasibility and efficiency of implementation, the

The relative repeatability standard deviation is the relative standard deviation of test results obtained under repeatability conditions. Repeatability conditions are conditions where test

can be reliably quantified with an acceptable level of precision and accuracy.

**European Union Reference Laboratory for GM Food and Feed (EU-RL GMFF)** 

close collaboration with European Network of GMO Laboratories (ENGL).

which was then used to generate entire transgenic plants

does not occur naturally by mating and/or natural recombination"

Countries.

**Genetically Modified (GM) event** 

**Limit of Detection (LOD)** 

**Limit of Quantification (LOQ)** 

**National Reference Laboratory (NRL)** 

member states are gathered in the ENGL.

associated unitary costs (e.g. cost/sample) of the method.

**Precision - Relative Repeatability Standard Deviation (RSDr%)** 

**Melting temperature (Tm)** 

validation.

**Practicability** 

**Genetically Modified Organism (GMO)** 

The robustness of a method is a measure of its capacity to remain unaffected by small, but deliberate deviations from the experimental conditions described in the procedure.

#### **Specificity**

Specificity is a property of a method to respond exclusively to the characteristic or analyte of interest.

### **Threshold cycle (Ct)**

The threshold cycle reflects the cycle number at which the fluorescence generated within a reaction crosses the threshold. It is inversely correlated to the logarithm of the initial copy number. The Ct value assigned to a particular well thus reflects the point during the reaction at which a sufficient number of amplicons has been accumulated.

#### **Trueness**

The trueness is defined as the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. The measure of trueness is usually expressed in terms of bias.

## **9. Acknowledgement**

The authors like to acknowledge Lievens Antoon for his critical review of the manuscript and De Keersmaecker Sigrid for her help with figures and layout. This work of the GMOlab was financially supported by four projects: the British Food Standard Agency (FSA, contract G03032) and the German Federal Office of Consumer Protection and Food Safety (BVL) through the Project GMOseek, under the European ERA-NET consortium SAFEFOODERA; the GMODETEC project (RT-06/6) of the Belgian Federal Ministry ''Health, Food Chain Safety and Environment'; the European Commission through the Integrated Project Co-Extra (Contract No. 007158), under the 6th Framework Program and by the Belgian Science Policy projects SPSD I & II of the Belgian Federal Ministry of Science Policy.

Development of a Molecular Platform

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**19** 

*1,2,3,4,5,7Iran 6Turkey* 

**Overview of Real-Time PCR Principles** 

*1Laboratory of Genetics, Legal Medicine Organization of Tabriz, Tabriz,* 

*5Faculty of Medicine, Shahid Behesti University of Medical Sciences, Tehran,* 

*7Department of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz* 

Real-time PCR is based on the revolutionary method of PCR, developed by Kary Mullis in the 1980s, which allows researchers to amplify specic pieces of DNA more than a billionfold (Saiki, Scharf et al. 1985; Mullis and Faloona 1987; Mullis 1990). PCR-based strategies have propelled molecular biology forward by enabling researchers to manipulate DNA more easily, thereby facilitating both common procedures, such as cloning, and huge endeavors such as the Human Genome Project (Olson, Hood et al. 1989; Ausubel, Brent et al. 2005). Real-time PCR represents yet another technological leap forward that has opened up new and powerful applications for researchers throughout the world. This is in part because the enormous sensitivity of PCR has been coupled to the precision afforded by "real-time"

Higuchi and co-workers (Higuchi, Dollinger et al. 1992; Higuchi, Fockler et al. 1993) at Roche Molecular Systems and Chiron accomplished the rst demonstration of real-time PCR. By including a common uorescent dye called ethidium bromide (EtBr) in the PCR and running the reaction under ultraviolet light, which causes EtBr to uoresce, they could visualize and record the accumulation of DNA with a video camera. It has been known since 1966 that EtBr increases its uorescence upon binding of nucleic acids (Le Pecq and Paoletti 1966), but only by combining this uorescent chemistry with PCR and real-time videography could real-time PCR be born as it was in the early 1990s. Subsequently, this

monitoring of PCR products as they are generated (Valasek and Repa 2005).

**1. Introduction** 

 \*

Corresponding Author

*4Department of Biotechnology, School of Allied Medical Sciences,* 

Mahmood Khosravi3, Atefeh Namipashaki4, Vahid Mehri Soofiany5,

*3Hematology Department of Medicine Faculty, Guilan University of Medical Sciences, Rasht,* 

Morteza Seifi1,\*, Asghar Ghasemi1, Siamak Heidarzadeh2,

Ali Alizadeh Khosroshahi6 and Nasim Danaei7

*2Division of Microbiology, School of Public Health, Tehran University of Medical Sciences, Tehran,* 

*Tehran University of Medical Sciences, Tehran,* 

*6Jarrah Pasha Medicine Faculty of Istanbul, Istanbul,* 

selection. *Proceedings of the National Academy of Sciences USA*, Vol. 85, No. 15, pp. 5536-5540


## **Overview of Real-Time PCR Principles**

Morteza Seifi1,\*, Asghar Ghasemi1, Siamak Heidarzadeh2, Mahmood Khosravi3, Atefeh Namipashaki4, Vahid Mehri Soofiany5, Ali Alizadeh Khosroshahi6 and Nasim Danaei7 *1Laboratory of Genetics, Legal Medicine Organization of Tabriz, Tabriz, 2Division of Microbiology, School of Public Health, Tehran University of Medical Sciences, Tehran, 3Hematology Department of Medicine Faculty, Guilan University of Medical Sciences, Rasht, 4Department of Biotechnology, School of Allied Medical Sciences, Tehran University of Medical Sciences, Tehran, 5Faculty of Medicine, Shahid Behesti University of Medical Sciences, Tehran, 6Jarrah Pasha Medicine Faculty of Istanbul, Istanbul, 7Department of Health and Nutrition, Tabriz University of Medical Sciences, Tabriz 1,2,3,4,5,7Iran 6Turkey* 

## **1. Introduction**

404 Polymerase Chain Reaction

Van den Bulcke, M., Lievens, A., Leunda, A., Mbongolo Mbella, E., Barbau-Piednoir, E. &

Van den Bulcke, M., Lievens, A., Barbau-Piednoir, E., Mbongolo Mbella, G., Roosens, N.,

*Chemistry*, Vol. 396, No. 6, pp. 2113-2123, DOI 10.1007/s00216-009-3286-7 Wehrmann, A., Van, V.A., Opsomer, C., Botterman, J. & Schulz, A. (1996). The similarities of

*Deutsche Lebensmittel-Rundschau*, Vol. 94, No. 5, pp. 159-161

5536-5540

PCT/EP2008/051059

selection. *Proceedings of the National Academy of Sciences USA*, Vol. 85, No. 15, pp.

Sneyers, M. (2008). Transgenic plant event detection. PATENT

Sneyers, M. & Leunda Casi, A. (2010). A theoretical introduction to "Combinatory SYBR®Green qPCR Screening", a matrix-based approach for the detection of materials derived from genetically modified plants. *Analytical Bioanalytical* 

bar and pat gene products make them equally applicable for plant engineers. *Nature Biotechnology*, Vol. 14, No. 10, pp. 1274-1278, doi:10.1038/nbt1096-1274 Wurz, A., Rüggeberg, H., Brodmann, P., Waiblinger, H.U. & Pietsch, K., (1998). DNA-

Extraktionmethode für den nachweis gentechnisch veränderter Soja in Sojalecithin.

Real-time PCR is based on the revolutionary method of PCR, developed by Kary Mullis in the 1980s, which allows researchers to amplify specic pieces of DNA more than a billionfold (Saiki, Scharf et al. 1985; Mullis and Faloona 1987; Mullis 1990). PCR-based strategies have propelled molecular biology forward by enabling researchers to manipulate DNA more easily, thereby facilitating both common procedures, such as cloning, and huge endeavors such as the Human Genome Project (Olson, Hood et al. 1989; Ausubel, Brent et al. 2005). Real-time PCR represents yet another technological leap forward that has opened up new and powerful applications for researchers throughout the world. This is in part because the enormous sensitivity of PCR has been coupled to the precision afforded by "real-time" monitoring of PCR products as they are generated (Valasek and Repa 2005).

Higuchi and co-workers (Higuchi, Dollinger et al. 1992; Higuchi, Fockler et al. 1993) at Roche Molecular Systems and Chiron accomplished the rst demonstration of real-time PCR. By including a common uorescent dye called ethidium bromide (EtBr) in the PCR and running the reaction under ultraviolet light, which causes EtBr to uoresce, they could visualize and record the accumulation of DNA with a video camera. It has been known since 1966 that EtBr increases its uorescence upon binding of nucleic acids (Le Pecq and Paoletti 1966), but only by combining this uorescent chemistry with PCR and real-time videography could real-time PCR be born as it was in the early 1990s. Subsequently, this

<sup>\*</sup> Corresponding Author

Overview of Real-Time PCR Principles 407

both aromatic systems, which convert electronic excitation energy into heat that dissipates to the surrounding solvent. On the other hand the dyes become brightly fluorescent when they bind to DNA, presumably to the minor groove, and rotation around the methine bond is restricted (Nygren, Svanvik et al. 1998). In PCR the fluorescence of these dyes increases with the amount of double stranded product formed, though not strictly in proportion because the dye fluorescence depends on the dye: base binding ratio, which decreases during the course of the reaction. The dye fluorescence depends also to some degree on the DNA sequence. But a certain amount of a particular double-stranded DNA target, in the absence of significant amounts of other double-stranded DNAs, gives rise to the same fluorescence every time. Hence, the dyes are excellent for quantitative real-time PCR when samples are compared at the same level of fluorescence in absence of interfering DNA. Although minor groove binding dyes show preference for runs of AT base-pairs (Jansen, Norde´n et al. 1993), asymmetric cyanines are considered sequence non-specific reporters in real-time PCR. They give rise to fluorescence signal in the presence of any double stranded DNA including undesired primer–dimer products. Primer–dimer formation interferes with the formation of specific products because of competition of the two reactions for reagents and may lead to erroneous readouts. It is therefore good practice to control for primer–dimer formation. This can be done by melting curve analysis after completing the PCR. The temperature is then gradually increased and the fluorescence is measured as function of temperature. The fluorescence decreases gradually with increasing temperature because of increased thermal motion which allows for more internal rotation in the bound dye (Nygren, Svanvik et al. 1998). However, when the temperature is reached at which the double stranded DNA strand separates the dye comes off and the fluorescence drops abruptly (Ririe, Rasmussen et al. 1997). This temperature, referred to as the melting temperature, Tm, is easiest determined as the maximum of the negative first derivative of the melting curve. Since primer–dimer products typically are shorter than the targeted product, they melt at a lower temperature and their

presence is easily recognized by melting curve analysis (Kubista, Andrade et al. 2006).

hence, the fluorescence of the system (Kubista, Andrade et al. 2006).

Labeled primers and probes are based on nucleic acids or some of their synthetic analogues such as the peptide nucleic acids (PNA) (Egholm, Buchardt et al. 1992) and the locked nucleic acids (LNA) (Costa, Ernault et al. 2004). The dye labels are of two kinds: (i) fluorophores with intrinsically strong fluorescence, such as fluorescein and rhodamine derivatives (Sjöback, Nygren et al. 1995), which through structural design are brought into contact with a quencher molecule, and (ii) fluorophores that change their fluorescence properties upon binding nucleic acids. Examples of probes with two dyes are the hydrolysis probes, popularly called Taqman probes (Holland, Abramson et al. 1991), which can be based either on regular oligonucleotides or on LNA (Braasch and Corey 2001), Molecular Beacons (Tyagi and Kramer 1996; Tyagi, Bratu et al. 1998), Hybridization probes (Caplin, Rasmussen et al. 1999), and the Lion probes (http://www.biotools.net). The dyes form a donor–acceptor pair, where the donor dye is excited and transfers its energy to the acceptor molecule if it is in proximity. Originally the acceptor molecule was also a dye, but today quencher molecules are more popular (Wilson and Johansson 2003). Energy transfer and quenching are distance dependent and structural rearrangement of the probe, or, in the case of hydrolysis probes, degradation, change the distance between the donor and acceptor and,

Probes based on a single dye, whose fluorescence changes upon binding target DNA include the LightUp probes (Svanvik, Westman et al. 2000), AllGlo probes

technology quickly matured into a competitive market, becoming commercially widespread and scientically inuential (Valasek and Repa 2005).

Real-time PCR instrumentation was rst made commercially available by Applied Biosystems in 1996, after which several other companies added new machines to the market. Presently, Applied Biosystems, BioGene, Bioneer, Bio-Rad, Cepheid, Corbett Research, Idaho Technology, MJ Research, Roche Applied Science, and Stratagene all offer instrumentation lines for real-time PCR (BioInformatics 2003).

Widespread use has also resulted in a multiplicity of names for the technology, each with a different shade of meaning. Real-time PCR simply refers to amplication of DNA (by PCR) that ismonitored while the amplication is occurring. The benet of this real-time capability is that it allows the researcher to better determine the amount of starting DNA in the sample before the amplication by PCR. Present day real-time methods generally involve uorogenic probes that "light up" to show the amount of DNA present at each cycle of PCR. "Kinetic PCR" refers to this process as well. "Quantitative PCR" refers to the ability to quantify the starting amount of a specic sequence of DNA. This term predates real-time PCR because it can refer to any PCR procedure, including earlier gel-based end-point assays, that attempts to quantify the starting amount of nucleic acid. Rarely, one might see the term "quantitative uorescent PCR" to designate that the quantication was accomplished via measuring output from a uorogenic probe, although this is redundant because all of the present chemistries for real-time PCR are uorescent. In addition, if reverse transcriptase enzymes are used before PCR amplication in any of the above situations, then "RT-PCR" replaces "PCR" in the term. Today, the two most common terms, real-time and quantitative, are often used interchangeably or in combination, because real-time PCR is quickly becoming the method of choice to quantify nucleic acids (Valasek and Repa 2005).

The basic goal of real-time PCR is to precisely distinguish and measure specic nucleic acid sequences in a sample even if there is only a very small quantity. Real-time PCR amplies a specic target sequence ina sample then monitors theamplication progress using uorescent technology. During amplication, how quickly the uorescent signal reaches a threshold level correlates with the amount of original target sequence, thereby enabling quantication. In addition, the nal product can be further characterized by subjecting it to increasing temperatures to determine when the double-stranded product "melts." This melting point is a unique property dependent on product length and nucleotide composition. To accomplish these tasks, conventional PCR has been coupled to state-of-the-art uorescent chemistries and instrumentation to becomereal-time PCR (Valasek and Repa 2005).

## **2. The chemistries of real-time PCR**

Today fluorescence is exclusively used as the detection method in real-time PCR. Both sequence specific probes and non-specific labels are available as reporters. In his initial work Higuchi used the common nucleic acid stain ethidium bromide, which becomes fluorescent upon intercalating into DNA (Higuchi, Dollinger et al. 1992). Classical intercalators, however, interfere with the polymerase reaction, and asymmetric cyanine dyes such as SYBR Green I and BEBO have become more popular (Bengtsson, Karlsson et al. 2003; Zipper, Brunner et al. 2004). Asymmetric cyanines have two aromatic systems containing nitrogen, one of which is positively charged, connected by amethine bridge. These dyes have virtually no fluorescence when they are free in solution due to vibrations engaging

technology quickly matured into a competitive market, becoming commercially widespread

Real-time PCR instrumentation was rst made commercially available by Applied Biosystems in 1996, after which several other companies added new machines to the market. Presently, Applied Biosystems, BioGene, Bioneer, Bio-Rad, Cepheid, Corbett Research, Idaho Technology, MJ Research, Roche Applied Science, and Stratagene all offer

Widespread use has also resulted in a multiplicity of names for the technology, each with a different shade of meaning. Real-time PCR simply refers to amplication of DNA (by PCR) that ismonitored while the amplication is occurring. The benet of this real-time capability is that it allows the researcher to better determine the amount of starting DNA in the sample before the amplication by PCR. Present day real-time methods generally involve uorogenic probes that "light up" to show the amount of DNA present at each cycle of PCR. "Kinetic PCR" refers to this process as well. "Quantitative PCR" refers to the ability to quantify the starting amount of a specic sequence of DNA. This term predates real-time PCR because it can refer to any PCR procedure, including earlier gel-based end-point assays, that attempts to quantify the starting amount of nucleic acid. Rarely, one might see the term "quantitative uorescent PCR" to designate that the quantication was accomplished via measuring output from a uorogenic probe, although this is redundant because all of the present chemistries for real-time PCR are uorescent. In addition, if reverse transcriptase enzymes are used before PCR amplication in any of the above situations, then "RT-PCR" replaces "PCR" in the term. Today, the two most common terms, real-time and quantitative, are often used interchangeably or in combination, because real-time PCR is quickly becoming

The basic goal of real-time PCR is to precisely distinguish and measure specic nucleic acid sequences in a sample even if there is only a very small quantity. Real-time PCR amplies a specic target sequence ina sample then monitors theamplication progress using uorescent technology. During amplication, how quickly the uorescent signal reaches a threshold level correlates with the amount of original target sequence, thereby enabling quantication. In addition, the nal product can be further characterized by subjecting it to increasing temperatures to determine when the double-stranded product "melts." This melting point is a unique property dependent on product length and nucleotide composition. To accomplish these tasks, conventional PCR has been coupled to state-of-the-art uorescent

Today fluorescence is exclusively used as the detection method in real-time PCR. Both sequence specific probes and non-specific labels are available as reporters. In his initial work Higuchi used the common nucleic acid stain ethidium bromide, which becomes fluorescent upon intercalating into DNA (Higuchi, Dollinger et al. 1992). Classical intercalators, however, interfere with the polymerase reaction, and asymmetric cyanine dyes such as SYBR Green I and BEBO have become more popular (Bengtsson, Karlsson et al. 2003; Zipper, Brunner et al. 2004). Asymmetric cyanines have two aromatic systems containing nitrogen, one of which is positively charged, connected by amethine bridge. These dyes have virtually no fluorescence when they are free in solution due to vibrations engaging

chemistries and instrumentation to becomereal-time PCR (Valasek and Repa 2005).

**2. The chemistries of real-time PCR** 

and scientically inuential (Valasek and Repa 2005).

instrumentation lines for real-time PCR (BioInformatics 2003).

the method of choice to quantify nucleic acids (Valasek and Repa 2005).

both aromatic systems, which convert electronic excitation energy into heat that dissipates to the surrounding solvent. On the other hand the dyes become brightly fluorescent when they bind to DNA, presumably to the minor groove, and rotation around the methine bond is restricted (Nygren, Svanvik et al. 1998). In PCR the fluorescence of these dyes increases with the amount of double stranded product formed, though not strictly in proportion because the dye fluorescence depends on the dye: base binding ratio, which decreases during the course of the reaction. The dye fluorescence depends also to some degree on the DNA sequence. But a certain amount of a particular double-stranded DNA target, in the absence of significant amounts of other double-stranded DNAs, gives rise to the same fluorescence every time. Hence, the dyes are excellent for quantitative real-time PCR when samples are compared at the same level of fluorescence in absence of interfering DNA. Although minor groove binding dyes show preference for runs of AT base-pairs (Jansen, Norde´n et al. 1993), asymmetric cyanines are considered sequence non-specific reporters in real-time PCR. They give rise to fluorescence signal in the presence of any double stranded DNA including undesired primer–dimer products. Primer–dimer formation interferes with the formation of specific products because of competition of the two reactions for reagents and may lead to erroneous readouts. It is therefore good practice to control for primer–dimer formation. This can be done by melting curve analysis after completing the PCR. The temperature is then gradually increased and the fluorescence is measured as function of temperature. The fluorescence decreases gradually with increasing temperature because of increased thermal motion which allows for more internal rotation in the bound dye (Nygren, Svanvik et al. 1998). However, when the temperature is reached at which the double stranded DNA strand separates the dye comes off and the fluorescence drops abruptly (Ririe, Rasmussen et al. 1997). This temperature, referred to as the melting temperature, Tm, is easiest determined as the maximum of the negative first derivative of the melting curve. Since primer–dimer products typically are shorter than the targeted product, they melt at a lower temperature and their presence is easily recognized by melting curve analysis (Kubista, Andrade et al. 2006).

Labeled primers and probes are based on nucleic acids or some of their synthetic analogues such as the peptide nucleic acids (PNA) (Egholm, Buchardt et al. 1992) and the locked nucleic acids (LNA) (Costa, Ernault et al. 2004). The dye labels are of two kinds: (i) fluorophores with intrinsically strong fluorescence, such as fluorescein and rhodamine derivatives (Sjöback, Nygren et al. 1995), which through structural design are brought into contact with a quencher molecule, and (ii) fluorophores that change their fluorescence properties upon binding nucleic acids. Examples of probes with two dyes are the hydrolysis probes, popularly called Taqman probes (Holland, Abramson et al. 1991), which can be based either on regular oligonucleotides or on LNA (Braasch and Corey 2001), Molecular Beacons (Tyagi and Kramer 1996; Tyagi, Bratu et al. 1998), Hybridization probes (Caplin, Rasmussen et al. 1999), and the Lion probes (http://www.biotools.net). The dyes form a donor–acceptor pair, where the donor dye is excited and transfers its energy to the acceptor molecule if it is in proximity. Originally the acceptor molecule was also a dye, but today quencher molecules are more popular (Wilson and Johansson 2003). Energy transfer and quenching are distance dependent and structural rearrangement of the probe, or, in the case of hydrolysis probes, degradation, change the distance between the donor and acceptor and, hence, the fluorescence of the system (Kubista, Andrade et al. 2006).

Probes based on a single dye, whose fluorescence changes upon binding target DNA include the LightUp probes (Svanvik, Westman et al. 2000), AllGlo probes

Overview of Real-Time PCR Principles 409

necessary to obtain reasonable amplification efficiencies. But they are easier to design than hairpin forming probes and an 80% success rate was recently reported (Kubista 2004).

In summary, a 'good'probe, independent of chemistry, should have low background fluorescence, high fluorescence upon target formation (high signal to noise ratio), and high target specificity. The dyes'excitation and emission spectra are important parameters to consider when designing multiplex reactions. Spectral overlap in excitation and emission

SYBR green I binds to the minor groove of dsDNA, emitting 1,000-fold greater uorescence than when it is free in solution (Wittwer, Herrmann et al. 1997). Therefore, the greater the amount of dsDNA present in the reaction tube, the greater the amount of DNA binding and uorescent signal from SYBR green I. Thus any amplication of DNA in the reaction tube is

The minor groove binding asymmetric cyanine dye BEBO is tested as sequence nonspecific label in real-time PCR. The Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 mM generated sufficient Fluorescence signal without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects. The dye has absorbance and emission wavelengths that can be detected on the FAM channel on most common real-time PCR platforms, and shows a strong fluorescence increase when bound to dsDNA. BEBO can be used as an unspecific dye for real-time PCR applications or other applications where staining of dsDNA is wanted (Bengtsson, Karlsson et al. 2003).

The unsymmetrical cyanine dyes BOXTO and its positive divalent derivative BOXTO-PRO were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition, sufficient fluorescent signal was acquired when 0.5–1.0 µM BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO

Hydrolysis probes (also called 5/-nuclease probes because the 5/-exonuclease activity of DNA polymerase cleaves the probe) offer an alternative approach to the problem of specicity. These are likely the most widely used uorogenic probe format (Mackay 2004) and are exemplied by TaqMan probes. In terms of structure, hydrolysis probes are sequence- specic dually uorophore-labeled DNA oligonucleotides (Valasek and Repa 2005). One uorophore is termed the quencher and the other is the reporter. When the quencher and reporter are in close proximity, that is, they are both attached to the same

should be minimized to keep cross-talk to a minimum (Kubista, Andrade et al. 2006).

**2.1 SYBR green I** 

**2.2 BEBO** 

**2.3 BOXTO** 

and SG (Ahmad 2007).

**2.4 5' nuclease (TaqMan) probes** 

measured (Valasek and Repa 2005).

(http://www.allelogic.com), Displacement probes (Li, Qingge et al. 2002), and the Simple probes (http://www.idahotech.com/itbiochem/simpleprobes.html).

Chemical modifications and alterations of the oligonucleotide backbone are employed in some probes to improve the binding properties to the target template. This makes it possible to use shorter probes, which is advantageous for the detection of targets with short conserved regions such as retroviruses. LightUp probes have a neutral peptide nucleic acid (PNA) backbone that binds to DNA with greater affinity than normal oligonucleotides (Kubista, Andrade et al. 2006).

The LightUp probes are 10–12 bases, which is short compared to normal oligonucleotide probes that are usually at least 25 bases (http://www.lightup.se). LNA-probes make use of modified nucleotides to enhance binding affinity. MGBprobes are hydrolysis probes with a minor groove binding molecule attached to the end of the probe to increase affinity for DNA, which makes it possible to use shorter probes (Kutyavin, Afonina et al. 2000). Examples of modified primers include: Scorpion primers (Whitcombe, Theaker et al. 1999), LUX primers (Nazarenko, Lowe et al. 2002), Ampliflour primers (Uehara, Nardone et al. 1999), and the QZyme system (BD QZymeTM Assays for Quantitative PCR, 2003). As long as a single target is detected per sample there is not much of a difference in using a dye or a probe. Assay specificity is in both cases determined by the primers. Probes do not detect primer–dimer products, but using non-optimized probe assays is hiding the problem under the rug. If primer–dimers form they cause problems whether they are seen or not. In probe based assays, particularly when high CT values are obtained, one should verify the absence of competing primer–dimer products (Kubista, Andrade et al. 2006). The traditional way is by gel electrophoresis. Recently, an alternative approach was proposed based on the BOXTO dye. BOXTO is a sequence non-specific doublestranded DNA binding dye that has distinct spectral characteristics to fluorescein and can be used in combination with FAM based probes. The BOXTO and the probe signals are detected in different channels of the real-time PCR instrument. While the probe reflects formation of the targeted product as usual, the BOXTO dye also reports the presence of any competing primer–dimer products, which can be identified by melting curve analysis (Lind, Stahlberg et al. 2006). The great advantage of probes is for multiplexing, where several products are amplified in the same tube and detected in parallel (Wittwer, Herrmann et al. 2001). Today multiplexing is mainly used to relate expression of reporter genes to that of an exogenous control gene in diagnostic applications (Mackya 2004), and for single nucleotide polymorphism (SNP) and mutation detection studies (Mhlanga and Malmberg 2001; Mattarucchi, Marsoni et al. 2005). Multiplex assays are more difficult to design because when products accumulate the parallel PCR reactions compete for reagents. To minimize competition limiting amounts of primers must be used. Also, primer design is harder, because complementarity must be avoided between all the primers. Multiplex assays can be based either on probes or on labeled primers, where labeled primers usually give rise to signal from primer–dimer products, while probes do not. The different probing technologies have their advantages and limitations. Dyes are cheaper than probes but they do not distinguish between products. Hairpin forming probes have the highest specificity, because the formation of the hairpin competes with the binding to mismatched targets. This makes them most suitable for SNP and multi-site variation (MSV) analysis (Bonnet, Tyagi et al. 1999). Hydrolysis probes require two-step PCR to function properly, which is not optimal for the polymerase reaction, and short amplicons are necessary to obtain reasonable amplification efficiencies. But they are easier to design than hairpin forming probes and an 80% success rate was recently reported (Kubista 2004).

In summary, a 'good'probe, independent of chemistry, should have low background fluorescence, high fluorescence upon target formation (high signal to noise ratio), and high target specificity. The dyes'excitation and emission spectra are important parameters to consider when designing multiplex reactions. Spectral overlap in excitation and emission should be minimized to keep cross-talk to a minimum (Kubista, Andrade et al. 2006).

## **2.1 SYBR green I**

408 Polymerase Chain Reaction

(http://www.allelogic.com), Displacement probes (Li, Qingge et al. 2002), and the Simple

Chemical modifications and alterations of the oligonucleotide backbone are employed in some probes to improve the binding properties to the target template. This makes it possible to use shorter probes, which is advantageous for the detection of targets with short conserved regions such as retroviruses. LightUp probes have a neutral peptide nucleic acid (PNA) backbone that binds to DNA with greater affinity than normal oligonucleotides

The LightUp probes are 10–12 bases, which is short compared to normal oligonucleotide probes that are usually at least 25 bases (http://www.lightup.se). LNA-probes make use of modified nucleotides to enhance binding affinity. MGBprobes are hydrolysis probes with a minor groove binding molecule attached to the end of the probe to increase affinity for DNA, which makes it possible to use shorter probes (Kutyavin, Afonina et al. 2000). Examples of modified primers include: Scorpion primers (Whitcombe, Theaker et al. 1999), LUX primers (Nazarenko, Lowe et al. 2002), Ampliflour primers (Uehara, Nardone et al. 1999), and the QZyme system (BD QZymeTM Assays for Quantitative PCR, 2003). As long as a single target is detected per sample there is not much of a difference in using a dye or a probe. Assay specificity is in both cases determined by the primers. Probes do not detect primer–dimer products, but using non-optimized probe assays is hiding the problem under the rug. If primer–dimers form they cause problems whether they are seen or not. In probe based assays, particularly when high CT values are obtained, one should verify the absence of competing primer–dimer products (Kubista, Andrade et al. 2006). The traditional way is by gel electrophoresis. Recently, an alternative approach was proposed based on the BOXTO dye. BOXTO is a sequence non-specific doublestranded DNA binding dye that has distinct spectral characteristics to fluorescein and can be used in combination with FAM based probes. The BOXTO and the probe signals are detected in different channels of the real-time PCR instrument. While the probe reflects formation of the targeted product as usual, the BOXTO dye also reports the presence of any competing primer–dimer products, which can be identified by melting curve analysis (Lind, Stahlberg et al. 2006). The great advantage of probes is for multiplexing, where several products are amplified in the same tube and detected in parallel (Wittwer, Herrmann et al. 2001). Today multiplexing is mainly used to relate expression of reporter genes to that of an exogenous control gene in diagnostic applications (Mackya 2004), and for single nucleotide polymorphism (SNP) and mutation detection studies (Mhlanga and Malmberg 2001; Mattarucchi, Marsoni et al. 2005). Multiplex assays are more difficult to design because when products accumulate the parallel PCR reactions compete for reagents. To minimize competition limiting amounts of primers must be used. Also, primer design is harder, because complementarity must be avoided between all the primers. Multiplex assays can be based either on probes or on labeled primers, where labeled primers usually give rise to signal from primer–dimer products, while probes do not. The different probing technologies have their advantages and limitations. Dyes are cheaper than probes but they do not distinguish between products. Hairpin forming probes have the highest specificity, because the formation of the hairpin competes with the binding to mismatched targets. This makes them most suitable for SNP and multi-site variation (MSV) analysis (Bonnet, Tyagi et al. 1999). Hydrolysis probes require two-step PCR to function properly, which is not optimal for the polymerase reaction, and short amplicons are

probes (http://www.idahotech.com/itbiochem/simpleprobes.html).

(Kubista, Andrade et al. 2006).

SYBR green I binds to the minor groove of dsDNA, emitting 1,000-fold greater uorescence than when it is free in solution (Wittwer, Herrmann et al. 1997). Therefore, the greater the amount of dsDNA present in the reaction tube, the greater the amount of DNA binding and uorescent signal from SYBR green I. Thus any amplication of DNA in the reaction tube is measured (Valasek and Repa 2005).

## **2.2 BEBO**

The minor groove binding asymmetric cyanine dye BEBO is tested as sequence nonspecific label in real-time PCR. The Fluorescence intensity of BEBO increases upon binding to double-stranded DNA allowing emission to be measured at the end of the elongation phase in the PCR cycle. BEBO concentrations between 0.1 and 0.4 mM generated sufficient Fluorescence signal without inhibiting the PCR. A comparison with the commonly used reporter dye SYBR Green I shows that the two dyes behave similarly in all important aspects. The dye has absorbance and emission wavelengths that can be detected on the FAM channel on most common real-time PCR platforms, and shows a strong fluorescence increase when bound to dsDNA. BEBO can be used as an unspecific dye for real-time PCR applications or other applications where staining of dsDNA is wanted (Bengtsson, Karlsson et al. 2003).

## **2.3 BOXTO**

The unsymmetrical cyanine dyes BOXTO and its positive divalent derivative BOXTO-PRO were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition, sufficient fluorescent signal was acquired when 0.5–1.0 µM BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG (Ahmad 2007).

#### **2.4 5' nuclease (TaqMan) probes**

Hydrolysis probes (also called 5/-nuclease probes because the 5/-exonuclease activity of DNA polymerase cleaves the probe) offer an alternative approach to the problem of specicity. These are likely the most widely used uorogenic probe format (Mackay 2004) and are exemplied by TaqMan probes. In terms of structure, hydrolysis probes are sequence- specic dually uorophore-labeled DNA oligonucleotides (Valasek and Repa 2005). One uorophore is termed the quencher and the other is the reporter. When the quencher and reporter are in close proximity, that is, they are both attached to the same

Overview of Real-Time PCR Principles 411

wavelength and this third wavelength is detected. If the two dyes do not align together because there is no specific DNA for them to bind, then FRET does not occur between the two dyes because the distances between the dyes are too great. A design detail of FRET hybridization probes is the 3' end of the second (downstream) probe is phosphorylated to prevent it from being used as a primer by Taq during PCR amplification. The two probes encompass a region of 40 to 50 DNA base pairs, providing exquisite specificity (Espy, Uhl et al. 2006). FRET hybridization probe technology permits melting curve analysis of the amplification product. If the temperature is slowly raised, eventually the probes will no longer be able to anneal to the target PCR product and the FRET signal will be lost. The temperature at which half the FRET signal is lost is referred to as the melting temperature of the probe system (Espy, Uhl et al. 2006). The Tm depends on the guanine plus cytosine content and oligonucleotide length. In contrast to TaqMan probes, a single nucleotide polymorphism in the target DNA under a hybridization FRET probe will still generate a signal, but the melting curve will display a lower Tm. The lowered Tm can be characteristic for a specific polymorphism underneath the probes; however, a lowered Tm can also be the result of any sequence difference under the probes. The target PCR product is detected and the altered Tm informs the user there is a difference in the sequence being detected. Generally, more than three base pair differences under a FRET hybridization probe prevent hybridization at typical annealing temperatures and are not detected (Espy, Uhl et al. 2006). This trait of FRET hybridization probes is advantageous in cases where the genome of the organism is known to mutate at a high frequency, such as with viruses. When a single or limited number (<3) of known polymorphisms occur between two similar targets, FRET hybridization probes can also be used for discriminating strains of organisms (Espy, Uhl et al. 2006). Like molecular beacons, there is not a specific thermocycling temperature requirement for FRET hybridization probes. Molecular beacons and FRET hybridization probes, unlike TaqMan probes, are both recycled (conserved) in each round of PCR temperature cycle. Also, for Molecular beacons and FRET hybridization probes, unlike TaqMan probes, fluorescent signal does not accumulate as PCR product accumulates after

Scorpions combine the detection probe with the upstream PCR primer (Whitcombe, Theaker et al. 1999) and consist of a fluorophore on the 5′ end, followed by a complementary stemloop structure (also containing the specific probe sequence), quencher dye, DNA polymerase blocker (a nonamplifiable monomer that prevents DNA polymerase extension), and finally a PCR primer on the 3′ end. The probe sequence contained within the hairpin allows the scorpion to anneal to the template strand, which separates the quencher for the fluorophore and results in increased fluorescence. Because sequence-specific priming and probing is a unimolecular event, scorpions perform better than bimolecular methods under conditions of rapid cycling such as the LightCycler (Thelwell, Millington et al. 2000). Cycling is performed at a temperature optimal for DNA polymerase activity instead of the reduced temperature necessary for the 5′ nuclease assay. Scorpions are specific enough for allele discrimination and may be multiplexed easily (Thelwell, Millington et al. 2000). The scorpion chemistry has been improved with the creation of duplex scorpions in which the reporter dye/probe and quencher fragment are on separate, complementary molecules (Solinas, Brown et al. 2001). The duplex scorpions still bind in a unimolecular event, but because the reporter and quenchers are on separate molecules, they yield greater signal

each PCR cycle (Espy, Uhl et al. 2006).

**2.7 Scorpions** 

short oligonucleotide; the quencher absorbs the signal from the reporter (Valasek and Repa 2005). This is an example of uorescence resonance energy transfer (also called Forster transfer) in which energy is transferred from a "donor" (the reporter) to an "acceptor" (the quencher) uorophore. During amplication, the oligonucleotide is broken apart by the action of DNA polymerase (5/-nuclease activity) and the reporter and quencher separate, allowing the reporter's energy and uorescent signal to be liberated. Thus destruction or hydrolysis of the oligonucleotide results in an increase of reporter signal and corresponds with the specic amplication of DNA (Valasek and Repa 2005). Examples of common quencher uorophores include TAMRA, DABCYL, and BHQ, whereas reporters are more numerous (e.g., FAM, VIC, NED, etc). Hydrolysis probes afford similar precision as SYBR green I (Wilhelm, Pingoud et al. 2003), but they give greater insurance regarding the specicity because only sequence-specic amplication is measured. In addition, hydrolysis probes allow for simple identication of point mutations within the amplicon using melting curve analysis (Valasek and Repa 2005).

#### **2.5 Molecular beacons**

Molecular beacons are similar to TaqMan probes but are not designed to be cleaved by the 5' nuclease activity of Taq polymerase. These probes have a fluorescent dye on the 5' end and a quencher dye on the 3' end of the oligonucleotide probe. A region at each end of the molecular beacon probe is designed to be complementary to itself, so at low temperatures, the ends anneal, creating a hairpin structure. This integral annealing property positions the two dyes in close proximity, quenching the fluorescence from the reporter dye (Espy, Uhl et al. 2006). The central region of the probe is designed to be complementary to a region of the PCR amplification product. At high temperatures, both the PCR amplification product and probe are single stranded. As the temperature of the PCR is lowered, the central region of the molecular beacon probe binds to the PCR product and forces the separation of the fluorescent reporter dye from the quenching dye. The effects of the quencher dye are obviated and a light signal from the reporter dye can be detected. If no PCR amplification product is available for binding, the probe reanneals to itself, forcing the reporter dye and quencher dye together, preventing fluorescent signal (Espy, Uhl et al. 2006). Typically, a single molecular beacon is used for detection of a PCR amplification product and multiple beacon probes with different reporter dyes are used for single nucleotide polymorphism detection. By selection of appropriate PCR temperatures and/or extension of the probe length, molecular beacons will bind to the target PCR product when an unknown nucleotide polymorphism is present but at a slight cost of reduced specificity. There is not a specific temperature thermocycling requirement for molecular beacons, so temperature optimization of the PCR is simplified (Espy, Uhl et al. 2006).

#### **2.6 FRET hybridization probes**

FRET hybridization probes, also referred to as LightCyclerprobes; represent a third type of probe detection format commonly used with real-time PCR testing platforms. FRET hybridization probes are two DNA probes designed to anneal next to each other in a headto-tail configuration on the PCR product. The upstream probe has a fluorescent dye on the 3' end and the downstream probe has an acceptor dye on the 5' end. If both probes anneal to the target PCR product, fluorescence from the 3' dye is absorbed by the adjacent acceptor dye on the 5' end of the second probe. The second dye is excited and emits light at a third

short oligonucleotide; the quencher absorbs the signal from the reporter (Valasek and Repa 2005). This is an example of uorescence resonance energy transfer (also called Forster transfer) in which energy is transferred from a "donor" (the reporter) to an "acceptor" (the quencher) uorophore. During amplication, the oligonucleotide is broken apart by the action of DNA polymerase (5/-nuclease activity) and the reporter and quencher separate, allowing the reporter's energy and uorescent signal to be liberated. Thus destruction or hydrolysis of the oligonucleotide results in an increase of reporter signal and corresponds with the specic amplication of DNA (Valasek and Repa 2005). Examples of common quencher uorophores include TAMRA, DABCYL, and BHQ, whereas reporters are more numerous (e.g., FAM, VIC, NED, etc). Hydrolysis probes afford similar precision as SYBR green I (Wilhelm, Pingoud et al. 2003), but they give greater insurance regarding the specicity because only sequence-specic amplication is measured. In addition, hydrolysis probes allow for simple identication of point mutations within the amplicon using melting

Molecular beacons are similar to TaqMan probes but are not designed to be cleaved by the 5' nuclease activity of Taq polymerase. These probes have a fluorescent dye on the 5' end and a quencher dye on the 3' end of the oligonucleotide probe. A region at each end of the molecular beacon probe is designed to be complementary to itself, so at low temperatures, the ends anneal, creating a hairpin structure. This integral annealing property positions the two dyes in close proximity, quenching the fluorescence from the reporter dye (Espy, Uhl et al. 2006). The central region of the probe is designed to be complementary to a region of the PCR amplification product. At high temperatures, both the PCR amplification product and probe are single stranded. As the temperature of the PCR is lowered, the central region of the molecular beacon probe binds to the PCR product and forces the separation of the fluorescent reporter dye from the quenching dye. The effects of the quencher dye are obviated and a light signal from the reporter dye can be detected. If no PCR amplification product is available for binding, the probe reanneals to itself, forcing the reporter dye and quencher dye together, preventing fluorescent signal (Espy, Uhl et al. 2006). Typically, a single molecular beacon is used for detection of a PCR amplification product and multiple beacon probes with different reporter dyes are used for single nucleotide polymorphism detection. By selection of appropriate PCR temperatures and/or extension of the probe length, molecular beacons will bind to the target PCR product when an unknown nucleotide polymorphism is present but at a slight cost of reduced specificity. There is not a specific temperature thermocycling requirement for molecular beacons, so temperature optimization

FRET hybridization probes, also referred to as LightCyclerprobes; represent a third type of probe detection format commonly used with real-time PCR testing platforms. FRET hybridization probes are two DNA probes designed to anneal next to each other in a headto-tail configuration on the PCR product. The upstream probe has a fluorescent dye on the 3' end and the downstream probe has an acceptor dye on the 5' end. If both probes anneal to the target PCR product, fluorescence from the 3' dye is absorbed by the adjacent acceptor dye on the 5' end of the second probe. The second dye is excited and emits light at a third

curve analysis (Valasek and Repa 2005).

of the PCR is simplified (Espy, Uhl et al. 2006).

**2.6 FRET hybridization probes** 

**2.5 Molecular beacons** 

wavelength and this third wavelength is detected. If the two dyes do not align together because there is no specific DNA for them to bind, then FRET does not occur between the two dyes because the distances between the dyes are too great. A design detail of FRET hybridization probes is the 3' end of the second (downstream) probe is phosphorylated to prevent it from being used as a primer by Taq during PCR amplification. The two probes encompass a region of 40 to 50 DNA base pairs, providing exquisite specificity (Espy, Uhl et al. 2006). FRET hybridization probe technology permits melting curve analysis of the amplification product. If the temperature is slowly raised, eventually the probes will no longer be able to anneal to the target PCR product and the FRET signal will be lost. The temperature at which half the FRET signal is lost is referred to as the melting temperature of the probe system (Espy, Uhl et al. 2006). The Tm depends on the guanine plus cytosine content and oligonucleotide length. In contrast to TaqMan probes, a single nucleotide polymorphism in the target DNA under a hybridization FRET probe will still generate a signal, but the melting curve will display a lower Tm. The lowered Tm can be characteristic for a specific polymorphism underneath the probes; however, a lowered Tm can also be the result of any sequence difference under the probes. The target PCR product is detected and the altered Tm informs the user there is a difference in the sequence being detected. Generally, more than three base pair differences under a FRET hybridization probe prevent hybridization at typical annealing temperatures and are not detected (Espy, Uhl et al. 2006). This trait of FRET hybridization probes is advantageous in cases where the genome of the organism is known to mutate at a high frequency, such as with viruses. When a single or limited number (<3) of known polymorphisms occur between two similar targets, FRET hybridization probes can also be used for discriminating strains of organisms (Espy, Uhl et al. 2006). Like molecular beacons, there is not a specific thermocycling temperature requirement for FRET hybridization probes. Molecular beacons and FRET hybridization probes, unlike TaqMan probes, are both recycled (conserved) in each round of PCR temperature cycle. Also, for Molecular beacons and FRET hybridization probes, unlike TaqMan probes, fluorescent signal does not accumulate as PCR product accumulates after each PCR cycle (Espy, Uhl et al. 2006).

#### **2.7 Scorpions**

Scorpions combine the detection probe with the upstream PCR primer (Whitcombe, Theaker et al. 1999) and consist of a fluorophore on the 5′ end, followed by a complementary stemloop structure (also containing the specific probe sequence), quencher dye, DNA polymerase blocker (a nonamplifiable monomer that prevents DNA polymerase extension), and finally a PCR primer on the 3′ end. The probe sequence contained within the hairpin allows the scorpion to anneal to the template strand, which separates the quencher for the fluorophore and results in increased fluorescence. Because sequence-specific priming and probing is a unimolecular event, scorpions perform better than bimolecular methods under conditions of rapid cycling such as the LightCycler (Thelwell, Millington et al. 2000). Cycling is performed at a temperature optimal for DNA polymerase activity instead of the reduced temperature necessary for the 5′ nuclease assay. Scorpions are specific enough for allele discrimination and may be multiplexed easily (Thelwell, Millington et al. 2000). The scorpion chemistry has been improved with the creation of duplex scorpions in which the reporter dye/probe and quencher fragment are on separate, complementary molecules (Solinas, Brown et al. 2001). The duplex scorpions still bind in a unimolecular event, but because the reporter and quenchers are on separate molecules, they yield greater signal

Overview of Real-Time PCR Principles 413

step of PCR, the probe hybridizes to the target with the help of the minor groove binder. The probe thus becomes linearized, separating the reporter and quencher and allowing the reporter to fluoresce. The resulting fluorescent signal is proportional to the amount of

qPCR assays using Amplifluor chemistry employ two target-specific primers and one universal primer called the UniPrimer. The first target-specific primer contains a 5' extension sequence called the Z-sequence that is also found at the 3' end of the UniPrimer. The UniPrimer forms a hairpin structure (Bio-Rad Laboratories 2006). A fluorescent reporter and a quencher are attached at the 5' and the 3' ends of the stem structure, respectively. In the hairpin conformation, the reporter fluorescence is quenched due to its proximity to the quencher. During the first amplification cycle, the first target-specific primer (with the Zsequence) hybridizes to the template and is extended. During the second amplification cycle, the second target-specific primer is used to synthesize a new target template that contains a sequence complementary to the Z-sequence. The product from the second amplification cycle can then serve as the template for the UniPrimer. In the third amplification cycle, the extended UniPrimer serves as a template for the next amplification cycle (Bio-Rad Laboratories 2006). In the fourth cycle, extension of the template through the hairpin region of the UniPrimer causes the UniPrimer to open up and adopt a linear configuration, which allows the reporter to fluoresce. Exponential amplification using the second target-specific primer and the UniPrimer occurs in subsequent amplification cycles. The resulting fluorescent signal is proportional to

amplified product in the sample (Bio-Rad Laboratories 2006).

the amount of amplified product in the sample (Bio-Rad Laboratories 2006).

**3. Design and optimization of SYBR Green I reactions** 

The steps for developing a SYBR Green I assay are:

1. Primer design and amplicon design

qPCR assays using BD QZyme primers employ a target-specific zymogene primer, a targetspecific reverse primer, and a universal oligonucleotide substrate. The oligonucleotide contains a fluorescent reporter on the 5' end and a quencher on the 3' end. When oligonucleotide substrate is intact, the fluorescence of the reporter is quenched by the quencher due to their proximity (Bio-Rad Laboratories 2006). The zymogene primer contains a sequence that encodes a catalytic DNA. During the first amplification cycle, the zymogene primer is extended. In the second cycle, the product of the first cycle is used as the template by the target-specific reverse primer, which is extended to create a new target sequence containing a catalytic DNA region. In the subsequent annealing step, the fluorescently labeled oligonucleotide substrate hybridizes to the catalytic DNA sequence and is cleaved. This cleavage separates the reporter from the quencher, resulting in a fluorescent signal that is proportional to the amount of amplified product in the sample

A SYBR Green I assay uses a pair of PCR primers that amplifies a specific region within the target sequence of interest and includes SYBR Green 1 for detecting the amplified product.

**2.12 Amplifluor primer** 

**2.13 BD QZyme primer** 

(Bio-Rad Laboratories 2006).

intensity because the reporter and quencher can separate completely (Wong and Medrano 2005).

## **2.8 Sunrise™ primers**

Created by Oncor (Gaithersburg, MD, USA), Sunrise primers are similar to scorpions in that they combine both the PCR primer and detection mechanism in the same molecule (Nazarenko, Bhatnagar et al. 1997). These probes consist of a dual-labeled (reporter and quencher fluorophores) hairpin loop on the 5′ end, with the 3′ end acting as the PCR primer. When unbound, the hairpin is intact, causing reporter quenching via FRET. Upon integration into the newly formed PCR product, the reporter and quencher are held far enough apart to allow reporter emission (Wong and Medrano 2005).

## **2.9 LUX™ fluorogenic primers**

Light upon extension (LUX) primers (Invitrogen, Carlsbad, CA, USA) are self-quenched single-fluorophore labeled primers almost identical to Sunrise primers. However, rather than using a quencher fluorophore, the secondary structure of the 3′ end reduces initial fluorescence to a minimal amount (Nazarenko, Lowe et al. 2002). Because this chemistry does not require a quencher dye, it is much less expensive than dual-labeled probes. While this system relies on only two oligonucleotides for specificity, unlike the SYBR Green I platform in which a dissociation curve is used to detect erroneous amplification, no such convenient detection exists for the LUX platform. Agarose gels must be run to ensure the presence of a single PCR product, a step that is extremely important not only for the LUX primers but also for the Sunrise primers and scorpions because PCR priming and probe binding are not independent in these chemistries (Wong and Medrano 2005).

#### **2.10 Light-up probes**

Light-up probes are peptide nucleic acids (PNAs) that use thiazole orange as the fluorophor. Upon hybridisation with DNA, duplex or triplex structures are formed with increased fluorescence intensity of the fluorophor. A quencher is not required. This technique is limited by unspecific fluorescence, which increases during PCR and therefore restricts the achievable sensitivity (Isacsson, Cao et al. 2000; Svanvik, Stahlberg et al. 2000;Svanvik, Westman et al. 2000). Some other formats use the increasing quench as indicator for product accumulation (Crockett and Wittwer 2001; Kurata, Kanagawa et al. 2001). In this case, the fluorescence is quenched by a guanine residue of the PCR product. These probes are comparatively inexpensive and easy to construct; however, measurement of the decrease of a signal is problematic, especially during the early exponential phase in which only very few probes are quenched (Wilhelm and Pingoud 2003).

#### **2.11 Eclipse probe**

qPCR assays using an Eclipse probe employ two primers and a sequence-specific oligonucleotide probe. The probe is complementary to a sequence within the amplicon and contains a fluorescent reporter at the 3' end, a quencher at the 5' end, and a minor groove binder (Bio-Rad Laboratories 2006). The unhybridized probe adopts a conformation that brings the reporter and quencher together, quenching the reporter. During the annealing step of PCR, the probe hybridizes to the target with the help of the minor groove binder. The probe thus becomes linearized, separating the reporter and quencher and allowing the reporter to fluoresce. The resulting fluorescent signal is proportional to the amount of amplified product in the sample (Bio-Rad Laboratories 2006).

## **2.12 Amplifluor primer**

412 Polymerase Chain Reaction

intensity because the reporter and quencher can separate completely (Wong and Medrano

Created by Oncor (Gaithersburg, MD, USA), Sunrise primers are similar to scorpions in that they combine both the PCR primer and detection mechanism in the same molecule (Nazarenko, Bhatnagar et al. 1997). These probes consist of a dual-labeled (reporter and quencher fluorophores) hairpin loop on the 5′ end, with the 3′ end acting as the PCR primer. When unbound, the hairpin is intact, causing reporter quenching via FRET. Upon integration into the newly formed PCR product, the reporter and quencher are held far

Light upon extension (LUX) primers (Invitrogen, Carlsbad, CA, USA) are self-quenched single-fluorophore labeled primers almost identical to Sunrise primers. However, rather than using a quencher fluorophore, the secondary structure of the 3′ end reduces initial fluorescence to a minimal amount (Nazarenko, Lowe et al. 2002). Because this chemistry does not require a quencher dye, it is much less expensive than dual-labeled probes. While this system relies on only two oligonucleotides for specificity, unlike the SYBR Green I platform in which a dissociation curve is used to detect erroneous amplification, no such convenient detection exists for the LUX platform. Agarose gels must be run to ensure the presence of a single PCR product, a step that is extremely important not only for the LUX primers but also for the Sunrise primers and scorpions because PCR priming and probe

Light-up probes are peptide nucleic acids (PNAs) that use thiazole orange as the fluorophor. Upon hybridisation with DNA, duplex or triplex structures are formed with increased fluorescence intensity of the fluorophor. A quencher is not required. This technique is limited by unspecific fluorescence, which increases during PCR and therefore restricts the achievable sensitivity (Isacsson, Cao et al. 2000; Svanvik, Stahlberg et al. 2000;Svanvik, Westman et al. 2000). Some other formats use the increasing quench as indicator for product accumulation (Crockett and Wittwer 2001; Kurata, Kanagawa et al. 2001). In this case, the fluorescence is quenched by a guanine residue of the PCR product. These probes are comparatively inexpensive and easy to construct; however, measurement of the decrease of a signal is problematic, especially during the early exponential phase in which only very few

qPCR assays using an Eclipse probe employ two primers and a sequence-specific oligonucleotide probe. The probe is complementary to a sequence within the amplicon and contains a fluorescent reporter at the 3' end, a quencher at the 5' end, and a minor groove binder (Bio-Rad Laboratories 2006). The unhybridized probe adopts a conformation that brings the reporter and quencher together, quenching the reporter. During the annealing

enough apart to allow reporter emission (Wong and Medrano 2005).

binding are not independent in these chemistries (Wong and Medrano 2005).

2005).

**2.8 Sunrise™ primers** 

**2.9 LUX™ fluorogenic primers** 

**2.10 Light-up probes** 

**2.11 Eclipse probe** 

probes are quenched (Wilhelm and Pingoud 2003).

qPCR assays using Amplifluor chemistry employ two target-specific primers and one universal primer called the UniPrimer. The first target-specific primer contains a 5' extension sequence called the Z-sequence that is also found at the 3' end of the UniPrimer. The UniPrimer forms a hairpin structure (Bio-Rad Laboratories 2006). A fluorescent reporter and a quencher are attached at the 5' and the 3' ends of the stem structure, respectively. In the hairpin conformation, the reporter fluorescence is quenched due to its proximity to the quencher. During the first amplification cycle, the first target-specific primer (with the Zsequence) hybridizes to the template and is extended. During the second amplification cycle, the second target-specific primer is used to synthesize a new target template that contains a sequence complementary to the Z-sequence. The product from the second amplification cycle can then serve as the template for the UniPrimer. In the third amplification cycle, the extended UniPrimer serves as a template for the next amplification cycle (Bio-Rad Laboratories 2006). In the fourth cycle, extension of the template through the hairpin region of the UniPrimer causes the UniPrimer to open up and adopt a linear configuration, which allows the reporter to fluoresce. Exponential amplification using the second target-specific primer and the UniPrimer occurs in subsequent amplification cycles. The resulting fluorescent signal is proportional to the amount of amplified product in the sample (Bio-Rad Laboratories 2006).

### **2.13 BD QZyme primer**

qPCR assays using BD QZyme primers employ a target-specific zymogene primer, a targetspecific reverse primer, and a universal oligonucleotide substrate. The oligonucleotide contains a fluorescent reporter on the 5' end and a quencher on the 3' end. When oligonucleotide substrate is intact, the fluorescence of the reporter is quenched by the quencher due to their proximity (Bio-Rad Laboratories 2006). The zymogene primer contains a sequence that encodes a catalytic DNA. During the first amplification cycle, the zymogene primer is extended. In the second cycle, the product of the first cycle is used as the template by the target-specific reverse primer, which is extended to create a new target sequence containing a catalytic DNA region. In the subsequent annealing step, the fluorescently labeled oligonucleotide substrate hybridizes to the catalytic DNA sequence and is cleaved. This cleavage separates the reporter from the quencher, resulting in a fluorescent signal that is proportional to the amount of amplified product in the sample (Bio-Rad Laboratories 2006).

## **3. Design and optimization of SYBR Green I reactions**

A SYBR Green I assay uses a pair of PCR primers that amplifies a specific region within the target sequence of interest and includes SYBR Green 1 for detecting the amplified product. The steps for developing a SYBR Green I assay are:

1. Primer design and amplicon design

Overview of Real-Time PCR Principles 415

The optimal annealing temperature can easily be assessed on qPCR instruments that have a temperature gradient feature, such as the MiniOpticon™, MyiQ™, DNA Engine Opticon®,

A gradient feature allows you to test a range of annealing temperatures simultaneously, so

To find the optimal annealing temperature for reaction, recommend testing a range of

Because SYBR Green I binds to all dsDNA, it is necessary to check the specificity of your qPCR assay by analyzing the reaction product(s). To do this, use the melt-curve function on your real-time instrument and also run products on an agarose gel. An optimized SYBR Green I qPCR reaction should have a single peak in the melt curve, corresponding to the

Nonspecific products that may have been co-amplified with the specific product can be identified by melt-curve analysis. In this example, the specific product is the peak with a Tm of 89°C and corresponds to the upper band on the gel. The nonspecific product is the peak with a Tm of 78°C and corresponds to the lower band in the gel. By comparing the gel image with the melt curve, you can identify peaks in the melt curve that correspond to specific product, additional nonspecific bands, and primer-dimers. If nonspecific products such as primer-dimers are detected by melt-curve analysis, recommend that redesign primers (Bio-

The efficiency, reproducibility, and dynamic range of a SYBR Green I assay can be determined by constructing a standard curve using serial dilutions of a known template. The efficiency of the assay should be 90–105%, the R2 of the standard curve should be >0.980

It is important to note that the range of template concentrations used for the standard curve must encompass the entire range of template concentration of the test samples to show that results from the test samples are within the linear dynamic range of the assay. If the test samples give results outside of the range of the standard curves, one of the following must

1. Construct a wider standard curve covering the test sample concentrations and perform

2. If the test samples give a lower CT than the highest concentration of standards used in

3. If the test samples give a higher CT than the lowest concentration of standards used in the standard curve, repeat the assay using larger amounts of the test samples

Steps of to determine the performance of your SYBR Green I qPCR assay:

a. Identify the optimal annealing temperature for your assay b. Construct a standard curve to evaluate assay performance

Opticon™ 2, iCycleriQ®, Chromo4™, and iQ™5 systems.

optimization reactions can be performed in a single experiment.

**F) Assay Performance Evaluation Using Standard Curves:** 

or r > |–0.990|, and the CT values of the replicates should be similar.

analysis to ensure that the assay is linear in that new range

the standard curve, repeat the assay using diluted test samples

annealing temperatures above and below the calculated Tm of the primers.

**E) Annealing Temperature Optimization:** 

single band on the agarose gel.

Rad Laboratories 2006).

be performed:

#### 2. Assay validation and optimization

## **A) Primer and Amplicon Design:**

A successful real-time PCR reaction requires efficient and specific amplification of the product. Both primers and target sequence can affect this efficiency. Therefore, care must be taken when choosing a target sequence and designing primers. A number of free and commercially available software programs are available for this purpose. One popular webbased program for primer design is Primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3\_www.cgi). A commercially available program such as Beacon Designer software performs both primer design and amplicon selection (Bio-Rad Laboratories 2006).

#### **B) Guidelines of amplicon design:**


#### **C) Parameters of primer design:**


#### **D) Assay Validation and Optimization:**

Components a SYBR Green I qPCR reaction:


Preformulated real-time PCR master mixes containing buffer, DNA polymerase, dNTPs, and SYBR Green I dye are available from several vendors.

Optimized SYBR Green I qPCR reactions should be sensitive and specific and should exhibit good amplification efficiency over a broad dynamic range (Bio-Rad Laboratories 2006).

A successful real-time PCR reaction requires efficient and specific amplification of the product. Both primers and target sequence can affect this efficiency. Therefore, care must be taken when choosing a target sequence and designing primers. A number of free and commercially available software programs are available for this purpose. One popular webbased program for primer design is Primer3 (http://frodo.wi.mit.edu/cgibin/primer3/primer3\_www.cgi). A commercially available program such as Beacon Designer software performs both primer design and amplicon selection (Bio-Rad Laboratories 2006).

1. Design amplicon to be 75–200 bp.Shorteramplicons are typically amplified with higher efficiency. An amplicon should be at least 75 bp to easily distinguish it from any

2. Avoid secondary structure if possible. Use programs such as mfold http://www.bioinfo.rpi.edu/applications/mfold/) to predict whether an amplicon will form any secondary structure at annealing temperature. See Real-Time PCR:

2. Maintain a melting temperature (Tm) between 50ºC and 65ºC. We calculate Tm values using the nearest-neighbor method with values of 50 mM for salt concentration and 300

3. Avoid secondary structure; adjust primer locations outside of the target sequence

6. Check sequence of forward and reverse primers to ensure no 3' complementarity (avoid

7. Verify specificity using tools such as the Basic Local Alignment Search Tool

Preformulated real-time PCR master mixes containing buffer, DNA polymerase, dNTPs,

Optimized SYBR Green I qPCR reactions should be sensitive and specific and should exhibit good amplification efficiency over a broad dynamic range (Bio-Rad Laboratories 2006).

General Considerations (Bio-Rad bulletin 2593) for more details

3. Avoid templates with long (>4) repeats of single bases

1. Design primers with a GC content of 50–60%

nM for oligonucleotide concentration

4. Avoid repeats of Gs or Cs longer than three bases

(http://www.ncbi.nlm.nih.gov/blast/)

and SYBR Green I dye are available from several vendors.

secondary structure if required

5. Place Gs and Cs on ends of primers

**D) Assay Validation and Optimization:**  Components a SYBR Green I qPCR reaction:

1. PCR master mix with SYBR Green I

2. Template 3. Primers

primer-dimer formation)

2. Assay validation and optimization

**A) Primer and Amplicon Design:** 

**B) Guidelines of amplicon design:** 

primer-dimers that might form

4. Maintain a GC content of 50–60%

**C) Parameters of primer design:** 

Steps of to determine the performance of your SYBR Green I qPCR assay:


## **E) Annealing Temperature Optimization:**

The optimal annealing temperature can easily be assessed on qPCR instruments that have a temperature gradient feature, such as the MiniOpticon™, MyiQ™, DNA Engine Opticon®, Opticon™ 2, iCycleriQ®, Chromo4™, and iQ™5 systems.

A gradient feature allows you to test a range of annealing temperatures simultaneously, so optimization reactions can be performed in a single experiment.

To find the optimal annealing temperature for reaction, recommend testing a range of annealing temperatures above and below the calculated Tm of the primers.

Because SYBR Green I binds to all dsDNA, it is necessary to check the specificity of your qPCR assay by analyzing the reaction product(s). To do this, use the melt-curve function on your real-time instrument and also run products on an agarose gel. An optimized SYBR Green I qPCR reaction should have a single peak in the melt curve, corresponding to the single band on the agarose gel.

Nonspecific products that may have been co-amplified with the specific product can be identified by melt-curve analysis. In this example, the specific product is the peak with a Tm of 89°C and corresponds to the upper band on the gel. The nonspecific product is the peak with a Tm of 78°C and corresponds to the lower band in the gel. By comparing the gel image with the melt curve, you can identify peaks in the melt curve that correspond to specific product, additional nonspecific bands, and primer-dimers. If nonspecific products such as primer-dimers are detected by melt-curve analysis, recommend that redesign primers (Bio-Rad Laboratories 2006).

### **F) Assay Performance Evaluation Using Standard Curves:**

The efficiency, reproducibility, and dynamic range of a SYBR Green I assay can be determined by constructing a standard curve using serial dilutions of a known template. The efficiency of the assay should be 90–105%, the R2 of the standard curve should be >0.980 or r > |–0.990|, and the CT values of the replicates should be similar.

It is important to note that the range of template concentrations used for the standard curve must encompass the entire range of template concentration of the test samples to show that results from the test samples are within the linear dynamic range of the assay. If the test samples give results outside of the range of the standard curves, one of the following must be performed:


Overview of Real-Time PCR Principles 417

extension. Typical TaqMan probes for nucleic acid quantification are designed to have a Tm of 60–70°C. An optimized TaqMan assay should be sensitive and specific, and should

In short, construct a standard curve using dilutions of a known template and use this curve to determine the efficiency of the assay along with R2 or r of the regression line. The efficiency of the reaction should be between 90 and 105%, the R2 should be >0.980 or r > |– 0.990|, and the replicates should give similar CT values. If the assay performs within these specifications, you are ready to start your experiment. If the assay performs outside these specifications, we suggest that you redesign your primers and TaqMan probe. It is important to note that the range of template concentrations used for the standard curve must encompass the entire range of template concentrations of the test samples to demonstrate that results from the test samples are within the dynamic range of the assay (Bio-Rad Laboratories 2006). If test samples give results outside the range of the standard

1. Construct a wider standard curve covering the test sample concentrations and perform

2. If the test samples give a lower CT than the highest concentration of standards used in

3. If the test samples give a higher CT than the lowest concentration of standards used in the standard curve, repeat the assay using larger amounts of the test samples

A critical requirement for real-time PCR technology is the ability to detect the uorescent signal and record the progress of the PCR. Because uorescent chemistries require both a specic input of energy for excitation and a detection of a particular emission wavelength, the instrumentation must be able to do both simultaneously and at the desired wavelengths. Thus the chemistries and instrumentation are intimately linked (Valasek and Repa 2005).

At present, there are three basic ways in which real-time instrumentation can supply the excitation energy for uorophores: by lamp, light-emitting diode (LED), or laser. Lamps are classied as broad-spectrum emission devices, whereas LEDs and lasers are narrow spectrum. Instruments that utilize lamps (tungsten halogen or quartz tungsten halogen) may also include lters to restrict the emitted light to specic excitation wavelengths. Instruments using lamps include Applied Biosystem's ABI Prism 7000, Stratagene's Mx4000 and Mx3000P, and Bio-Rad's iCycleriQ. LED systems include Roche's LightCycler, Cepheid's SmartCycler, Corbett's Rotor-Gene, and MJ Research's DNA Engine Opticon 2. The ABI Prism 7900HT is the sole machine to use a laser for excitation (Valasek and Repa 2005). To collect data, the emission energies must also be detected at the appropriate wavelengths. Detectors include charge-coupled device cameras, photomultiplier tubes, or other types of photodetectors. Narrow wavelength lters or channels are generally employed to allow only the desired wavelength(s) to pass to the photodetector to be measured. Usually, multiple discrete wavelengths can be measured at once, which allows for multiplexing, i.e., running multiple assays in a single reaction tube (Valasek and Repa 2005). Another portion of the instrumentation consists of a thermocycler to carry out PCR. Of particular importance for real-time PCR is the ability of the thermocycler to maintain a

exhibit good amplification efficiency over a broad dynamic range.

curve, one of the three following steps must be performed:

**5. The instrumentation of real-time PCR** 

analysis to ensure that the assay is linear in that new range

the standard curve, repeat the assay using diluted test samples

## **4. Design and optimization of TaqManProbe reactions**

A TaqMan assay uses a pair of PCR primers and a dual-labeled target-specific fluorescent probe. The steps for developing a TaqMan assay are:


#### **A. Primer and Probe Design:**

As with any qPCR reaction, TaqMan-based assays require efficient and specific amplification of the product. Typically, the primers are designed to have an annealing temperature between 55 and 60oC. We recommend using software such as Beacon Designer for designing your TaqMan primers and TaqMan probe.. Because the dual-labeled probe is the most costly component of a TaqMan assay, suggested that order the two primers and validate their performance using SYBR Green I before ordering the dual-labeled probe.

The TaqMan probe should have a Tm 5–10°C higher than that of the primers. In most cases, the probe should be <30 nucleotides and must not contain a G at its 5' end because this could quench the fluorescent signal even after hydrolysis. Choose a sequence within the target that has a GC content of 30–80%, and design the probe to anneal to the strand that has more Gs than Cs (so the probe contains more Cs than Gs).

An important aspect of designing a TaqMan probe is reporter and quencher selection. We recommend using FAM-labeled probes when designing singleplex reactions, because they are inexpensive and readily available, perform well, and can be detected by all instruments currently on the market.

Another important consideration for obtaining accurate real-time qPCR data is probe quality. Even a perfectly designed probe can fail if the probe is improperly synthesized or purified. Improper removal of uncoupled fluorescent label, inefficient coupling, and/or poor quenching can produce high fluorescent background or noise. A low signal-to-noise ratio results in decreased sensitivity and a smaller linear dynamic range. Two probes with identical sequences and identical fluorophore labels can be measurably different when synthesized by different suppliers or even at different times by the same supplier.

## **B. Assay Validation and Optimization:**

A TaqMan probe-based qPCR reaction contains the following components:


Preformulated PCR master mixes containing buffer, DNA polymerase, and dNTPs are commercially available from several vendors. For TaqMan assays, we recommend using iQ™ supermix with 300 nM of each of the two primers and 200 nM of probe(s). TaqMan assays require careful attention to temperature conditions. A typical TaqMan protocol contains a denaturation step followed by a combined annealing and extension step at 55– 60°C, instead of the traditional three-step PCR cycle of denaturation, annealing, and extension. This is to ensure that the probe remains bound to its target during primer

A TaqMan assay uses a pair of PCR primers and a dual-labeled target-specific fluorescent

As with any qPCR reaction, TaqMan-based assays require efficient and specific amplification of the product. Typically, the primers are designed to have an annealing temperature between 55 and 60oC. We recommend using software such as Beacon Designer for designing your TaqMan primers and TaqMan probe.. Because the dual-labeled probe is the most costly component of a TaqMan assay, suggested that order the two primers and validate their performance using SYBR Green I before ordering the dual-labeled probe.

The TaqMan probe should have a Tm 5–10°C higher than that of the primers. In most cases, the probe should be <30 nucleotides and must not contain a G at its 5' end because this could quench the fluorescent signal even after hydrolysis. Choose a sequence within the target that has a GC content of 30–80%, and design the probe to anneal to the strand that has

An important aspect of designing a TaqMan probe is reporter and quencher selection. We recommend using FAM-labeled probes when designing singleplex reactions, because they are inexpensive and readily available, perform well, and can be detected by all instruments

Another important consideration for obtaining accurate real-time qPCR data is probe quality. Even a perfectly designed probe can fail if the probe is improperly synthesized or purified. Improper removal of uncoupled fluorescent label, inefficient coupling, and/or poor quenching can produce high fluorescent background or noise. A low signal-to-noise ratio results in decreased sensitivity and a smaller linear dynamic range. Two probes with identical sequences and identical fluorophore labels can be measurably different when

Preformulated PCR master mixes containing buffer, DNA polymerase, and dNTPs are commercially available from several vendors. For TaqMan assays, we recommend using iQ™ supermix with 300 nM of each of the two primers and 200 nM of probe(s). TaqMan assays require careful attention to temperature conditions. A typical TaqMan protocol contains a denaturation step followed by a combined annealing and extension step at 55– 60°C, instead of the traditional three-step PCR cycle of denaturation, annealing, and extension. This is to ensure that the probe remains bound to its target during primer

synthesized by different suppliers or even at different times by the same supplier.

A TaqMan probe-based qPCR reaction contains the following components:

**4. Design and optimization of TaqManProbe reactions** 

probe. The steps for developing a TaqMan assay are:

more Gs than Cs (so the probe contains more Cs than Gs).

a. Primer and probe design

**A. Primer and Probe Design:** 

currently on the market.

1. PCR master mix 2. Template 3. Primers 4. Probe(s)

**B. Assay Validation and Optimization:** 

b. Assay validation and optimization

extension. Typical TaqMan probes for nucleic acid quantification are designed to have a Tm of 60–70°C. An optimized TaqMan assay should be sensitive and specific, and should exhibit good amplification efficiency over a broad dynamic range.

In short, construct a standard curve using dilutions of a known template and use this curve to determine the efficiency of the assay along with R2 or r of the regression line. The efficiency of the reaction should be between 90 and 105%, the R2 should be >0.980 or r > |– 0.990|, and the replicates should give similar CT values. If the assay performs within these specifications, you are ready to start your experiment. If the assay performs outside these specifications, we suggest that you redesign your primers and TaqMan probe. It is important to note that the range of template concentrations used for the standard curve must encompass the entire range of template concentrations of the test samples to demonstrate that results from the test samples are within the dynamic range of the assay (Bio-Rad Laboratories 2006). If test samples give results outside the range of the standard curve, one of the three following steps must be performed:


## **5. The instrumentation of real-time PCR**

A critical requirement for real-time PCR technology is the ability to detect the uorescent signal and record the progress of the PCR. Because uorescent chemistries require both a specic input of energy for excitation and a detection of a particular emission wavelength, the instrumentation must be able to do both simultaneously and at the desired wavelengths. Thus the chemistries and instrumentation are intimately linked (Valasek and Repa 2005).

At present, there are three basic ways in which real-time instrumentation can supply the excitation energy for uorophores: by lamp, light-emitting diode (LED), or laser. Lamps are classied as broad-spectrum emission devices, whereas LEDs and lasers are narrow spectrum. Instruments that utilize lamps (tungsten halogen or quartz tungsten halogen) may also include lters to restrict the emitted light to specic excitation wavelengths. Instruments using lamps include Applied Biosystem's ABI Prism 7000, Stratagene's Mx4000 and Mx3000P, and Bio-Rad's iCycleriQ. LED systems include Roche's LightCycler, Cepheid's SmartCycler, Corbett's Rotor-Gene, and MJ Research's DNA Engine Opticon 2. The ABI Prism 7900HT is the sole machine to use a laser for excitation (Valasek and Repa 2005). To collect data, the emission energies must also be detected at the appropriate wavelengths. Detectors include charge-coupled device cameras, photomultiplier tubes, or other types of photodetectors. Narrow wavelength lters or channels are generally employed to allow only the desired wavelength(s) to pass to the photodetector to be measured. Usually, multiple discrete wavelengths can be measured at once, which allows for multiplexing, i.e., running multiple assays in a single reaction tube (Valasek and Repa 2005). Another portion of the instrumentation consists of a thermocycler to carry out PCR. Of particular importance for real-time PCR is the ability of the thermocycler to maintain a

Overview of Real-Time PCR Principles 419

This could reduce the time of analysis to result from 3-4 hours to 1.5 hours. On the other hand, if sensitivity is the most important issue, these machines, with their smaller reaction volume and consequently lower sensitivity, wouldn't be the first choice. The ABI 7700 and Bio-Rad -I-Cycler IQ® have a 96 well format, enabling higher throughput than other systems. The 384-well plates, as designed by ABI for use in the 7900 HT system, can further enhance through put. For diagnostic application, internal control of nucleic acid isolation and PCR inhibition, it is essential to obtain valid results. This can be achieved using the system that enables multi-color detection, such as the I-Cycler IQ® and the Smart Cycler®. Recently, a multi-color format of the Light Cycler® is also present in market. Multiplex realtime PCRs can be developed for three different targets and an internal control by using the four detection wavelengths possible in multicolor detection. As a matter of fact, the choice of which real time system to use depends on the range of application required. To achieve meaningful results, each assay must be validated and optimized for the particular system

There are many methods in molecular biology for measuring quantities of target nucleic acid sequences. However, most of these methods exhibit one or more of the following shortcomings: they are time consuming, labor intensive, insufciently sensitive, nonquantitative, require the use of radioactivity, or have a substantial probability of cross contamination (Reischl, Wittwer et al. 2002). These methods include but are not limited to Northern and Southern hybridizations, HPLC, scintillation proximity assay, PCR-ELISA, RNase protection assay, in situ hybridization, and various gel electrophoresis PCR endpoint systems (Valasek and Repa 2005). Real-time PCR has distinct advantages over these earlier methods for several reasons. Perhaps the most important is its ability to quantify nucleic acids over an extraordinarily wide dynamic range (at least 5 log units). This is coupled to extreme sensitivity, allowing the detection of less than ve copies (perhaps only one copy in some cases) of a target sequence, making it possible to analyze small samples like clinical biopsies or miniscule lysates from laser capture microdissection. With appropriate internal standards and calculations, mean variation coefcients are 1–2%, allowing reproducible analysis of subtle gene expression changes even at low levels of expression (Klein 2002; Luu-The, Paquet et al. 2005). In addition, all real-time platforms are relatively quick, with some affording high-throughput automation. Finally, real-time PCR is performed in a closed reaction vessel that requires no post-PCR manipulations, thereby minimizing the chances for cross contamination in the laboratory (Valasek and Repa 2005).

There are several limitations to real-time PCR methods. The majority of these are present in all PCR or RT-PCR-based techniques. Real-time PCR is susceptible to PCR inhibition bycompounds present in certain biological samples. For example, clinical and forensic uses for real-timePCR may be affected by inhibitors found in certain body uidssuch as hemoglobin or urea (Wilson 1997). Food microbiological applications may encounter organic and phenolic inhibitors (Wilson 1997). To circumvent this problem, alternative DNA polymerases (e.g., T, Pwo, Tth, etc.) that are resistant to particular inhibitors can be used. Other limitations primarily concern real-time PCR- based analysis of gene expression

chosen (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005).

**6. Advantages of real-time PCR quantitation** 

**7. Limitations of real-time PCR quantitation** 

consistent temperature among all sample wells, as any differences in temperature could lead to different PCR amplication efciencies. This is accomplished by using a heating block (Peltier based or resistive), heated air, or a combination of the two. As one might expect, heating blocks generally change temperature more slowly than heated air, resulting in longer thermocycling times. For example, Roche's LightCycler models utilizing heated air can perform 40 cycles in 30 min, whereas Applied Biosystem's ABI Prism 7900HT utilizing a Peltier-based heating block take s 1 h 45 min (Valasek and Repa 2005). Real-time instrumentation certainly would not be complete without appropriate computer hardware and data-acquisition and analysis software. Software platforms try to simplify analysis of real-time PCR data by offering graphical output of assay results including amplication and dissociation (melting point) curves. The amplication curve gives data regarding the kinetics of amplication of the target sequence, whereas the dissociation curve reveals the characteristics of the nal amplied product (Valasek and Repa 2005).

## **5.1 Comparison of the different systems**

Essentially, each real time PCR instrument consists of a computer-controlled thermocycler integrated with fluorescent detection system and dedicated software to analyze the result. Some systems can detect four different wave lengths (I-cycler, Mx4000 [stratagene] and Smart Cycler®, Version 2.0 Light Cycler®) whereas others can detect two different wavelengths (Light Cycler®). The Light Cycler® and Smart Cycler® are capable of performing rapid-cycle real time PCR because the reaction is set-up in capillaries or especially designated tubes. Both have optimized heating- cooling characteristic. A complete amplification protocol can be performed in 30-45 minutes (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005). The Smart Cycler® is a combination of 16 individual, one tube real time PCR units. It is capable of performing a different PCR program on each of 16 reaction tubes. This is very useful for a rapid optimization of the assay as many variables can be tested at the same time. The Bio-Rad I-cycler IQ® instrument can perform real time amplification with a temperature gradient for specific PCR steps, allowing the optimization of real time PCR assay. The spectrofluorometers in the thermal cycler have a number of differences. Laser-based systems are tuned to excite each fluorophore at a specific wavelength and provide maximum efficiency. Lamp-based systems provide a broad excitation range that can be filtered to work with a number of fluorophores. The laser source not only gives brighter illumination to the fluorophore signal, but also produces less background noise(Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005).

In conclusion, real time PCR is a powerful advancement of the basic PCR technique. The important steps in deciding which particular assay format to use are related to the type of data required. The requirement for a research laboratory is quite distinct from those of a diagnostic laboratory. For the latter, probe confirmation of the PCR product is an essential part of the assay, whereas SYBR green detection may be sufficient for many other applications such as quantifying expression of a gene. All of the real-time PCR machines analyzed are capable of detecting PCR product in real time and a specific assay can be made optionally on every system. However, there are some decisions to be made when selecting among different formats. The choice of system is dependent on individual laboratory needs (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005). Considering diagnostic applications, the Light Cycler® or Smart Cycler® may obtain faster results for urgent assays.

consistent temperature among all sample wells, as any differences in temperature could lead to different PCR amplication efciencies. This is accomplished by using a heating block (Peltier based or resistive), heated air, or a combination of the two. As one might expect, heating blocks generally change temperature more slowly than heated air, resulting in longer thermocycling times. For example, Roche's LightCycler models utilizing heated air can perform 40 cycles in 30 min, whereas Applied Biosystem's ABI Prism 7900HT utilizing a Peltier-based heating block take s 1 h 45 min (Valasek and Repa 2005). Real-time instrumentation certainly would not be complete without appropriate computer hardware and data-acquisition and analysis software. Software platforms try to simplify analysis of real-time PCR data by offering graphical output of assay results including amplication and dissociation (melting point) curves. The amplication curve gives data regarding the kinetics of amplication of the target sequence, whereas the dissociation curve reveals the

Essentially, each real time PCR instrument consists of a computer-controlled thermocycler integrated with fluorescent detection system and dedicated software to analyze the result. Some systems can detect four different wave lengths (I-cycler, Mx4000 [stratagene] and Smart Cycler®, Version 2.0 Light Cycler®) whereas others can detect two different wavelengths (Light Cycler®). The Light Cycler® and Smart Cycler® are capable of performing rapid-cycle real time PCR because the reaction is set-up in capillaries or especially designated tubes. Both have optimized heating- cooling characteristic. A complete amplification protocol can be performed in 30-45 minutes (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005). The Smart Cycler® is a combination of 16 individual, one tube real time PCR units. It is capable of performing a different PCR program on each of 16 reaction tubes. This is very useful for a rapid optimization of the assay as many variables can be tested at the same time. The Bio-Rad I-cycler IQ® instrument can perform real time amplification with a temperature gradient for specific PCR steps, allowing the optimization of real time PCR assay. The spectrofluorometers in the thermal cycler have a number of differences. Laser-based systems are tuned to excite each fluorophore at a specific wavelength and provide maximum efficiency. Lamp-based systems provide a broad excitation range that can be filtered to work with a number of fluorophores. The laser source not only gives brighter illumination to the fluorophore signal, but also produces less

background noise(Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005).

In conclusion, real time PCR is a powerful advancement of the basic PCR technique. The important steps in deciding which particular assay format to use are related to the type of data required. The requirement for a research laboratory is quite distinct from those of a diagnostic laboratory. For the latter, probe confirmation of the PCR product is an essential part of the assay, whereas SYBR green detection may be sufficient for many other applications such as quantifying expression of a gene. All of the real-time PCR machines analyzed are capable of detecting PCR product in real time and a specific assay can be made optionally on every system. However, there are some decisions to be made when selecting among different formats. The choice of system is dependent on individual laboratory needs (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005). Considering diagnostic applications, the Light Cycler® or Smart Cycler® may obtain faster results for urgent assays.

characteristics of the nal amplied product (Valasek and Repa 2005).

**5.1 Comparison of the different systems** 

This could reduce the time of analysis to result from 3-4 hours to 1.5 hours. On the other hand, if sensitivity is the most important issue, these machines, with their smaller reaction volume and consequently lower sensitivity, wouldn't be the first choice. The ABI 7700 and Bio-Rad -I-Cycler IQ® have a 96 well format, enabling higher throughput than other systems. The 384-well plates, as designed by ABI for use in the 7900 HT system, can further enhance through put. For diagnostic application, internal control of nucleic acid isolation and PCR inhibition, it is essential to obtain valid results. This can be achieved using the system that enables multi-color detection, such as the I-Cycler IQ® and the Smart Cycler®. Recently, a multi-color format of the Light Cycler® is also present in market. Multiplex realtime PCRs can be developed for three different targets and an internal control by using the four detection wavelengths possible in multicolor detection. As a matter of fact, the choice of which real time system to use depends on the range of application required. To achieve meaningful results, each assay must be validated and optimized for the particular system chosen (Myi ; Giulietti, Overbergh et al. 2001; Soheili and Samiei 2005).

## **6. Advantages of real-time PCR quantitation**

There are many methods in molecular biology for measuring quantities of target nucleic acid sequences. However, most of these methods exhibit one or more of the following shortcomings: they are time consuming, labor intensive, insufciently sensitive, nonquantitative, require the use of radioactivity, or have a substantial probability of cross contamination (Reischl, Wittwer et al. 2002). These methods include but are not limited to Northern and Southern hybridizations, HPLC, scintillation proximity assay, PCR-ELISA, RNase protection assay, in situ hybridization, and various gel electrophoresis PCR endpoint systems (Valasek and Repa 2005). Real-time PCR has distinct advantages over these earlier methods for several reasons. Perhaps the most important is its ability to quantify nucleic acids over an extraordinarily wide dynamic range (at least 5 log units). This is coupled to extreme sensitivity, allowing the detection of less than ve copies (perhaps only one copy in some cases) of a target sequence, making it possible to analyze small samples like clinical biopsies or miniscule lysates from laser capture microdissection. With appropriate internal standards and calculations, mean variation coefcients are 1–2%, allowing reproducible analysis of subtle gene expression changes even at low levels of expression (Klein 2002; Luu-The, Paquet et al. 2005). In addition, all real-time platforms are relatively quick, with some affording high-throughput automation. Finally, real-time PCR is performed in a closed reaction vessel that requires no post-PCR manipulations, thereby minimizing the chances for cross contamination in the laboratory (Valasek and Repa 2005).

#### **7. Limitations of real-time PCR quantitation**

There are several limitations to real-time PCR methods. The majority of these are present in all PCR or RT-PCR-based techniques. Real-time PCR is susceptible to PCR inhibition bycompounds present in certain biological samples. For example, clinical and forensic uses for real-timePCR may be affected by inhibitors found in certain body uidssuch as hemoglobin or urea (Wilson 1997). Food microbiological applications may encounter organic and phenolic inhibitors (Wilson 1997). To circumvent this problem, alternative DNA polymerases (e.g., T, Pwo, Tth, etc.) that are resistant to particular inhibitors can be used. Other limitations primarily concern real-time PCR- based analysis of gene expression

Overview of Real-Time PCR Principles 421

During relative quantitation, changes in sample gene expression are measured based on either an external standard or a reference sample, also known as a calibrator (Livak and Schmittgen 2001). When using a calibrator, the results are expressed as a target/reference ratio. There are numerous mathematical models available to calculate the mean normalized gene expression from relative quantitationassays. Depending onthe method employed, these can yield different results and thus discrepant measures of standard error (Liu and Saint 2002; Muller, Janovjak et al. 2002). Table 1 shows a comparison of the different

For quantitative analysis, the amplification curves are evaluated. The amplification process is monitored either through the fluorescence of dsDNA-specific dyes (like SYBR Green I) or ofsequence-specific probes. Each curve consists of at least three distinct phases: 1) an initial lag phase in which no product accumulation can be measured, 2) an exponential phase, and 3) a plateau phase (Wilhelm and Pingoud 2003). The exponential phase in principle could be extrapolated to the start of the reaction (Cycle 0) to calculate the template copy number, but the error would be too high. The template copy number can be estimated with greater precision from the number of cycles needed for the signal to reach an arbitrary threshold. The threshold must intersect the signal curve in its exponential phase, in which the signal increase correlates with product accumulation. The intersection point is the so-called threshold value (CT) or crossing point (CP). This point may be between two successive cycles (i.e. it may be a fractional number). For exact quantifications, the efficiency of the amplification reaction must be known. It is crucial that the amplification efficiencies of standards and unknowns are identical (Wilhelm and Pingoud 2003). The efficiency can be estimated from the CT values of samples with known template concentrations ('standards')

where*p*is a proportionality factor to relate PCR product concentration and signal intensity, *N*0is the amount of template, *ɛ*is the amplification efficiency (1 ≤ *ɛ* ≤ 2; ɛ= 2 means 100%

 *c*=- (log*ɛ*)-1(log*N*0 + log*p*- log*S*) (2)

 *c*=*m*log*N*0+ *b* (3) This equation describes the linear relationship between the CTvalues determined and the log of the template concentration (*N*0). The parameters *m*and *b*can be determined by a regression analysis of the CT values of the standards. When solved for *N*0, this equation serves as a

 *N*0 = 10(CT*-b*)/m (4)

With *m*= - (log*ɛ*)-1 and *b* = - (logɛ)-1(log*p*-log*S*), Equation 2 simplifies to Equation 3:

calibration curve for the calculation of the unknowns according to Equation 4:

*S*= *pN0ɛ<sup>c</sup>* (1)

methods, with an explanation of each method to follow (Wong and Medrano 2005).

**2. Relative quantitation** 

**9. Quantitative analyses** 

as described below (Wilhelm and Pingoud 2003).

efficiency) and *c*is the cycle number. Solving for c results in Equation 2:

During the exponential phase, the signal *S*can be described by Equation 1:

(Bustin 2000; Bustin 2002;Bustin and Nolan 2004). Because of the necessary use of RNA in an extra enzymatic step, more problems have the opportunity to occur. RNA itself is extremely labile compared with DNA, and therefore isolation must be carefully performed to ensure both the integrity of the RNA itself and the removal of contaminating nucleases, genomic DNA, and RT or PCR inhibitors. This can be a problem with any sample source, but clinical samples are of special concern because inconsistencies in sample size, collection, storage, and transport can lead to a variable quality of RNA templates. Conversion of RNA to cDNA during the RT reaction is also subject to variability because multiple reverse transcriptase enzymes with different characteristics exist, and different classes of oligonucleotides (e.g., random, poly-dT, or gene specic primers) can be used to prime RT (Valasek and Repa 2005). Probably the largest present limitation of real-time PCR, however, is not inherent in the technology but rather resides in human error: improper assay development, incorrect data analysis, or unwarranted conclusions. In our experience using real-time PCR for gene expression analysis, real-time PCR primer sets must be designed and validated by stringent criteria to ensure specicity and accuracy of the results. For microbiology, false positives or negatives must be considered when designing an assay to detect pathogens. Amplication and melting curves must be visually inspected while independent calculations based on these curves should be double-checked for accuracy. Real-time PCR gene expression analysis measures mRNA levels and, therefore, only suggests possible changes in protein levels or function rather than demonstrating them. And although there is a tight connection between gene expression and gene product function (Brown and Botstein 1999)(8), this is certainly not always the case, and formal demonstration may be needed for a given research project. Of course, conclusions based on data derived from real-time PCR are best utilized when the biological context is well understood (Bustin 2002).

## **8. Types of real-time quantification**

#### **1. Absolute Quantitation**

Absolute quantitation uses serially diluted standards of known concentrations to generate a standard curve. The standard curve produces a linear relationship between Ct and initial amounts of total RNA or cDNA, allowing the determination of the concentration of unknowns based on their Ct values (Heid, Stevens et al. 1996). This method assumes all standards and samples have approximately equal amplification efficiencies (Souaze, Ntodou-Thome et al. 1996). In addition, the concentration of serial dilutions should encompass the levels in the experimental samples and stay within the range of accurately quantifiable and detectable levels specific for both the real-time PCR machine and assay.The PCR standard is a fragment of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), or cRNA bearing the target sequence (Wong and Medrano 2005). A simple protocol for constructing a cRNA standard for one-step PCR can be found in Fronhoffs et al. (Fronhoffs, Totzke et al. 2002), while a DNA standard for two-step real-time PCR can be synthesized by cloning the target sequence into a plasmid (Gerard, Olsson et al. 1998), purifying a conventional PCR product (Liss 2002), or directly synthesizing the target nucleic acid. The standard used must be a pure species. DNA standards have been shown to have a larger quantification range and greater sensitivity, reproducibility, and stability than RNA standards (Pfaffl, Tichopad et al. 2004). However, a DNA standard cannot be used for a onestep real-time RT-PCR due to the absence of a control for the reverse transcription efficiency (Giulietti, Overbergh et al. 2001).

#### **2. Relative quantitation**

420 Polymerase Chain Reaction

(Bustin 2000; Bustin 2002;Bustin and Nolan 2004). Because of the necessary use of RNA in an extra enzymatic step, more problems have the opportunity to occur. RNA itself is extremely labile compared with DNA, and therefore isolation must be carefully performed to ensure both the integrity of the RNA itself and the removal of contaminating nucleases, genomic DNA, and RT or PCR inhibitors. This can be a problem with any sample source, but clinical samples are of special concern because inconsistencies in sample size, collection, storage, and transport can lead to a variable quality of RNA templates. Conversion of RNA to cDNA during the RT reaction is also subject to variability because multiple reverse transcriptase enzymes with different characteristics exist, and different classes of oligonucleotides (e.g., random, poly-dT, or gene specic primers) can be used to prime RT (Valasek and Repa 2005). Probably the largest present limitation of real-time PCR, however, is not inherent in the technology but rather resides in human error: improper assay development, incorrect data analysis, or unwarranted conclusions. In our experience using real-time PCR for gene expression analysis, real-time PCR primer sets must be designed and validated by stringent criteria to ensure specicity and accuracy of the results. For microbiology, false positives or negatives must be considered when designing an assay to detect pathogens. Amplication and melting curves must be visually inspected while independent calculations based on these curves should be double-checked for accuracy. Real-time PCR gene expression analysis measures mRNA levels and, therefore, only suggests possible changes in protein levels or function rather than demonstrating them. And although there is a tight connection between gene expression and gene product function (Brown and Botstein 1999)(8), this is certainly not always the case, and formal demonstration may be needed for a given research project. Of course, conclusions based on data derived from real-time PCR are best utilized

Absolute quantitation uses serially diluted standards of known concentrations to generate a standard curve. The standard curve produces a linear relationship between Ct and initial amounts of total RNA or cDNA, allowing the determination of the concentration of unknowns based on their Ct values (Heid, Stevens et al. 1996). This method assumes all standards and samples have approximately equal amplification efficiencies (Souaze, Ntodou-Thome et al. 1996). In addition, the concentration of serial dilutions should encompass the levels in the experimental samples and stay within the range of accurately quantifiable and detectable levels specific for both the real-time PCR machine and assay.The PCR standard is a fragment of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), or cRNA bearing the target sequence (Wong and Medrano 2005). A simple protocol for constructing a cRNA standard for one-step PCR can be found in Fronhoffs et al. (Fronhoffs, Totzke et al. 2002), while a DNA standard for two-step real-time PCR can be synthesized by cloning the target sequence into a plasmid (Gerard, Olsson et al. 1998), purifying a conventional PCR product (Liss 2002), or directly synthesizing the target nucleic acid. The standard used must be a pure species. DNA standards have been shown to have a larger quantification range and greater sensitivity, reproducibility, and stability than RNA standards (Pfaffl, Tichopad et al. 2004). However, a DNA standard cannot be used for a onestep real-time RT-PCR due to the absence of a control for the reverse transcription efficiency

when the biological context is well understood (Bustin 2002).

**8. Types of real-time quantification** 

**1. Absolute Quantitation** 

(Giulietti, Overbergh et al. 2001).

During relative quantitation, changes in sample gene expression are measured based on either an external standard or a reference sample, also known as a calibrator (Livak and Schmittgen 2001). When using a calibrator, the results are expressed as a target/reference ratio. There are numerous mathematical models available to calculate the mean normalized gene expression from relative quantitationassays. Depending onthe method employed, these can yield different results and thus discrepant measures of standard error (Liu and Saint 2002; Muller, Janovjak et al. 2002). Table 1 shows a comparison of the different methods, with an explanation of each method to follow (Wong and Medrano 2005).

#### **9. Quantitative analyses**

For quantitative analysis, the amplification curves are evaluated. The amplification process is monitored either through the fluorescence of dsDNA-specific dyes (like SYBR Green I) or ofsequence-specific probes. Each curve consists of at least three distinct phases: 1) an initial lag phase in which no product accumulation can be measured, 2) an exponential phase, and 3) a plateau phase (Wilhelm and Pingoud 2003). The exponential phase in principle could be extrapolated to the start of the reaction (Cycle 0) to calculate the template copy number, but the error would be too high. The template copy number can be estimated with greater precision from the number of cycles needed for the signal to reach an arbitrary threshold. The threshold must intersect the signal curve in its exponential phase, in which the signal increase correlates with product accumulation. The intersection point is the so-called threshold value (CT) or crossing point (CP). This point may be between two successive cycles (i.e. it may be a fractional number). For exact quantifications, the efficiency of the amplification reaction must be known. It is crucial that the amplification efficiencies of standards and unknowns are identical (Wilhelm and Pingoud 2003). The efficiency can be estimated from the CT values of samples with known template concentrations ('standards') as described below (Wilhelm and Pingoud 2003).

During the exponential phase, the signal *S*can be described by Equation 1:

$$S = pN\_{0\mathcal{E}}c \tag{1}$$

where*p*is a proportionality factor to relate PCR product concentration and signal intensity, *N*0is the amount of template, *ɛ*is the amplification efficiency (1 ≤ *ɛ* ≤ 2; ɛ= 2 means 100% efficiency) and *c*is the cycle number.

Solving for c results in Equation 2:

$$c = \text{-- (log\varepsilon)}\cdot\text{l}\text{(logN}\_0 + \text{logp-}\log S) \tag{2}$$

With *m*= - (log*ɛ*)-1 and *b* = - (logɛ)-1(log*p*-log*S*), Equation 2 simplifies to Equation 3:

$$c \equiv \text{mlog}\text{N}\_0 + b \tag{3}$$

This equation describes the linear relationship between the CTvalues determined and the log of the template concentration (*N*0). The parameters *m*and *b*can be determined by a regression analysis of the CT values of the standards. When solved for *N*0, this equation serves as a calibration curve for the calculation of the unknowns according to Equation 4:

$$N\_0 = \text{10} \{ ^{\text{C}}\text{T}^{\text{-}6} \}^{\text{(\text{m})}} \tag{4}$$

Overview of Real-Time PCR Principles 423

Fig. 1. Real-time PCR detection chemistries. Probe sequences are shown in blue while target DNA sequences are shown in black. Primers are indicated by horizontal arrowheads. Not all

 *N*0 = *ɛ* (b-CT) (6) The maximum value for *ɛ* is 2.0 (i.e. the amount of product is doubled in each cycle). The experimental value for *ɛ* usually varies between 1.5 and 1.9. Lower efficiencies limit the sensitivity of the assay but allow quantifications with higher precisions. Therefore, reactions

*ɛ* = 10-1/m (5)

unlabeled PCR primers are shown. Oligo, oligonucleotide.

By inserting *ɛ* back into Equation 4, one obtains Equation 6:

The efficiency can be calculated from the parameter m by using Equation 5:


Table 1. Characteristics of Relative Quantitation Methods


Table 2. Characteristics of Detection Chemistries

Table 1. Characteristics of Relative Quantitation Methods

Table 2. Characteristics of Detection Chemistries

Fig. 1. Real-time PCR detection chemistries. Probe sequences are shown in blue while target DNA sequences are shown in black. Primers are indicated by horizontal arrowheads. Not all unlabeled PCR primers are shown. Oligo, oligonucleotide.

The efficiency can be calculated from the parameter m by using Equation 5:

$$\mathbf{a}\_{\mathcal{E}} = \mathbf{10^{1/m}} \tag{5}$$

By inserting *ɛ* back into Equation 4, one obtains Equation 6:

$$N\_0 = \varepsilon \cdot \mathbb{M} \cdot \mathbb{C}\_T \tag{6}$$

The maximum value for *ɛ* is 2.0 (i.e. the amount of product is doubled in each cycle). The experimental value for *ɛ* usually varies between 1.5 and 1.9. Lower efficiencies limit the sensitivity of the assay but allow quantifications with higher precisions. Therefore, reactions

Overview of Real-Time PCR Principles 425

The problem of where to set the threshold makes relativequantifications difficult if not impossible. However, a simple trickcan be used to combine the advantages of both methods: thereaction mixtures are prepared in duplicate (Gibson, Heid et al. 1996). To one of thesemixtures, the probe specific for the competitor sequence isadded, whereas the probe specific for the sample sequence isadded to the other mixture. This process is carried out for a seriesof reactions with different amounts of competitor added. Withthis procedure, two calibration lines are obtained and theintersection of the two lines is the equivalence point

Melting curves represent the temperature dependence of the fluorescence. They are recorded subsequent to the amplification of the target sequence by PCR. The detection can be performed either with dsDNA-specific dyes like SYBR Green I or with sequence-specific probes such as the molecular beacons and the hybridisation probes (scorpion and sunrise primers cannot be used for melting curve analysis because they are integrated into the PCR products; TaqMan probes cannot be used for melting curve analyses either, since their signal generation depends on the hydrolysis of the probe). Melting curves of sequence-specific probes are used for genotyping, resolving single base mismatches between target sequence and probe (Lay and Wittwer 1997; Whitcombe, Brownie et al. 1998), whereas SYBR Green I is used most frequently for product characterization (Ririe, Rasmussen et al. 1997). It has been reported that melting curves measured with SYBR Green I can also be utilized for genotyping of insertion/deletion polymorphisms and of single nucleotide polymorphisms

In melting curves, the signal decreases gradually as a result of a temperature-dependent quench and more abruptly at a certain temperature because of the melting of the products

(dsDNA or ssDNA/probe hybrid). The melting temperature (Tm) of a product is defined as the temperature at which the steepest decrease of signal occurs. This can be identified conveniently as the peak value(s) (global or local maxima) in the negative derivative of the melting curve. Additionally, the area under the curve (AUC) of the peaks is proportional to the amount of product. Therefore, melting curve analysis may be used for quantifications with internal standardization when the Tm values of sample and competitor products are significantly different (Al-Robaiy, Rupf et al. 2001). However, well-performed normalization is required to reduce the systematic error due to the temperature dependent quench. This quench also limits the sensitivity of melting curve analyses. At present, there is only one software package available that can remove the quench effects from the data (Wilhelm,

With SYBR Green I, the amplification of the correct target sequence can be confirmed. In most cases, nonspecific products have different lengths and therefore deviating melting temperatures (Ririe, Rasmussen et al. 1997).Hybridisation probes, molecular beacons and TaqMan probes are used for mutation detection (Lay and Wittwer 1997; Bernard, Ajioka et al. 1998; Bernard and Wittwer 2000), genotyping (Whitcombe, Brownie et al. 1998; Ulvik and Ueland 2001; Grant, Steinlicht et al. 2002; Randen, Sørensen et al. 2003) and SNP screening

(Sasvari‐Szekely, Gerstner et al. 2000; Mhlanga and Malmberg 2001).

(Wilhelm and Pingoud 2003).

**10. Melting curve analyses** 

Pingoud et al. 2003).

(SNPs) (Akey, Sosnoski et al. 2001; Lin, Tseng et al. 2001).

should be optimized for high efficiency. The effect of the efficiency on the precision, however, is not pronounced.

With more than six orders of magnitude, the dynamic range of this procedure is extraordinarily high (Marcucci, Livak et al. 1998; Verhagen, Willemse et al. 2000;Sails, Fox et al. 2003). The accuracy of this technique is limited by the precision of the determination of the CT values. The error of the CT values results from the signal noise and the CT calculation method. In highly optimized assays, standard errors of less than ± 0.2 cycles can be achieved. By assuming an amplification efficiency of 2 (i.e. 100 %), this implies that the minimum relative error for the quantification is about 10- 20%. The effects of different analysis and calculation methods and the effects of amplification-independent signal trends on the accuracy and precision of quantifications by realtime PCR are described in detail in papers by Lui et al. and Wilhelm et al (Liu and Saint 2002; Wilhelm, Pingoud et al. 2003).

Quantification is relative to the standard used. Only when the absolute concentration of the template molecules in the standard sample is known can the results be absolute. However, in most cases, determination of absolute concentrations is not required. That real-time PCR allows absolute quantification is demonstrated in principle by the reported determination of genome sizes (Wilhelm, Pingoud et al. 2003).

All quantifications by PCR are relative, either to a standard or to a reference gene. Interestingly, Equation 6 nicely illustrates the relative character of the quantifications using a dilution series of a standard; the meaning of the parameter b is the expected CT value of a sample with 'one' copy (or any other unit as defined by the operator). The difference of this value minus the CT value determined for the unknown sample (∆CT = *b* - CT) is a direct measure for the relative difference in template concentrations of the unknown and standard (Wilhelm and Pingoud 2003).

To analyse relative changes in transcript levels, the chosen standard is usually a reference transcript, for example from a housekeeping gene, itself with unknown template concentration. The calculation of ∆CT values between reference and sample transcript in a reference and a test sample then provides a simple tool to estimate relative changes. The derivation, assumptions and applications of the so-called 2∆∆CT method are described elsewhere by Livak et al (Livak and Schmittgen 2001). The results of this method are only semiquantitative because the efficiency *ɛ* is assumed to be 2.0 in all experiments and for all templates, which is at best an optimistic estimate. More precise results are obtained with a procedure introduced by Pfaffl et al., which includes a measured value for *ɛ* (Pfaffl 2001; Wilhelm and Pingoud 2003).

In general, care must also be taken for accurate quantifications with external standardization, especially with respect to polymerase inhibitors, which may be present in differentconcentrations in the unknowns and standards. This problem is circumvented by internal standardization. Here, an analytically distinguishable standard template ('competitor') is added to the sample and co-amplified in the same reaction (Gilliland, Perrin et al. 1990; Goerke, Bayer et al. 2001). The direct and simultaneous quantitative analysis of both products in realtime PCR also poses problems. These difficulties are mostly due to the fact that different fluorophors have to be used to distinguish the sequences of competitor and sample. As a result of different FRET and quantum efficiencies, the CT values obtained for competitor and sample are not directly comparable.

should be optimized for high efficiency. The effect of the efficiency on the precision,

With more than six orders of magnitude, the dynamic range of this procedure is extraordinarily high (Marcucci, Livak et al. 1998; Verhagen, Willemse et al. 2000;Sails, Fox et al. 2003). The accuracy of this technique is limited by the precision of the determination of the CT values. The error of the CT values results from the signal noise and the CT calculation method. In highly optimized assays, standard errors of less than ± 0.2 cycles can be achieved. By assuming an amplification efficiency of 2 (i.e. 100 %), this implies that the minimum relative error for the quantification is about 10- 20%. The effects of different analysis and calculation methods and the effects of amplification-independent signal trends on the accuracy and precision of quantifications by realtime PCR are described in detail in papers by Lui et al. and Wilhelm et al (Liu and Saint 2002; Wilhelm, Pingoud et al. 2003).

Quantification is relative to the standard used. Only when the absolute concentration of the template molecules in the standard sample is known can the results be absolute. However, in most cases, determination of absolute concentrations is not required. That real-time PCR allows absolute quantification is demonstrated in principle by the reported determination of

All quantifications by PCR are relative, either to a standard or to a reference gene. Interestingly, Equation 6 nicely illustrates the relative character of the quantifications using a dilution series of a standard; the meaning of the parameter b is the expected CT value of a sample with 'one' copy (or any other unit as defined by the operator). The difference of this value minus the CT value determined for the unknown sample (∆CT = *b* - CT) is a direct measure for the relative difference in template concentrations of the unknown and standard

To analyse relative changes in transcript levels, the chosen standard is usually a reference transcript, for example from a housekeeping gene, itself with unknown template concentration. The calculation of ∆CT values between reference and sample transcript in a reference and a test sample then provides a simple tool to estimate relative changes. The derivation, assumptions and applications of the so-called 2∆∆CT method are described elsewhere by Livak et al (Livak and Schmittgen 2001). The results of this method are only semiquantitative because the efficiency *ɛ* is assumed to be 2.0 in all experiments and for all templates, which is at best an optimistic estimate. More precise results are obtained with a procedure introduced by Pfaffl et al., which includes a measured value for *ɛ* (Pfaffl 2001;

In general, care must also be taken for accurate quantifications with external standardization, especially with respect to polymerase inhibitors, which may be present in differentconcentrations in the unknowns and standards. This problem is circumvented by internal standardization. Here, an analytically distinguishable standard template ('competitor') is added to the sample and co-amplified in the same reaction (Gilliland, Perrin et al. 1990; Goerke, Bayer et al. 2001). The direct and simultaneous quantitative analysis of both products in realtime PCR also poses problems. These difficulties are mostly due to the fact that different fluorophors have to be used to distinguish the sequences of competitor and sample. As a result of different FRET and quantum efficiencies, the CT values obtained

however, is not pronounced.

genome sizes (Wilhelm, Pingoud et al. 2003).

(Wilhelm and Pingoud 2003).

Wilhelm and Pingoud 2003).

for competitor and sample are not directly comparable.

The problem of where to set the threshold makes relativequantifications difficult if not impossible. However, a simple trickcan be used to combine the advantages of both methods: thereaction mixtures are prepared in duplicate (Gibson, Heid et al. 1996). To one of thesemixtures, the probe specific for the competitor sequence isadded, whereas the probe specific for the sample sequence isadded to the other mixture. This process is carried out for a seriesof reactions with different amounts of competitor added. Withthis procedure, two calibration lines are obtained and theintersection of the two lines is the equivalence point (Wilhelm and Pingoud 2003).

## **10. Melting curve analyses**

Melting curves represent the temperature dependence of the fluorescence. They are recorded subsequent to the amplification of the target sequence by PCR. The detection can be performed either with dsDNA-specific dyes like SYBR Green I or with sequence-specific probes such as the molecular beacons and the hybridisation probes (scorpion and sunrise primers cannot be used for melting curve analysis because they are integrated into the PCR products; TaqMan probes cannot be used for melting curve analyses either, since their signal generation depends on the hydrolysis of the probe). Melting curves of sequence-specific probes are used for genotyping, resolving single base mismatches between target sequence and probe (Lay and Wittwer 1997; Whitcombe, Brownie et al. 1998), whereas SYBR Green I is used most frequently for product characterization (Ririe, Rasmussen et al. 1997). It has been reported that melting curves measured with SYBR Green I can also be utilized for genotyping of insertion/deletion polymorphisms and of single nucleotide polymorphisms (SNPs) (Akey, Sosnoski et al. 2001; Lin, Tseng et al. 2001).

In melting curves, the signal decreases gradually as a result of a temperature-dependent quench and more abruptly at a certain temperature because of the melting of the products

(dsDNA or ssDNA/probe hybrid). The melting temperature (Tm) of a product is defined as the temperature at which the steepest decrease of signal occurs. This can be identified conveniently as the peak value(s) (global or local maxima) in the negative derivative of the melting curve. Additionally, the area under the curve (AUC) of the peaks is proportional to the amount of product. Therefore, melting curve analysis may be used for quantifications with internal standardization when the Tm values of sample and competitor products are significantly different (Al-Robaiy, Rupf et al. 2001). However, well-performed normalization is required to reduce the systematic error due to the temperature dependent quench. This quench also limits the sensitivity of melting curve analyses. At present, there is only one software package available that can remove the quench effects from the data (Wilhelm, Pingoud et al. 2003).

With SYBR Green I, the amplification of the correct target sequence can be confirmed. In most cases, nonspecific products have different lengths and therefore deviating melting temperatures (Ririe, Rasmussen et al. 1997).Hybridisation probes, molecular beacons and TaqMan probes are used for mutation detection (Lay and Wittwer 1997; Bernard, Ajioka et al. 1998; Bernard and Wittwer 2000), genotyping (Whitcombe, Brownie et al. 1998; Ulvik and Ueland 2001; Grant, Steinlicht et al. 2002; Randen, Sørensen et al. 2003) and SNP screening (Sasvari‐Szekely, Gerstner et al. 2000; Mhlanga and Malmberg 2001).

Overview of Real-Time PCR Principles 427

analysis (Worm, Aggerholm et al. 2001; Akey, Akey et al. 2002), which simplifies the

In brief, the advantages of real-time PCR are exploited in clinical diagnosis and the monitoring of infectious diseases and tumors. The technique is applied for the analysis of age dependent diseases, cytokine and tissue-specific expression, forensic samples, epigenetic factors like DNA methylation and for food monitoring. The field of applications is still growing rapidly, which suggests that real-time PCR will become one of the most important

Gene expression analysis at the messenger RNA (mRNA) level has become increasingly important in biological research. Generally we detect RNAs to determine if differences protein expression could be explained at the transcriptional level. In particular, measurement of mRNA is needed in situations where quantification of the protein is difficult or cumbersome. Most recently, mRNA expression analysis is being used to provide insight into complex regulatory networks and to identify genes relevant to new biological processes or implicated in diseases (Hendriks-Balk, Michel et al. 2007).Common methods for RNA detection include: Northern blotting, in situ hybridization, qualitative RTPCR, RNase protection assay, competitive RT-PCR, microarray analysis, and quantitative real-time PCR. The specificity, wide dynamic range , ease-of-use , requiring a minimal amount of RNA, no post-PCR handling and avoiding the use of radioactivity, has made the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) the method of choice for quantitating RNA levels (Radonic, Thulke et al. 2004). The technique has two main steps: CDNA synthesis by reverse transcription of mRNA and subsequent quantification of specific CDNAs by real-time PCR. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity so these specification has led to made it the "gold standard" for mRNA quantification (Huggett, Dheda et al. 2005). Most gene expression assays are based on the comparison of two or more samples and require uniform sampling conditions for this comparison to be valid. Unfortunately, many factors can contribute to variability in the analysis of samples, making the results difficult to reproduce between experiments. During the preparation of CDNA for real-time PCR analysis there is significant potential for small errors to accumulate. For example, differences in sample size, RNA extraction efficiency, pippetting accuracy and reverse transcription efficiency will all add variability to your samples (Huggett, Dheda et al. 2005). Not only can the quantity and quality of RNA extracted from multiple samples vary, but even replicates can vary dramatically due to factors such as sample degradation, extraction efficiency, and contamination. On the other hand, since many biological samples contain inhibitors of the RT and/or the PCR step, it is crucial to assess the presence of any inhibitors of polymerase activity in RT and PCR. so it is clear that we need to incorporate some normalization method to control for errors. The identification of a valid reference for data normalisation remains the most stubborn of problems and none of the solutions proposed are ideal. Normalization methods range from ensuring that a similar sample size is chosen to using an internal housekeeping or reference gene (Table

analysis of epigenetic variations of the genome and developmental processes.

techniques in molecular life sciences and medicine (Wilhelm and Pingoud 2003).

**12. Normalization** 

3) (Huggett, Dheda et al. 2005).

## **11. Applications**

Real-time PCR is used for absolute and relative quantifications of DNA and RNA template molecules and for genotyping in a variety of applications (Wilhelm and Pingoud 2003).

Quantitative real-time PCR is used to determine viral loads (Mackay, Arden et al. 2002),gene expression (Bustin 2000; Goerke, Bayer et al. 2001), titers of germs and contaminations (infood, blood, other body fluids and tissues) (Locatelli, Urso et al. 2000; Hernandez, Rio et al. 2001; Norton 2002), allele imbalances (Ruiz-Ponte, Loidi et al. 2000) and the degrees of amplification and deletion ofgenes (Chiang, Wei et al. 1999; Nigro, Takahashi et al. 2001).

Real-time PCR is also becoming increasingly important in thediagnosis of tumors, such as for the detection and monitoringof minimal residual diseases (Marcucci, Livak et al. 1998; Elmaagacli, Beelen et al. 2000; Amabile, Giannini et al. 2001; Krauter, Heil et al. 2001; Krauter, Hoellge et al. 2001), the identification of micrometastases in colorectal cancer (Bustin, Gyselman et al. 1999), neuroblastoma (Cheung and Cheung 2001) and prostate cancer (Gelmini, Tricarico et al. 2001). It has been used to quantify amplifications of oncogenes (Bieche, Laurendeau et al. 1999; Lehmann, Glöckner et al. 2000; Lyon, Millson et al. 2001; Konigshoff, Wilhelm et al. 2003) as well as deletions of tumor suppressor genes in tumor samples (Wilhelm and Pingoud 2003). Also, the response of human cancer to drugs has been studied (Au, Chim et al. 2002; Miyoshi, Ando et al. 2002;Reimer, Koczan et al. 2002). Other clinically relevant applications are cytokine mRNA profiling in immune response (Hempel, Smith et al. 2002; Stordeur, Poulin et al. 2002) and tissue-specific gene expression analysis (Bustin 2002; Poola 2003; Prieto-Alamo, Cabrera-Luque et al. 2003).

Also, the results of DNA chip experiments are validated by real-time PCR quantifications (Miyazato, Ueno et al. 2001; Rickman, Bobek et al. 2001;Crnogorac-Jurcevic, Efthimiou et al. 2002).

Chimerism analysis is possible when sequence-specific probes are utilized to differentiate and quantify alleles. High dynamic ranges can be achieved with allele-specific real-time PCR (Shively, Chang et al. 2003). Robust chimerism analyses with extremely large dynamic ranges based on insertion/deletion polymorphisms and on SNPs are also possible (Wilhelm, Reuter et al. 2002; Maas, Schaap et al. 2003). Genetic chimerisms have been monitored by Ychromosome-specific real-time PCR for sex-mismatched transplantations (Fehse, Chukhlovin et al. 2001; Byrne, Huang et al. 2002; Elmaagacli 2002) and by allele-specific realtime PCR (Maas, Schaap et al. 2003; Shively, Chang et al. 2003). This combination of allelespecific amplification with real-time PCR has been shown to reveal detection limits of down to 0.01% for SNPs (Maas, Schaap et al. 2003). Real-time PCR is increasingly used in forensic analyses (Andreasson, Gyllensten et al. 2002; von Wurmb-Schwark, Higuchi et al. 2002; Ye, Parra et al. 2002), but also to monitor disease- or age-related accumulation of deletions in the mitochondrial genome (Mehmet, Ahmed et al. 2001; He, Chinnery et al. 2002).

Melting curve analyses are used for real-time competitive PCR (Al-Robaiy, Rupf et al. 2001; Lyon, Millson et al. 2001), gene dosage tests (Ruiz-Ponte, Loidi et al. 2000) and genotyping and SNP detection (Bullock, Bruns et al. 2002; Burian, Grosch et al. 2002; Randen, Sørensen et al. 2003). These applications will have a particularly strong impact on pharmacogenetics (Palladino, Kay et al. 2003). Profiling of DNA methylation is also possible by melting curve analysis (Worm, Aggerholm et al. 2001; Akey, Akey et al. 2002), which simplifies the analysis of epigenetic variations of the genome and developmental processes.

In brief, the advantages of real-time PCR are exploited in clinical diagnosis and the monitoring of infectious diseases and tumors. The technique is applied for the analysis of age dependent diseases, cytokine and tissue-specific expression, forensic samples, epigenetic factors like DNA methylation and for food monitoring. The field of applications is still growing rapidly, which suggests that real-time PCR will become one of the most important techniques in molecular life sciences and medicine (Wilhelm and Pingoud 2003).

## **12. Normalization**

426 Polymerase Chain Reaction

Real-time PCR is used for absolute and relative quantifications of DNA and RNA template molecules and for genotyping in a variety of applications (Wilhelm and Pingoud 2003).

Quantitative real-time PCR is used to determine viral loads (Mackay, Arden et al. 2002),gene expression (Bustin 2000; Goerke, Bayer et al. 2001), titers of germs and contaminations (infood, blood, other body fluids and tissues) (Locatelli, Urso et al. 2000; Hernandez, Rio et al. 2001; Norton 2002), allele imbalances (Ruiz-Ponte, Loidi et al. 2000) and the degrees of amplification and deletion ofgenes (Chiang, Wei et al. 1999; Nigro,

Real-time PCR is also becoming increasingly important in thediagnosis of tumors, such as for the detection and monitoringof minimal residual diseases (Marcucci, Livak et al. 1998; Elmaagacli, Beelen et al. 2000; Amabile, Giannini et al. 2001; Krauter, Heil et al. 2001; Krauter, Hoellge et al. 2001), the identification of micrometastases in colorectal cancer (Bustin, Gyselman et al. 1999), neuroblastoma (Cheung and Cheung 2001) and prostate cancer (Gelmini, Tricarico et al. 2001). It has been used to quantify amplifications of oncogenes (Bieche, Laurendeau et al. 1999; Lehmann, Glöckner et al. 2000; Lyon, Millson et al. 2001; Konigshoff, Wilhelm et al. 2003) as well as deletions of tumor suppressor genes in tumor samples (Wilhelm and Pingoud 2003). Also, the response of human cancer to drugs has been studied (Au, Chim et al. 2002; Miyoshi, Ando et al. 2002;Reimer, Koczan et al. 2002). Other clinically relevant applications are cytokine mRNA profiling in immune response (Hempel, Smith et al. 2002; Stordeur, Poulin et al. 2002) and tissue-specific gene expression analysis (Bustin 2002; Poola 2003; Prieto-Alamo, Cabrera-Luque et al. 2003).

Also, the results of DNA chip experiments are validated by real-time PCR quantifications (Miyazato, Ueno et al. 2001; Rickman, Bobek et al. 2001;Crnogorac-Jurcevic, Efthimiou et al.

Chimerism analysis is possible when sequence-specific probes are utilized to differentiate and quantify alleles. High dynamic ranges can be achieved with allele-specific real-time PCR (Shively, Chang et al. 2003). Robust chimerism analyses with extremely large dynamic ranges based on insertion/deletion polymorphisms and on SNPs are also possible (Wilhelm, Reuter et al. 2002; Maas, Schaap et al. 2003). Genetic chimerisms have been monitored by Ychromosome-specific real-time PCR for sex-mismatched transplantations (Fehse, Chukhlovin et al. 2001; Byrne, Huang et al. 2002; Elmaagacli 2002) and by allele-specific realtime PCR (Maas, Schaap et al. 2003; Shively, Chang et al. 2003). This combination of allelespecific amplification with real-time PCR has been shown to reveal detection limits of down to 0.01% for SNPs (Maas, Schaap et al. 2003). Real-time PCR is increasingly used in forensic analyses (Andreasson, Gyllensten et al. 2002; von Wurmb-Schwark, Higuchi et al. 2002; Ye, Parra et al. 2002), but also to monitor disease- or age-related accumulation of deletions in the mitochondrial genome (Mehmet, Ahmed et al. 2001; He, Chinnery et al.

Melting curve analyses are used for real-time competitive PCR (Al-Robaiy, Rupf et al. 2001; Lyon, Millson et al. 2001), gene dosage tests (Ruiz-Ponte, Loidi et al. 2000) and genotyping and SNP detection (Bullock, Bruns et al. 2002; Burian, Grosch et al. 2002; Randen, Sørensen et al. 2003). These applications will have a particularly strong impact on pharmacogenetics (Palladino, Kay et al. 2003). Profiling of DNA methylation is also possible by melting curve

**11. Applications** 

Takahashi et al. 2001).

2002).

2002).

Gene expression analysis at the messenger RNA (mRNA) level has become increasingly important in biological research. Generally we detect RNAs to determine if differences protein expression could be explained at the transcriptional level. In particular, measurement of mRNA is needed in situations where quantification of the protein is difficult or cumbersome. Most recently, mRNA expression analysis is being used to provide insight into complex regulatory networks and to identify genes relevant to new biological processes or implicated in diseases (Hendriks-Balk, Michel et al. 2007).Common methods for RNA detection include: Northern blotting, in situ hybridization, qualitative RTPCR, RNase protection assay, competitive RT-PCR, microarray analysis, and quantitative real-time PCR. The specificity, wide dynamic range , ease-of-use , requiring a minimal amount of RNA, no post-PCR handling and avoiding the use of radioactivity, has made the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) the method of choice for quantitating RNA levels (Radonic, Thulke et al. 2004). The technique has two main steps: CDNA synthesis by reverse transcription of mRNA and subsequent quantification of specific CDNAs by real-time PCR. It is in many cases the only method for measuring mRNA levels of vivo low copy number targets of interest for which alternative assays either do not exist or lack the required sensitivity so these specification has led to made it the "gold standard" for mRNA quantification (Huggett, Dheda et al. 2005). Most gene expression assays are based on the comparison of two or more samples and require uniform sampling conditions for this comparison to be valid. Unfortunately, many factors can contribute to variability in the analysis of samples, making the results difficult to reproduce between experiments. During the preparation of CDNA for real-time PCR analysis there is significant potential for small errors to accumulate. For example, differences in sample size, RNA extraction efficiency, pippetting accuracy and reverse transcription efficiency will all add variability to your samples (Huggett, Dheda et al. 2005). Not only can the quantity and quality of RNA extracted from multiple samples vary, but even replicates can vary dramatically due to factors such as sample degradation, extraction efficiency, and contamination. On the other hand, since many biological samples contain inhibitors of the RT and/or the PCR step, it is crucial to assess the presence of any inhibitors of polymerase activity in RT and PCR. so it is clear that we need to incorporate some normalization method to control for errors. The identification of a valid reference for data normalisation remains the most stubborn of problems and none of the solutions proposed are ideal. Normalization methods range from ensuring that a similar sample size is chosen to using an internal housekeeping or reference gene (Table 3) (Huggett, Dheda et al. 2005).

Overview of Real-Time PCR Principles 429

milliliter of blood in the latter group. Even cellular subpopulations of the same pathological origin can be highly heterogeneous. Tumor biopsies, in particular, are made up not just of normal and cancer epithelial cells, but there may be several subclones of epithelial cancer cells together with stromal, immune and vascular components (Vandesompele, De Preter et al. 2002; Bustin, Benes et al. 2005). This variability can give us misleading or meaningless result to solve this we can use laser capture microdissection to normalize against the dissected area which can report the target mRNA levels conveniently as copies per area or cell dissected. In in vitro cell culture, due to different morphologies or clumping up of cells, it's hard to determine sample size (cell number) .we can treat them with buffers and/or enzymes till they could be counted, however these treatments surely could affect gene expression. This approach could not be applied for solid tumors for which the amount of cells cannot be determined accurately. To work around this problem, it was suggested to standardize the RT-qPCR data between samples using the amount of genomic DNA as an indicator reflecting the number of cells in each sample. However, these approaches do not account for the degradation of RNA or the efficiency of RT and PCR. So, while ensuring a similar sample size is important it clearly is not sufficient on its own (Huggett, Dheda et al.

Another method for normalization is measuring the amount of genomic DNA (gDNA). This appears to be an ideal method as it does not require reverse transcription for detection by real-time PCR (Bustin 2002). However, this approach do not account for the degradation of RNA or the efficiency of RT and PCR. .Moreover, in the case of normalization with genomic DNA, the fact of working with tumor cells can present additional problems because they tend to have abnormal karyotype. Therefore, the ratio between the amount of DNA and cell number is variable (Huggett, Dheda et al. 2005). Another major problem with using this strategy is that RNA extraction procedures are usually not designed to purify DNA, so the extraction rate may vary between different samples, with DNA yields often being low. In conclude Normalization against genomic DNA is rarely used since it is difficult to coextract

The normalization of RT-qPCR results can be compared to the amount of total RNA used in the reverse transcription step. Not only does this facilitates normalization but circumvents problems associated with the linearity of the reverse transcriptase step. There are several methods for quantifying RNA; the most common is to measure the absorbance at 260 nm (A 260) with a UV spectrophotometer. The major advantage of this spectrometer, whose sensitivity is estimated at 5 ng/uL, is that it requires only 1 microL of sample, placed in direct contact with the optical system (Huggett, Dheda et al. 2005; Hendriks-Balk, Michel et al. 2007). However, contaminants absorbing at 260 nm, such as proteins, phenol or genomic DNA, can lead to overestimated results. Another optical system is flourimetry in which intercalating fluorescent nucleic acid is used, the kit RiboGreen ® Molecular Probes based on this principle. This is a more sensitive technique but does not discriminate RNA from DNA, and contaminants such as phenol can produce variable results (Huggett, Dheda et al. 2005). Since it is generally assumed that OD260 analysis is less accurate than the RiboGreen assay, we have compared RNA quantification data obtained using the RiboGreen assay with

with RNA and it may vary in copy number per cell (Huggett, Dheda et al. 2005).

2005).

**2. Normalization with genomic DNA** 

**3. Normalization with total RNA** 


(Huggett, Dheda et al. 2005)

Table 3. Comparison of the actual amount of RNA used in different reverse transcription reactions with the respective amount of HuPO

Comparison of the actual amount of RNA used in different reverse transcription reactions with the respective amount of HuPO

cDNA measured by real-time RT-PCR

#### **12.1 Methods of normalization**

#### **1. Standardizing Sample size**

The most basic method of normalization ensures that an experiment compares similar sample sizes and this is achieved by measuring tissue weight, volume or cell number. This method can reduce the experimental error of first stage of qRT-PCR. It seems to be straightforward, but we can't ensure that equal volume of different samples contain the same cellular material. Real-time RT-PCR experiments that rely on the extraction of RNA from complex tissue samples are averaging the data from numerous, variable subpopulations of cells of different lineage at different stages of differentiation (Bustin, Benes et al. 2005). This can be misleading, as is illustrated when sampling a similar volume of blood from HIV +ve patients. Patients with HIV that have less advanced immunosupression (CD4 counts X200 cells/ml) will yield a higher amount RNA than patients with CD4 counts p200 cells/ml. This is simply because there are fewer cells per

Table 3. Comparison of the actual amount of RNA used in different reverse transcription

Comparison of the actual amount of RNA used in different reverse transcription reactions

The most basic method of normalization ensures that an experiment compares similar sample sizes and this is achieved by measuring tissue weight, volume or cell number. This method can reduce the experimental error of first stage of qRT-PCR. It seems to be straightforward, but we can't ensure that equal volume of different samples contain the same cellular material. Real-time RT-PCR experiments that rely on the extraction of RNA from complex tissue samples are averaging the data from numerous, variable subpopulations of cells of different lineage at different stages of differentiation (Bustin, Benes et al. 2005). This can be misleading, as is illustrated when sampling a similar volume of blood from HIV +ve patients. Patients with HIV that have less advanced immunosupression (CD4 counts X200 cells/ml) will yield a higher amount RNA than patients with CD4 counts p200 cells/ml. This is simply because there are fewer cells per

(Huggett, Dheda et al. 2005)

reactions with the respective amount of HuPO

with the respective amount of HuPO cDNA measured by real-time RT-PCR

**12.1 Methods of normalization 1. Standardizing Sample size** 

milliliter of blood in the latter group. Even cellular subpopulations of the same pathological origin can be highly heterogeneous. Tumor biopsies, in particular, are made up not just of normal and cancer epithelial cells, but there may be several subclones of epithelial cancer cells together with stromal, immune and vascular components (Vandesompele, De Preter et al. 2002; Bustin, Benes et al. 2005). This variability can give us misleading or meaningless result to solve this we can use laser capture microdissection to normalize against the dissected area which can report the target mRNA levels conveniently as copies per area or cell dissected. In in vitro cell culture, due to different morphologies or clumping up of cells, it's hard to determine sample size (cell number) .we can treat them with buffers and/or enzymes till they could be counted, however these treatments surely could affect gene expression. This approach could not be applied for solid tumors for which the amount of cells cannot be determined accurately. To work around this problem, it was suggested to standardize the RT-qPCR data between samples using the amount of genomic DNA as an indicator reflecting the number of cells in each sample. However, these approaches do not account for the degradation of RNA or the efficiency of RT and PCR. So, while ensuring a similar sample size is important it clearly is not sufficient on its own (Huggett, Dheda et al. 2005).

#### **2. Normalization with genomic DNA**

Another method for normalization is measuring the amount of genomic DNA (gDNA). This appears to be an ideal method as it does not require reverse transcription for detection by real-time PCR (Bustin 2002). However, this approach do not account for the degradation of RNA or the efficiency of RT and PCR. .Moreover, in the case of normalization with genomic DNA, the fact of working with tumor cells can present additional problems because they tend to have abnormal karyotype. Therefore, the ratio between the amount of DNA and cell number is variable (Huggett, Dheda et al. 2005). Another major problem with using this strategy is that RNA extraction procedures are usually not designed to purify DNA, so the extraction rate may vary between different samples, with DNA yields often being low. In conclude Normalization against genomic DNA is rarely used since it is difficult to coextract with RNA and it may vary in copy number per cell (Huggett, Dheda et al. 2005).

#### **3. Normalization with total RNA**

The normalization of RT-qPCR results can be compared to the amount of total RNA used in the reverse transcription step. Not only does this facilitates normalization but circumvents problems associated with the linearity of the reverse transcriptase step. There are several methods for quantifying RNA; the most common is to measure the absorbance at 260 nm (A 260) with a UV spectrophotometer. The major advantage of this spectrometer, whose sensitivity is estimated at 5 ng/uL, is that it requires only 1 microL of sample, placed in direct contact with the optical system (Huggett, Dheda et al. 2005; Hendriks-Balk, Michel et al. 2007). However, contaminants absorbing at 260 nm, such as proteins, phenol or genomic DNA, can lead to overestimated results. Another optical system is flourimetry in which intercalating fluorescent nucleic acid is used, the kit RiboGreen ® Molecular Probes based on this principle. This is a more sensitive technique but does not discriminate RNA from DNA, and contaminants such as phenol can produce variable results (Huggett, Dheda et al. 2005). Since it is generally assumed that OD260 analysis is less accurate than the RiboGreen assay, we have compared RNA quantification data obtained using the RiboGreen assay with

Overview of Real-Time PCR Principles 431

total RNA assumes that the rRNA:mRNA ratio is the same in all groups, which might not always be the case. Moreover, rRNA is not present in purified mRNA and the high abundance of rRNA compared to mRNA makes it difficult to subtract the baseline value in realtime PCR analysis. Thus, markers of rRNA such as 18S or 28S rRNA might also be suboptimal as normalization factors in many settings (Hendriks-Balk, Michel et al. 2007). Also, it has been reported that rRNA transcription is affected by biological factors and drugs. An important parameter to consider when normalized relative to the RNA is the quality of it. Differences in the quality of the samples strongly depend on the extraction step and are the source of the most common variations in RT-qPCR. It is therefore important to use the same method of extraction for all samples analyzed (Vandesompele, De Preter et al. 2002). The quality of RNA is defined by both its purity (no contamination) and integrity (non-degraded RNA). Its purity was determined by measuring the absorbance at 230 (organic contaminants) and 280 nm (specific proteins), RNA is considered pure ratios A 260/ A 230 and A 260 / A 280 > 1.8. With regard to the integrity, the traditional method was to visualize the bands of 28S and 18S ribosomal RNA on a gel electrophoresis. Indeed, it is difficult to analyze directly the mRNA; they represent only 1% to 3% of total RNA. We must therefore consider that the degradation of ribosomal RNA, the majority, reflecting the degradation of mRNA. Thus, the 18S/28S ratio assesses the integrity of RNA; a ratio close to 2 is considered an indicator of RNA with little or no gradient. However, this method requires a large amount of RNA (0.5-2 mg), and is not sensitive enough to detect slight damage. Normalizing a sample against total RNA has the drawback of not controlling for variation inherent in the reverse transcription or PCR reactions and it ignores the efficiency of converting RNA into CDNA. Also rRNA cannot be used for normalization when quantifying targets from polyA-enriched samples (Huggett, Dheda et al. 2005). A final drawback when using total RNA for normalization is the lack of internal control for RT or PCR inhibitors. All quantitative methods assume that the RNA targets are reverse transcribed and subsequently amplified with similar efficiency but the reaction is extremely sensitive to the presence of inhibitors, which can be reagents used in the extraction step (salts, alcohols, phenols), or components copurification organic (urea, heme, heparin, immunoglobulin G). These compounds can also inhibit the PCR reaction. Thus, two reactions with an equal amount of RNA, but the efficiencies of RT and / or PCR are different, will yield results that cannot be compared. Different methods exist to assess the presence of inhibitors in biological samples. First, it is possible to compare the efficacy of PCR for different dilutions (1/20 and 1/80 for example) of a sample. An alternative is to add a defined amount of a synthetic single-stranded amplicon CDNA samples, and comparing its amplification compared to a control without CDNA. However, these methods are limited to verify the absence of PCR inhibitors, and do not evaluate the effectiveness of the reverse

transcription step (Bustin 2002).

**4. Normalization with an artificial molecule (spike)** 

An interesting solution to control the two enzymatic reactions (RT and PCR) is added to RNA extracted an exogenous RNA, which will compare the amplification between the different samples. This sequence control should show no similarity to the target RNA, we will use such a specific mRNA from a plant when studying gene expression in humans. The main criticism of using spikes is that, while they can be introduced prior to extraction, unlike the cellular RNAs they are not extracted from within the tissue. Consequently, there

OD260 analysis using a Genequant II (Pharmacia). The results (Fig. 2) suggest that both methods generate comparable results when the RNA concentration is not less than 100 ng/μl, with RiboGreen measurements lower than those obtained using the spectrophotometer. OD260 analysis becomes less reliable at lower RNA concentrations (Bustin 2002).

Fig. 2. Comparison of RNA quantification using the Genequant II and the RiboGreen fluorescent assay. RNA from 34 normal colon biopsies was quantitated using a standard Genequant II protocol which measures the absorbance at 260 nm. The same samples were then quantitated using a standard RiboGreen fluorescent assay. RT-PCR assays targeting the GHR were carried out and *C*t normalised against the respective concentrations determined by the two methods. The scatterplot shows a good correlation between the two methods (*r*2=0·8612) (Bustin 2002).

What is also important, but often overlooked, is the need to assess the quality of RNA because degraded RNAs may adversely affect results. The opportune development of Agilent's 2100 Bioanalyser and LabChip technology has provided a new standard of RNA quality control as well as permitting concomitant quantification of RNA. The analysis is not influenced by contamination of phenol or proteins, against the presence of genomic DNA requires a correction of the measurement of the concentration of RNA (Bustin, Benes et al. 2005). This is particularly important when the RNA has been extracted from 'dirty' tissue such as the colon (Vandesompele, De Preter et al. 2002). This technique allows characterizing the RNA in a concentration between 5 and 500 ng/uL. For each sample, the software determines the ratio 28S/18S and assigns a RIN (RNA Integrity Number) which takes into account the entire electropherogram. The value of RIN ranges from 1 to 10, with 1 being totally degraded RNA, and 10 to a high-quality RNA. In addition to being fast and allow high throughput, this technique requires only 1 microL of sample. It is the simplest method and objective qualitative analysis of RNA, its use is recommended (Schmittgen and Zakrajsek 2000; Bustin 2002). Similar in concept, but requiring an additional RT-PCR assay, is normalization against one of the rRNAs. rRNA levels may vary less under conditions that affect the expression of mRNAs and the use of rRNA has been claimed to be more reliable than that of several reference genes in rat livers and human skin fibroblasts (Bustin 2002; Huggett, Dheda et al. 2005). But, a drawback is that it primarily measures ribosomal RNA (rRNA) whereas real-time PCR aims to determine mRNA expression and normalization for

OD260 analysis using a Genequant II (Pharmacia). The results (Fig. 2) suggest that both methods generate comparable results when the RNA concentration is not less than 100 ng/μl, with RiboGreen measurements lower than those obtained using the spectrophotometer. OD260 analysis becomes less reliable at lower RNA concentrations

Fig. 2. Comparison of RNA quantification using the Genequant II and the RiboGreen fluorescent assay. RNA from 34 normal colon biopsies was quantitated using a standard Genequant II protocol which measures the absorbance at 260 nm. The same samples were then quantitated using a standard RiboGreen fluorescent assay. RT-PCR assays targeting the GHR were carried out and *C*t normalised against the respective concentrations determined by the two methods. The scatterplot shows a good correlation between the two methods

What is also important, but often overlooked, is the need to assess the quality of RNA because degraded RNAs may adversely affect results. The opportune development of Agilent's 2100 Bioanalyser and LabChip technology has provided a new standard of RNA quality control as well as permitting concomitant quantification of RNA. The analysis is not influenced by contamination of phenol or proteins, against the presence of genomic DNA requires a correction of the measurement of the concentration of RNA (Bustin, Benes et al. 2005). This is particularly important when the RNA has been extracted from 'dirty' tissue such as the colon (Vandesompele, De Preter et al. 2002). This technique allows characterizing the RNA in a concentration between 5 and 500 ng/uL. For each sample, the software determines the ratio 28S/18S and assigns a RIN (RNA Integrity Number) which takes into account the entire electropherogram. The value of RIN ranges from 1 to 10, with 1 being totally degraded RNA, and 10 to a high-quality RNA. In addition to being fast and allow high throughput, this technique requires only 1 microL of sample. It is the simplest method and objective qualitative analysis of RNA, its use is recommended (Schmittgen and Zakrajsek 2000; Bustin 2002). Similar in concept, but requiring an additional RT-PCR assay, is normalization against one of the rRNAs. rRNA levels may vary less under conditions that affect the expression of mRNAs and the use of rRNA has been claimed to be more reliable than that of several reference genes in rat livers and human skin fibroblasts (Bustin 2002; Huggett, Dheda et al. 2005). But, a drawback is that it primarily measures ribosomal RNA (rRNA) whereas real-time PCR aims to determine mRNA expression and normalization for

(Bustin 2002).

(*r*2=0·8612) (Bustin 2002).

total RNA assumes that the rRNA:mRNA ratio is the same in all groups, which might not always be the case. Moreover, rRNA is not present in purified mRNA and the high abundance of rRNA compared to mRNA makes it difficult to subtract the baseline value in realtime PCR analysis. Thus, markers of rRNA such as 18S or 28S rRNA might also be suboptimal as normalization factors in many settings (Hendriks-Balk, Michel et al. 2007). Also, it has been reported that rRNA transcription is affected by biological factors and drugs. An important parameter to consider when normalized relative to the RNA is the quality of it. Differences in the quality of the samples strongly depend on the extraction step and are the source of the most common variations in RT-qPCR. It is therefore important to use the same method of extraction for all samples analyzed (Vandesompele, De Preter et al. 2002). The quality of RNA is defined by both its purity (no contamination) and integrity (non-degraded RNA). Its purity was determined by measuring the absorbance at 230 (organic contaminants) and 280 nm (specific proteins), RNA is considered pure ratios A 260/ A 230 and A 260 / A 280 > 1.8. With regard to the integrity, the traditional method was to visualize the bands of 28S and 18S ribosomal RNA on a gel electrophoresis. Indeed, it is difficult to analyze directly the mRNA; they represent only 1% to 3% of total RNA. We must therefore consider that the degradation of ribosomal RNA, the majority, reflecting the degradation of mRNA. Thus, the 18S/28S ratio assesses the integrity of RNA; a ratio close to 2 is considered an indicator of RNA with little or no gradient. However, this method requires a large amount of RNA (0.5-2 mg), and is not sensitive enough to detect slight damage. Normalizing a sample against total RNA has the drawback of not controlling for variation inherent in the reverse transcription or PCR reactions and it ignores the efficiency of converting RNA into CDNA. Also rRNA cannot be used for normalization when quantifying targets from polyA-enriched samples (Huggett, Dheda et al. 2005). A final drawback when using total RNA for normalization is the lack of internal control for RT or PCR inhibitors. All quantitative methods assume that the RNA targets are reverse transcribed and subsequently amplified with similar efficiency but the reaction is extremely sensitive to the presence of inhibitors, which can be reagents used in the extraction step (salts, alcohols, phenols), or components copurification organic (urea, heme, heparin, immunoglobulin G). These compounds can also inhibit the PCR reaction. Thus, two reactions with an equal amount of RNA, but the efficiencies of RT and / or PCR are different, will yield results that cannot be compared. Different methods exist to assess the presence of inhibitors in biological samples. First, it is possible to compare the efficacy of PCR for different dilutions (1/20 and 1/80 for example) of a sample. An alternative is to add a defined amount of a synthetic single-stranded amplicon CDNA samples, and comparing its amplification compared to a control without CDNA. However, these methods are limited to verify the absence of PCR inhibitors, and do not evaluate the effectiveness of the reverse transcription step (Bustin 2002).

#### **4. Normalization with an artificial molecule (spike)**

An interesting solution to control the two enzymatic reactions (RT and PCR) is added to RNA extracted an exogenous RNA, which will compare the amplification between the different samples. This sequence control should show no similarity to the target RNA, we will use such a specific mRNA from a plant when studying gene expression in humans. The main criticism of using spikes is that, while they can be introduced prior to extraction, unlike the cellular RNAs they are not extracted from within the tissue. Consequently, there

Overview of Real-Time PCR Principles 433

ΔΔCT ΔCT (control) - ΔCT (sample) Finally, the normalized ratio of expression of a target gene is determined by the formula: 2 -

Unlike the relative standard curve method, where the amplification efficiency (E) target genes and reference is directly taken into account when building ranges, the method of ΔΔCT is assumed that the efficiencies of the two genes are equal to 100% (E = 2, with each cycle of the exponential phase, the concentration of PCR products is doubled). However, a difference in PCR efficiency of 3% (ΔE = 0.03) between the two genes results in an error of 47% for the ratio of expression if E target <E ref and 209% if E target > E ref after 25 cycles. In addition, the error increases exponentially with larger variations of efficiency and a greater number of cycles. New models have been developed taking into account the efficiency of PCR target gene and reference gene. The most common is the model of Pfaffl, where the relative expression ratio (R) of a target gene between a sample and control is determined by

R = (������� )Δ� �������(������� ������� )

(���������� )Δ� ����������(������� ������� )

In this model of Pfaffl, the efficiency of PCR for a given gene is calculated from the construction of a calibration curve using the following formula: E = 10 [-1/ gradient]. This method gives a good estimate of effectiveness, although it is possible that it is overestimated. However, this approach assumes that the amplification efficiencies between the diluted samples are identical, creating a linear relationship between C T and amount of CDNA in the beginning. Therefore, some authors such as Liu and Saint have developed models that take into account standards of efficiency for each sample, the latter being determined by the kinetics of the amplification curve. However, with this kind of approach, the slightest error in the measurement of effectiveness is amplified and passed exponentially on the expression ratio calculated. The different models of normalization with reference genes therefore have all the advantages and disadvantages. At present, there is no timehonored method for the treatment of the results of RT-qPCR. Normalization to a reference gene is a simple method and frequently used because it can control many variables. An advantage of reference genes as compared to total or rRNA is that the reference gene is subject to the same conditions as the mRNA of interest (Bustin, Benes et al. 2005; Hendriks-Balk, Michel et al. 2007). What has become apparent over recent years is that there is no single reference gene for all experimental systems. Quantified errors related to the use of a single reference gene as more than three-fold in 25% and more than six-fold in 10% of samples. Today it is clear that reference genes must be carefully validated for each experimental situation and those new experimental conditions or different tissue samples require re-validation of the chosen reference genes (Balogh, Paragh et al. 2008). If inappropriate reference genes are used for normalization, the experimental results obtained can differ greatly from those using a validated reference gene. Validation of a reference gene requires removal of any non-specific variation in expression. This can be done using a

Next, the ΔΔCT between control and the sample is calculated:

ΔΔCT.

the following formula:

may be situations (e.g. if the samples differ histologically) when the spike may not be a good control for the extraction procedure. The stages required to generate the alien molecule may also not be feasible for small laboratories wanting to perform limited amounts of real-time RTPCR (Schmittgen and Zakrajsek 2000; Argyropoulos, Psallida et al. 2006).

#### **5. Normalization with reference genes**

Reference genes represent the by far most common method for normalizing qRT-PCR data. Reference genes are often referred to as housekeeping genes assuming that those genes are expressed at a constant level in various tissues at all stages of development and are unaffected by the experimental treatment (Hendriks-Balk, Michel et al. 2007; Balogh, Paragh et al. 2008). Use of this endogenous control theory allows controlling all stages of the experimental protocol; its expression reflects not only the quantity and quality of RNA used, but the efficiencies of the RT and PCR. An advantage of reference genes as compared to total or rRNA is that the reference gene is subject to the same conditions as the mRNA of interest (Hendriks-Balk, Michel et al. 2007). The most commonly used reference genes include βactin (ACTB), (GAPDH), (HPRT) and 18S rRNA. The other commonly used reference genes would be PGK1, B2M, GAPD, HMBS, HPRT1, RPL13A, SDHA, TBP, UBC and YWHAZ (Vandesompele, De Preter et al. 2002). The initial concentration of a target is usually derived from the CT (cycle threshold), which is the number of amplification cycles where the amplification curve crosses the threshold line. This line is placed at the exponential phase, so as to be clearly distinguishable from background noise. For each sample, the CT obtained for the genes of interest and reference must be converted to normalized expression ratio. For this, various options are available, they are integrated in the software provided with the various qPCR instruments or described in the literature. The relative standard curve method requires the construction, for the target gene and reference gene, a range made from a series of dilutions of a reference sample. These ranges to obtain standard curves, obtained by expressing the CT as a function of log of the initial concentration of cDNA. Concentration values for each point of the range can be set arbitrarily in accordance with the dilution factors. Therefore, the relative amount of a target is determined by the CT by interpolation with the standard curve. The standard expression of a gene of interest is determined by the following formula:

#### R= ೃೌೡ ೌೠ ೞ ோ௧௩ ௨௧ ௧

In addition, a calibrator is typically used. This is a sample used as a basis for comparing results. The normalized ratio of each sample is divided by the normalized ratio of the calibrator. Thus, the calibrator becomes the reference 1x, and all other samples are expressed as a ratio relative to the calibrator. The method of ΔΔC T uses a mathematical formule to calculate the ratio of expression of a target gene between two samples, normalized with reference gene. First, the differences ΔC T between the values of CT target gene and reference gene were determined for the test sample and control.

ΔCT (sample) = CT (target sample) - CT (reference sample)

ΔCt (control) = CT (target control) - CT (reference control)

may be situations (e.g. if the samples differ histologically) when the spike may not be a good control for the extraction procedure. The stages required to generate the alien molecule may also not be feasible for small laboratories wanting to perform limited amounts of real-time

Reference genes represent the by far most common method for normalizing qRT-PCR data. Reference genes are often referred to as housekeeping genes assuming that those genes are expressed at a constant level in various tissues at all stages of development and are unaffected by the experimental treatment (Hendriks-Balk, Michel et al. 2007; Balogh, Paragh et al. 2008). Use of this endogenous control theory allows controlling all stages of the experimental protocol; its expression reflects not only the quantity and quality of RNA used, but the efficiencies of the RT and PCR. An advantage of reference genes as compared to total or rRNA is that the reference gene is subject to the same conditions as the mRNA of interest (Hendriks-Balk, Michel et al. 2007). The most commonly used reference genes include βactin (ACTB), (GAPDH), (HPRT) and 18S rRNA. The other commonly used reference genes would be PGK1, B2M, GAPD, HMBS, HPRT1, RPL13A, SDHA, TBP, UBC and YWHAZ (Vandesompele, De Preter et al. 2002). The initial concentration of a target is usually derived from the CT (cycle threshold), which is the number of amplification cycles where the amplification curve crosses the threshold line. This line is placed at the exponential phase, so as to be clearly distinguishable from background noise. For each sample, the CT obtained for the genes of interest and reference must be converted to normalized expression ratio. For this, various options are available, they are integrated in the software provided with the various qPCR instruments or described in the literature. The relative standard curve method requires the construction, for the target gene and reference gene, a range made from a series of dilutions of a reference sample. These ranges to obtain standard curves, obtained by expressing the CT as a function of log of the initial concentration of cDNA. Concentration values for each point of the range can be set arbitrarily in accordance with the dilution factors. Therefore, the relative amount of a target is determined by the CT by interpolation with the standard curve. The standard expression of a gene of interest is determined by the

> R= ೃೌೡ ೌೠ ೞ ோ௧௩ ௨௧ ௧

In addition, a calibrator is typically used. This is a sample used as a basis for comparing results. The normalized ratio of each sample is divided by the normalized ratio of the calibrator. Thus, the calibrator becomes the reference 1x, and all other samples are expressed as a ratio relative to the calibrator. The method of ΔΔC T uses a mathematical formule to calculate the ratio of expression of a target gene between two samples, normalized with reference gene. First, the differences ΔC T between the values of CT target gene and reference

ΔCT (sample) = CT (target sample) - CT (reference sample)

ΔCt (control) = CT (target control) - CT (reference control)

gene were determined for the test sample and control.

RTPCR (Schmittgen and Zakrajsek 2000; Argyropoulos, Psallida et al. 2006).

**5. Normalization with reference genes** 

following formula:

Next, the ΔΔCT between control and the sample is calculated:

$$\Delta\Delta\text{C}\_{\text{T}}\Delta\text{C}\_{\text{T}}\text{(control)} - \Delta\text{C}\_{\text{T}}\text{(sample)}$$

Finally, the normalized ratio of expression of a target gene is determined by the formula: 2 - ΔΔCT.

Unlike the relative standard curve method, where the amplification efficiency (E) target genes and reference is directly taken into account when building ranges, the method of ΔΔCT is assumed that the efficiencies of the two genes are equal to 100% (E = 2, with each cycle of the exponential phase, the concentration of PCR products is doubled). However, a difference in PCR efficiency of 3% (ΔE = 0.03) between the two genes results in an error of 47% for the ratio of expression if E target <E ref and 209% if E target > E ref after 25 cycles. In addition, the error increases exponentially with larger variations of efficiency and a greater number of cycles. New models have been developed taking into account the efficiency of PCR target gene and reference gene. The most common is the model of Pfaffl, where the relative expression ratio (R) of a target gene between a sample and control is determined by the following formula:

$$\mathcal{R} = \frac{(E\_{\text{target}})^{\Delta \mathcal{C}} \text{T}^{\text{target}}(\text{control} \mid \text{-sample})}{(E\_{\text{reference}})^{\Delta \mathcal{C}} \text{T}^{\text{reference}}(\text{control} \mid \text{-sample})}$$

In this model of Pfaffl, the efficiency of PCR for a given gene is calculated from the construction of a calibration curve using the following formula: E = 10 [-1/ gradient]. This method gives a good estimate of effectiveness, although it is possible that it is overestimated. However, this approach assumes that the amplification efficiencies between the diluted samples are identical, creating a linear relationship between C T and amount of CDNA in the beginning. Therefore, some authors such as Liu and Saint have developed models that take into account standards of efficiency for each sample, the latter being determined by the kinetics of the amplification curve. However, with this kind of approach, the slightest error in the measurement of effectiveness is amplified and passed exponentially on the expression ratio calculated. The different models of normalization with reference genes therefore have all the advantages and disadvantages. At present, there is no timehonored method for the treatment of the results of RT-qPCR. Normalization to a reference gene is a simple method and frequently used because it can control many variables. An advantage of reference genes as compared to total or rRNA is that the reference gene is subject to the same conditions as the mRNA of interest (Bustin, Benes et al. 2005; Hendriks-Balk, Michel et al. 2007). What has become apparent over recent years is that there is no single reference gene for all experimental systems. Quantified errors related to the use of a single reference gene as more than three-fold in 25% and more than six-fold in 10% of samples. Today it is clear that reference genes must be carefully validated for each experimental situation and those new experimental conditions or different tissue samples require re-validation of the chosen reference genes (Balogh, Paragh et al. 2008). If inappropriate reference genes are used for normalization, the experimental results obtained can differ greatly from those using a validated reference gene. Validation of a reference gene requires removal of any non-specific variation in expression. This can be done using a

Overview of Real-Time PCR Principles 435

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## **13. Abbreviation**

ACTB: Beta actin B2M: Beta-2-microglobulin CT: Threshold Cycle FRET: Fluorescence Resonance Energy Transfer GAPD: Glyceraldehyde-3- phosphate dehydrogenase HMBS: Hydroxymethyl-bilane synthase HPRT: hypoxanthine ribosyltransferase HPRT1: Hypoxanthine phosphoribosyl-transferase 1 HRM: High Resolution Melting KPCR: Kinetic Polymerase Chain Reaction LNA: Locked Nucleic Acid LUX: Light Upon Extension PCR: Polymerase Chain Reaction PGK1: phosphoglycerokinase 1 PNA: Peptide Nucleic Acid Q-PCR (QRT-PCR): Quantitative Real-Time Polymerase Chain Reaction RPL13A: Ribosomal protein L13a RT PCR: Reverse transcription PCR SDHA: Succinate dehydrogenase complex, subunit A TBP: TATA box binding protein Tm: Melting Point UBC: Ubiquitin C YWHAZ: Tyrosine 3-monooxygenase/ tryptophan 5-monooxygenase activation protein, zeta polypeptide

## **14. References**


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**13. Abbreviation**  ACTB: Beta actin

B2M: Beta-2-microglobulin CT: Threshold Cycle

FRET: Fluorescence Resonance Energy Transfer GAPD: Glyceraldehyde-3- phosphate dehydrogenase

HPRT1: Hypoxanthine phosphoribosyl-transferase 1

SDHA: Succinate dehydrogenase complex, subunit A

HMBS: Hydroxymethyl-bilane synthase HPRT: hypoxanthine ribosyltransferase

KPCR: Kinetic Polymerase Chain Reaction

HRM: High Resolution Melting

RPL13A: Ribosomal protein L13a RT PCR: Reverse transcription PCR

TBP: TATA box binding protein

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Tm: Melting Point UBC: Ubiquitin C

zeta polypeptide

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LNA: Locked Nucleic Acid LUX: Light Upon Extension PCR: Polymerase Chain Reaction PGK1: phosphoglycerokinase 1 PNA: Peptide Nucleic Acid


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**20** 

\*P. S. Shwed

*Canada* 

**PCR Advances Towards the Identification of** 

*Biotechnology Laboratory, Environmental Health Sciences and Research Bureau,* 

Public health and safety, diagnostics and surveillance are aided by knowledge of the identity and genetic content of biotechnology microbes and their close relatives. Both types of information allow recognition and prediction of virulence and pathogenicity of microbes. PCR has played an important role in enabling the identification of micro-organisms and the distinction of pathogenic from non-pathogenic species, since the technical descriptions in the mid-1980s (Mullis et al., 1986; Mullis and Faloona, 1987). This DNA amplification technology allows the generation of large template quantity, a pre-requisite for cloning and for dideoxy DNA "Sanger" sequencing (Sanger et al., 1977). As such, PCR has been integral

During the last decade, PCR has remained a cornerstone in microbial genetic characterization. Marker sequencing remains a component of the polyphasic characterization of microbial genomes in which genetic, morphological and biochemical data are reconciled. At the same time, great progress has been made in single cell microbial genetics and PCR miniaturization has been implemented in second generation sequencing platforms (Metzker, 2010). Collectively, these developments have resulted in increased numbers of whole genome sequences from individual microbes of "unculturable" microorganisms and outbreak strains such as Shiga toxin–producing *E. coli* strain O104:H4 detected in Europe during 2011 (Mellmann et al., 2011). High throughput sequencing has allowed for insights into natural and human environments and their mixed bacterial

This chapter serves to highlight PCR advances that have enabled microbial identification during the last decade. At the level of single species, identifications can involve phylogenetic marker sequencing, or whole genome sequencing from individual cells or cultures. Mixed microbial populations, may be sorted, individually identified by sequencing

(c) Her Majesty the Queen in Right of Canada, 2012. Published by InTech under Creative Commons

in first generation and phylogenetic marker sequencing projects (Bottger, 1989).

populations (Hamady and Knight, 2009; Mardis, 2011; Sapkota et al., 2010).

**1. Introduction** 

Attribution 3.0 License

**Individual and Mixed Populations of** 

*Healthy Environments and Consumer Safety Branch, Health Canada,* 

*Environmental and Radiation Health Sciences Directorate,* 

**Biotechnology Microbes** 

Zipper, H., H. Brunner, et al. (2004). "Investigations on DNA intercalation and surface binding by SYBR Green I, its structure determination and methodological implications." *Nucleic Acids Res*32(12): e103.

## **PCR Advances Towards the Identification of Individual and Mixed Populations of Biotechnology Microbes**

\*P. S. Shwed

*Biotechnology Laboratory, Environmental Health Sciences and Research Bureau, Environmental and Radiation Health Sciences Directorate, Healthy Environments and Consumer Safety Branch, Health Canada, Canada* 

## **1. Introduction**

442 Polymerase Chain Reaction

Zipper, H., H. Brunner, et al. (2004). "Investigations on DNA intercalation and surface

implications." *Nucleic Acids Res*32(12): e103.

binding by SYBR Green I, its structure determination and methodological

Public health and safety, diagnostics and surveillance are aided by knowledge of the identity and genetic content of biotechnology microbes and their close relatives. Both types of information allow recognition and prediction of virulence and pathogenicity of microbes. PCR has played an important role in enabling the identification of micro-organisms and the distinction of pathogenic from non-pathogenic species, since the technical descriptions in the mid-1980s (Mullis et al., 1986; Mullis and Faloona, 1987). This DNA amplification technology allows the generation of large template quantity, a pre-requisite for cloning and for dideoxy DNA "Sanger" sequencing (Sanger et al., 1977). As such, PCR has been integral in first generation and phylogenetic marker sequencing projects (Bottger, 1989).

During the last decade, PCR has remained a cornerstone in microbial genetic characterization. Marker sequencing remains a component of the polyphasic characterization of microbial genomes in which genetic, morphological and biochemical data are reconciled. At the same time, great progress has been made in single cell microbial genetics and PCR miniaturization has been implemented in second generation sequencing platforms (Metzker, 2010). Collectively, these developments have resulted in increased numbers of whole genome sequences from individual microbes of "unculturable" microorganisms and outbreak strains such as Shiga toxin–producing *E. coli* strain O104:H4 detected in Europe during 2011 (Mellmann et al., 2011). High throughput sequencing has allowed for insights into natural and human environments and their mixed bacterial populations (Hamady and Knight, 2009; Mardis, 2011; Sapkota et al., 2010).

This chapter serves to highlight PCR advances that have enabled microbial identification during the last decade. At the level of single species, identifications can involve phylogenetic marker sequencing, or whole genome sequencing from individual cells or cultures. Mixed microbial populations, may be sorted, individually identified by sequencing

 (c) Her Majesty the Queen in Right of Canada, 2012. Published by InTech under Creative Commons Attribution 3.0 License

PCR Advances Towards the Identification

revealed (Stahl et al., 1984),(Stahl et al., 1985).

prokaryotic rRNA genes and are referred to as "universal".

specificity.

(Gevers et al., 2005)).

**2.1.2 PCR as a component of genomic methods** 

of Individual and Mixed Populations of Biotechnology Microbes 445

is composed of nine hypervariable regions separated by conserved regions and sequences are available for numerous organisms via public databases such as NCBI, the Ribosomal Database project (Cole et al., 2009) and Greengenes (Desantis et al., 2006). The nine different variable 16S rRNA regions are flanked by conserved nucleotide stretches in bacteria (Neefs et al., 1993) and these could be used as targets for PCR primers with near-universal

It was during the mid-1980's that PCR first enabled molecular microbial ecology studies involving the 16S rRNA gene. Pace and colleagues first amplified 16S rRNA from bulk nucleic acid extractions using nearly "universal" primers, in order to sequence, classify and compare these to phylogenetic trees (Pace, 1997) (Lane et al., 1985) (Woese, 1987). At this time it was observed that not all environmental microorganisms were capable of colony formation and that by sequencing cloned ribosomal DNA, new microbial species could be

Over the last few decades, a large number of primer sequences have been designed for amplification and sequencing of 16S RNA genes, as reviewed in (Baker et al., 2003). There are a number of databases available for the primer sequences. Some of these primers have been designed as taxa specific, whereas others have been designed to amplify all

16S rRNA sequences may offer limited taxonomic resolution, particularly for genera that feature close phylogenetic relationships. *B. cepacia* complex reference strains feature high similarity values (above 98%) which reflects a close phylogenetic relationship (Coenye and Vandamme, 2003). Also, up to 2% intraspecies diversity has been observed in *B. cepacia* rRNA sequences and they cannot be identified at the species level by simple comparison of 16S rRNA sequences. Similarly, for the *B. cereus* group, there is also insufficient divergence in 16S rRNA to allow for resolution of strains and species (Bavykin et al., 2004). In these cases, other global markers have been explored for strain discrimination such as the genes that encode: RNA polymerase subunits, DNA gyrases, heat shock and recA proteins and hisA. The strong functional and structural constraints for these gene products, limits the number of mutations that can occur in the genes and renders them useful as markers for relatedness.

Identification of distinct strains of a prokaryotic species can take place by multi-locus sequence typing, in which sequence mismatches in a small number of house keeping genes are analyzed (as reviewed in (Maiden, 2006)). In the case of prokaryotic identification of closely related species, a similar strategy designated multi-locus sequence analysis has been used for several studies and involves a two step process: rRNA sequencing in order to assign an unknown strain to a group (either genus or family), that in turn defines the particular genes and primers to be used for analysis. This two-tiered approach has allowed discrimination of *Burkholderia* strains and those of the *Bacillus cereus* group (reviewed in

During the last decade, various applications of DNA microarrays have been used to assess the risk of a particular microbe by enabling detection and/or identification at the species, subspecies or strain level, or presence of virulence genes (reviewed in (Shwed et al., 2007)). However, DNA amplification is rarely a technical component of these studies. However, as

or collectively sequenced using high throughput platforms. The potential and challenges of these new platforms, as well as their applications towards novel microbial strains that will be produced by synthetic biology approaches, will be discussed.

## **2. Current challenges in microbial identification**

Collectively, microbes occupy a vast range of ecological niches and feature intrinsic diverse metabolic potential. Microbial biotechnology has enabled the screening and enhancement of strains for commercial applications such as: preservation and harvest of natural resources (bio-pesticides and bio-mining of metals), environmental remediation (improved soil/air/water quality) and applications for sustainable development. Often, biotechnology microbes are used as single species, while other commercial products involve mixtures of a few or many different species and strains.

Bacterial strains, that feature desirable phenotypic traits, have been traditionally isolated by high-throughput screening, or strains have been improved by random mutagenesis and screening. Currently, consensus identification and classification of bacterial strains is carried out by a polyphasic approach. Phenotypic data (biochemical tests, fatty acid composition), genotypic data and phylogenetic information, that includes genetic information, derived from PCR amplification of marker genes, are reconciled (Vandamme et al., 1996).

Discrimination of beneficial and harmful species is challenging in a number of genera that contain closely related species: *Burkholderia, Bacillus, Acinetobacter and Pseudomonas*. For example, in the *Burkholderia* genus, *B. cepacia* is a non-pathogenic soil bacterium that is being developed for the application of phytoremediation (Barac et al., 2004) and clinically, *B. cepacia* bacteria have been associated with infections and cystic fibrosis, as reviewed in (Coenye and Vandamme, 2003). Another prime example concerns the *Bacillus cereus sensu lato* family of bacteria. This group comprises the *Bacillus cereus* species *sensu stricto*, *B. anthracis,* strains and subspecies of *B. thuringiensis*, *B. mycoides*, and *B. weihenstephanensis*. Most *Bacillus cereus* organisms are common soil bacteria that are pathogenic to insects and invertebrates. Some species may cause contamination problems in the dairy industry and paper mills and may also be a causative agent of food poisoning. Select strains of *Bacillus anthracis* and a few *Bacillus cereus sensu stricto* strains are the only ones reported to cause fatal pulmonary or intestinal infections (Dixon et al., 2000).

Bacterial mixtures, that have been created or isolated in order to carry out a function, pose technical challenges to polyphasic characterization. Phenotypic data is typically derived for individual microbial isolates. However, current culture techniques cannot support a substantial fraction of microbial species (Handelsman, 2004) and there is risk of bias towards culturable species. In these cases, molecular methods that directly acquire genetic information may remove this hindrance.

## **2.1 PCR towards microbial identification**

## **2.1.1 Phylogenetic marker amplification and analysis**

Of all global markers, small subunit ribosomal RNA (16S rRNA) encoding genes are the best characterized genes for microbial systematics. The 16S rRNA gene is ubiquitous, highly conserved, but possesses enough variability to allow taxa specific discrimination. The gene

or collectively sequenced using high throughput platforms. The potential and challenges of these new platforms, as well as their applications towards novel microbial strains that will

Collectively, microbes occupy a vast range of ecological niches and feature intrinsic diverse metabolic potential. Microbial biotechnology has enabled the screening and enhancement of strains for commercial applications such as: preservation and harvest of natural resources (bio-pesticides and bio-mining of metals), environmental remediation (improved soil/air/water quality) and applications for sustainable development. Often, biotechnology microbes are used as single species, while other commercial products involve mixtures of a

Bacterial strains, that feature desirable phenotypic traits, have been traditionally isolated by high-throughput screening, or strains have been improved by random mutagenesis and screening. Currently, consensus identification and classification of bacterial strains is carried out by a polyphasic approach. Phenotypic data (biochemical tests, fatty acid composition), genotypic data and phylogenetic information, that includes genetic information, derived

Discrimination of beneficial and harmful species is challenging in a number of genera that contain closely related species: *Burkholderia, Bacillus, Acinetobacter and Pseudomonas*. For example, in the *Burkholderia* genus, *B. cepacia* is a non-pathogenic soil bacterium that is being developed for the application of phytoremediation (Barac et al., 2004) and clinically, *B. cepacia* bacteria have been associated with infections and cystic fibrosis, as reviewed in (Coenye and Vandamme, 2003). Another prime example concerns the *Bacillus cereus sensu lato* family of bacteria. This group comprises the *Bacillus cereus* species *sensu stricto*, *B. anthracis,* strains and subspecies of *B. thuringiensis*, *B. mycoides*, and *B. weihenstephanensis*. Most *Bacillus cereus* organisms are common soil bacteria that are pathogenic to insects and invertebrates. Some species may cause contamination problems in the dairy industry and paper mills and may also be a causative agent of food poisoning. Select strains of *Bacillus anthracis* and a few *Bacillus cereus sensu stricto* strains are the only ones reported to cause

Bacterial mixtures, that have been created or isolated in order to carry out a function, pose technical challenges to polyphasic characterization. Phenotypic data is typically derived for individual microbial isolates. However, current culture techniques cannot support a substantial fraction of microbial species (Handelsman, 2004) and there is risk of bias towards culturable species. In these cases, molecular methods that directly acquire genetic

Of all global markers, small subunit ribosomal RNA (16S rRNA) encoding genes are the best characterized genes for microbial systematics. The 16S rRNA gene is ubiquitous, highly conserved, but possesses enough variability to allow taxa specific discrimination. The gene

from PCR amplification of marker genes, are reconciled (Vandamme et al., 1996).

be produced by synthetic biology approaches, will be discussed.

**2. Current challenges in microbial identification** 

fatal pulmonary or intestinal infections (Dixon et al., 2000).

**2.1.1 Phylogenetic marker amplification and analysis** 

information may remove this hindrance.

**2.1 PCR towards microbial identification** 

few or many different species and strains.

is composed of nine hypervariable regions separated by conserved regions and sequences are available for numerous organisms via public databases such as NCBI, the Ribosomal Database project (Cole et al., 2009) and Greengenes (Desantis et al., 2006). The nine different variable 16S rRNA regions are flanked by conserved nucleotide stretches in bacteria (Neefs et al., 1993) and these could be used as targets for PCR primers with near-universal specificity.

It was during the mid-1980's that PCR first enabled molecular microbial ecology studies involving the 16S rRNA gene. Pace and colleagues first amplified 16S rRNA from bulk nucleic acid extractions using nearly "universal" primers, in order to sequence, classify and compare these to phylogenetic trees (Pace, 1997) (Lane et al., 1985) (Woese, 1987). At this time it was observed that not all environmental microorganisms were capable of colony formation and that by sequencing cloned ribosomal DNA, new microbial species could be revealed (Stahl et al., 1984),(Stahl et al., 1985).

Over the last few decades, a large number of primer sequences have been designed for amplification and sequencing of 16S RNA genes, as reviewed in (Baker et al., 2003). There are a number of databases available for the primer sequences. Some of these primers have been designed as taxa specific, whereas others have been designed to amplify all prokaryotic rRNA genes and are referred to as "universal".

16S rRNA sequences may offer limited taxonomic resolution, particularly for genera that feature close phylogenetic relationships. *B. cepacia* complex reference strains feature high similarity values (above 98%) which reflects a close phylogenetic relationship (Coenye and Vandamme, 2003). Also, up to 2% intraspecies diversity has been observed in *B. cepacia* rRNA sequences and they cannot be identified at the species level by simple comparison of 16S rRNA sequences. Similarly, for the *B. cereus* group, there is also insufficient divergence in 16S rRNA to allow for resolution of strains and species (Bavykin et al., 2004). In these cases, other global markers have been explored for strain discrimination such as the genes that encode: RNA polymerase subunits, DNA gyrases, heat shock and recA proteins and hisA. The strong functional and structural constraints for these gene products, limits the number of mutations that can occur in the genes and renders them useful as markers for relatedness.

Identification of distinct strains of a prokaryotic species can take place by multi-locus sequence typing, in which sequence mismatches in a small number of house keeping genes are analyzed (as reviewed in (Maiden, 2006)). In the case of prokaryotic identification of closely related species, a similar strategy designated multi-locus sequence analysis has been used for several studies and involves a two step process: rRNA sequencing in order to assign an unknown strain to a group (either genus or family), that in turn defines the particular genes and primers to be used for analysis. This two-tiered approach has allowed discrimination of *Burkholderia* strains and those of the *Bacillus cereus* group (reviewed in (Gevers et al., 2005)).

#### **2.1.2 PCR as a component of genomic methods**

During the last decade, various applications of DNA microarrays have been used to assess the risk of a particular microbe by enabling detection and/or identification at the species, subspecies or strain level, or presence of virulence genes (reviewed in (Shwed et al., 2007)). However, DNA amplification is rarely a technical component of these studies. However, as

PCR Advances Towards the Identification

**4. Emerging PCR applications** 

(Arumugam et al., 2011; Dowd et al., 2008).

16S primer bias (von Mering et al., 2007).

of Individual and Mixed Populations of Biotechnology Microbes 447

generation of a shot gun genomic library, by the random fragmentation of DNA and the ligation of universal adaptor sequences. Afterwards, *in vitro* clonal amplification is carried out by one of two principal types of PCR, which generate template for sequencing. Table 1 shows how various commercial platforms use PCR to derive features that are sequenced.

Emulsion PCR is carried out as described above (section 3.0 and shown in Fig.1 A, B), with the exception that paramagnetic beads that are bound to one of the PCR primers on their surface, are used (Dressman et al., 2003). These beads allow the solid-phase capture of clonally amplified PCR amplicons from each emulsion PCR compartment. For some commercial pyrosequencing platforms, beads are then deposited on microfabricated arrays

Bridge PCR (Adessi et al., 2000; Fedurco et al., 2006) involves the use of spatially distributed oligonucleotides that are covalently attached to a support (shown in Fig. 1 C,D). A DNA library is hybridized as single stranded DNA to the support. Immobilized copies of the library are synthesized by extension from the immobilized primers. After denaturation, the template copies are able to loop and hybridize to an adjacent oligonucleotide on the support. Additional copies of the template are synthesized and the process is repeated on each

**Instrument PCR type Sequence Method Reference** 

In recent years, complex microbial communities, such as those of the human gut intestinal tract, or those associated with biofilm infections, have been analyzed by second generation sequencing of shot gun libraries derived from either metagenomic DNA, or PCR amplified variable 16S regions amplified from metagenomic DNA prepared from a microbial mixture

Second generation platforms allow economies of scale in sequencing. PCR amplified products can be characterized without cloning, which saves time and costs. Also, the estimated costs per megabase of derived sequence are lower for the new platforms compared to first generation sequencing (Shendure et. al. 2011). Lastly, multiplexed runs, derived from 16S rRNA coding sequences from several communities, are feasible by using

It has been proposed that sequencing of individual variable regions is sufficient for taxonomic differentiation of bacterial mixtures (Liu et al., 2007). The sequence read lengths of second generation platforms are generally short, but several new models have shown greater read lengths (Liu et al., 2008). On the other hand, direct sequencing of metagenomic DNA has been proposed to be less biased than that of PCR amplified DNA, due to lack of

454 Emulsion Pyrosequencing (Margulies et al., 2005) Illumina Bridge Polymerase (Fedurco et. al. 2006) SOLiD Emulsion Ligase (Shendure et al., 2005)

of picolitre scale wells to allow immobilization and *in situ* pyrosequencing.

template so that clonal clusters, each with about 2000 molecules are generated.

Table 1. PCR clonal amplification by second-generation sequencing instrument

**4.1 Second generation sequencing from microbial mixtures** 

unique sequence barcodes during amplification (Hamady et al., 2008).

will be described in section 3.1, novel PCR amplification strategies are a component of the workflow for high throughput sequencing platforms.

## **3. Miniaturization of PCR**

Arguably, the major PCR advancement of the last decade has been the development of miniaturized and parallelized platforms. Whereas previously PCR reactions were typically carried out at the microlitre scale, new configurations have enabled femtolitre scale reactions. In turn, higher throughput and cost efficiencies have been achieved.

One miniaturization has been achieved by reaction entrapment in thermodynamically stable "water in oil" nanoreactor microemulsion systems, such as reverse micelles, as described for enzymatic reactions (Klyachko and Levashov, 2003). These emulsions are easily prepared and stable under a wide variety of temperatures, pH and salt concentrations. The smallest droplets rival the scale of bacteria with diameters of less than one micrometre with volumes in the femtolitre scale.

Emulsion PCR was first reported for the directed evolution of heat-stable, heparin insensitive variants of *Taq* DNA polymerase (Ghadessy et al., 2001). The concept of emulsion PCR was to disperse template DNA into a water in oil emulsion such that most droplets contain a single template and only a few droplets contain more than one template. Amplification was carried out within the drops by PCR, so that each droplet generated an amplified number of clonal copies.

## **3.1 Convergence of miniaturized PCR with other technologies**

During the last decade, advancements have been made in the engineering of microfluidic scale devices that integrate multiple analytical steps into "laboratory on chip" systems (as reviewed in (Liu and Mathies, 2009)). These devices allow the generation and manipulation of aqueous microdroplets at high rates and with high fidelity manipulation in microfluidic channels. PCR-based genetic analysis and sequencing can now be carried out at the picolitre to nanolitre volume scale, with the advantages of decreased thermal cycling times and reagent consumption along with increased throughput.

Microfluidic droplet PCR has been reported to allow 1.5 million parallel amplifications for target enrichment of loci in the human genome (Tewhey et al., 2009). In this instance, microfluidic chips were designed to merge 20 picolitre droplets that contain about 3 picograms of biotinylated fragments of template DNA (2-4 kb) with droplets that contain a pair of PCR primers that amplify specific sequences. This platform allowed a yield of more than one million merged droplets that are subjected to PCR. At the end of the amplification reaction, the emulsion is broken. After centrifugation, the aqueous phase, that contains the PCR products from all the droplets, is subjected to a second generation sequencing strategy.

During the last decade, several commercial second-generation sequencing platforms have been developed and these feature cyclic array sequencing strategies, involving new variations of PCR. In both cases, amplification of densely arrayed amplicons is achieved, in order to serve as features for *in situ* sequencing and imaging-based sequence by synthesis data collection (more detailed descriptions of second generation sequencing platforms are reviewed in (Shendure et al., 2011)). Common to all strategies, the first step is the *in vitro*

will be described in section 3.1, novel PCR amplification strategies are a component of the

Arguably, the major PCR advancement of the last decade has been the development of miniaturized and parallelized platforms. Whereas previously PCR reactions were typically carried out at the microlitre scale, new configurations have enabled femtolitre scale

One miniaturization has been achieved by reaction entrapment in thermodynamically stable "water in oil" nanoreactor microemulsion systems, such as reverse micelles, as described for enzymatic reactions (Klyachko and Levashov, 2003). These emulsions are easily prepared and stable under a wide variety of temperatures, pH and salt concentrations. The smallest droplets rival the scale of bacteria with diameters of less than one micrometre with volumes

Emulsion PCR was first reported for the directed evolution of heat-stable, heparin insensitive variants of *Taq* DNA polymerase (Ghadessy et al., 2001). The concept of emulsion PCR was to disperse template DNA into a water in oil emulsion such that most droplets contain a single template and only a few droplets contain more than one template. Amplification was carried out within the drops by PCR, so that each droplet generated an

During the last decade, advancements have been made in the engineering of microfluidic scale devices that integrate multiple analytical steps into "laboratory on chip" systems (as reviewed in (Liu and Mathies, 2009)). These devices allow the generation and manipulation of aqueous microdroplets at high rates and with high fidelity manipulation in microfluidic channels. PCR-based genetic analysis and sequencing can now be carried out at the picolitre to nanolitre volume scale, with the advantages of decreased thermal cycling times and

Microfluidic droplet PCR has been reported to allow 1.5 million parallel amplifications for target enrichment of loci in the human genome (Tewhey et al., 2009). In this instance, microfluidic chips were designed to merge 20 picolitre droplets that contain about 3 picograms of biotinylated fragments of template DNA (2-4 kb) with droplets that contain a pair of PCR primers that amplify specific sequences. This platform allowed a yield of more than one million merged droplets that are subjected to PCR. At the end of the amplification reaction, the emulsion is broken. After centrifugation, the aqueous phase, that contains the PCR products from all the droplets, is subjected to a second generation sequencing strategy. During the last decade, several commercial second-generation sequencing platforms have been developed and these feature cyclic array sequencing strategies, involving new variations of PCR. In both cases, amplification of densely arrayed amplicons is achieved, in order to serve as features for *in situ* sequencing and imaging-based sequence by synthesis data collection (more detailed descriptions of second generation sequencing platforms are reviewed in (Shendure et al., 2011)). Common to all strategies, the first step is the *in vitro*

reactions. In turn, higher throughput and cost efficiencies have been achieved.

**3.1 Convergence of miniaturized PCR with other technologies** 

reagent consumption along with increased throughput.

workflow for high throughput sequencing platforms.

**3. Miniaturization of PCR** 

in the femtolitre scale.

amplified number of clonal copies.

generation of a shot gun genomic library, by the random fragmentation of DNA and the ligation of universal adaptor sequences. Afterwards, *in vitro* clonal amplification is carried out by one of two principal types of PCR, which generate template for sequencing. Table 1 shows how various commercial platforms use PCR to derive features that are sequenced.

Emulsion PCR is carried out as described above (section 3.0 and shown in Fig.1 A, B), with the exception that paramagnetic beads that are bound to one of the PCR primers on their surface, are used (Dressman et al., 2003). These beads allow the solid-phase capture of clonally amplified PCR amplicons from each emulsion PCR compartment. For some commercial pyrosequencing platforms, beads are then deposited on microfabricated arrays of picolitre scale wells to allow immobilization and *in situ* pyrosequencing.

Bridge PCR (Adessi et al., 2000; Fedurco et al., 2006) involves the use of spatially distributed oligonucleotides that are covalently attached to a support (shown in Fig. 1 C,D). A DNA library is hybridized as single stranded DNA to the support. Immobilized copies of the library are synthesized by extension from the immobilized primers. After denaturation, the template copies are able to loop and hybridize to an adjacent oligonucleotide on the support. Additional copies of the template are synthesized and the process is repeated on each template so that clonal clusters, each with about 2000 molecules are generated.


Table 1. PCR clonal amplification by second-generation sequencing instrument

## **4. Emerging PCR applications**

## **4.1 Second generation sequencing from microbial mixtures**

In recent years, complex microbial communities, such as those of the human gut intestinal tract, or those associated with biofilm infections, have been analyzed by second generation sequencing of shot gun libraries derived from either metagenomic DNA, or PCR amplified variable 16S regions amplified from metagenomic DNA prepared from a microbial mixture (Arumugam et al., 2011; Dowd et al., 2008).

Second generation platforms allow economies of scale in sequencing. PCR amplified products can be characterized without cloning, which saves time and costs. Also, the estimated costs per megabase of derived sequence are lower for the new platforms compared to first generation sequencing (Shendure et. al. 2011). Lastly, multiplexed runs, derived from 16S rRNA coding sequences from several communities, are feasible by using unique sequence barcodes during amplification (Hamady et al., 2008).

It has been proposed that sequencing of individual variable regions is sufficient for taxonomic differentiation of bacterial mixtures (Liu et al., 2007). The sequence read lengths of second generation platforms are generally short, but several new models have shown greater read lengths (Liu et al., 2008). On the other hand, direct sequencing of metagenomic DNA has been proposed to be less biased than that of PCR amplified DNA, due to lack of 16S primer bias (von Mering et al., 2007).

PCR Advances Towards the Identification

**4.2 PCR analysis of single cells** 

environments (reviewed in (Hongoh, 2010)).

**4.3 Whole genome sequencing from individual cells** 

individual microbes.

reference genome.

isothermal conditions (Dean et al., 2001).

of Individual and Mixed Populations of Biotechnology Microbes 449

Collectively, these identification approaches are limited by the use of reference databases of known species and genes from readily cultivated microbes. As a consequence, species within a microbial community that lack a reference sequence will remain unidentified.

The analysis of complex mixtures of environmental bacteria will benefit from microfluidic digital PCR analysis that involves single cell sorting from mixtures of bacteria. Single bacterial cells can be isolated by various technologies, including: optical tweezers, micromanipulation, FACS, serial dilutions, or laser capture microdissection. In turn, experimentation that involves retrieving "needles in a haystack", such as searches for microbes

Characterization of environmental bacteria of the 1 microlitre volume termite hindgut model, exemplify the potential of cell sorting and PCR. This microenvironment contains about 106-108 microbial cells, comprised of unculturable species not detected in other

Otteson et al. (Ottesen et al., 2006), applied a microfluidic digital PCR characterization approach for the termite bacteria. In this study, individual cells were partitioned in a microfluidic array panel and served as templates for the simultaneous amplification of both rRNA and metabolic genes of interest. The digital PCR aspect involved ensuring that the partitioning was into reactions that contained an average of one template (bacterial cell) or less (Sykes et al., 1992). Retrieved PCR products from individual chambers allowed sequence analysis of both genes by standard methods and allowed the determination of new bacterial species that contribute to metabolism. More recently, microfluidic digital PCR was used to associate particular viruses that infect the bacteria of the termite gut, without culturing either the viruses or the hosts (Tadmor et al., 2011). Here, amplification of both rRNA gene and a viral marker gene was carried out from a PCR array panel containing

Genomic sequences provide the most absolute indication of genetic variation and virulence potential for a bacterial strain. The documentation of the complete nucleic acid sequences of high priority beneficial and detrimental microorganisms in public databases are efforts that can greatly aid the identification of unknown strains. In studies involving closely related bacterial strains, shotgun library sequences can be assembled by mapping the reads to a

Direct single bacterial cell genome sequencing can be carried out by multiple displacement amplification, using individually lysed bacteria and the few femtograms of DNA present in bacterial cells in order to generate template for shotgun sequencing. This reaction involves the use of 29 DNA polymerase and random primers to amplify DNA templates under

Genomic sequencing from individual uncultured bacterial cells was first shown by Raghunathan et al., using *E. coli* cells that had been isolated by flow cytometry (Raghunathan et al., 2005). This report illustrated contamination as a technical challenge

featuring particular genes are facilitated by microfluidics technologies (Baker, 2010).

Fig. 1. PCR advancements towards second-generation sequencing **Panels A.B: Emulsion PCR** 

Panel A) A shot-gun DNA library is ligated to adaptors (blue and red bars), diluted, and PCR amplified in a water in oil emulsion, within aqueous microdroplets. The droplets contain streptavidin coated beads that carry one of the biotinylated PCR primers tethered to beads. Panel B) Where DNA is amplified in the presence of a bead, several thousand copies of the template will be captured.

#### **Panels C,D: Bridge PCR**

Panel C) A shot-gun DNA library is ligated to adaptors, made single stranded and hybridized to PCR primers that are immobilized with flexible linkers on a substrate. Bridge amplification occurs when primer extension occurs from immediately adjacent primers. Panel D) Immobilized clusters of about one thousand amplicons are formed after successive cycles of extension and denaturation.

The critical analytical step of taxonomic analyses of microbial diversity analysis is known as binning, where the sequences from a mixture of organisms are assigned phylogenetic groups. However, the outcome of binning results may range from kingdom level to genus level assignment, depending on the quality of data and the read length of data (Yang et al., 2010). One of the binning strategies in use is based on classification of DNA fragments based on sequence homology, using publically available reference databases such as Basic Local Alignment Search Tool (Huson et al., 2007; Meyer et al., 2008). The second strategy involves similarity to protein families and domains, such as in the phylogenetic algorithm CARMA (Krause et al., 2008).

Collectively, these identification approaches are limited by the use of reference databases of known species and genes from readily cultivated microbes. As a consequence, species within a microbial community that lack a reference sequence will remain unidentified.

## **4.2 PCR analysis of single cells**

448 Polymerase Chain Reaction

Fig. 1. PCR advancements towards second-generation sequencing

Panel A) A shot-gun DNA library is ligated to adaptors (blue and red bars), diluted, and PCR amplified in a water in oil emulsion, within aqueous microdroplets. The droplets contain streptavidin coated beads that carry one of the biotinylated PCR primers tethered to beads. Panel B) Where DNA is amplified in the presence of a bead, several thousand copies

Panel C) A shot-gun DNA library is ligated to adaptors, made single stranded and

hybridized to PCR primers that are immobilized with flexible linkers on a substrate. Bridge amplification occurs when primer extension occurs from immediately adjacent primers. Panel D) Immobilized clusters of about one thousand amplicons are formed after successive

The critical analytical step of taxonomic analyses of microbial diversity analysis is known as binning, where the sequences from a mixture of organisms are assigned phylogenetic groups. However, the outcome of binning results may range from kingdom level to genus level assignment, depending on the quality of data and the read length of data (Yang et al., 2010). One of the binning strategies in use is based on classification of DNA fragments based on sequence homology, using publically available reference databases such as Basic Local Alignment Search Tool (Huson et al., 2007; Meyer et al., 2008). The second strategy involves similarity to protein families and domains, such as in the phylogenetic algorithm CARMA

**Panels A.B: Emulsion PCR** 

of the template will be captured.

cycles of extension and denaturation.

**Panels C,D: Bridge PCR** 

(Krause et al., 2008).

The analysis of complex mixtures of environmental bacteria will benefit from microfluidic digital PCR analysis that involves single cell sorting from mixtures of bacteria. Single bacterial cells can be isolated by various technologies, including: optical tweezers, micromanipulation, FACS, serial dilutions, or laser capture microdissection. In turn, experimentation that involves retrieving "needles in a haystack", such as searches for microbes featuring particular genes are facilitated by microfluidics technologies (Baker, 2010).

Characterization of environmental bacteria of the 1 microlitre volume termite hindgut model, exemplify the potential of cell sorting and PCR. This microenvironment contains about 106-108 microbial cells, comprised of unculturable species not detected in other environments (reviewed in (Hongoh, 2010)).

Otteson et al. (Ottesen et al., 2006), applied a microfluidic digital PCR characterization approach for the termite bacteria. In this study, individual cells were partitioned in a microfluidic array panel and served as templates for the simultaneous amplification of both rRNA and metabolic genes of interest. The digital PCR aspect involved ensuring that the partitioning was into reactions that contained an average of one template (bacterial cell) or less (Sykes et al., 1992). Retrieved PCR products from individual chambers allowed sequence analysis of both genes by standard methods and allowed the determination of new bacterial species that contribute to metabolism. More recently, microfluidic digital PCR was used to associate particular viruses that infect the bacteria of the termite gut, without culturing either the viruses or the hosts (Tadmor et al., 2011). Here, amplification of both rRNA gene and a viral marker gene was carried out from a PCR array panel containing individual microbes.

## **4.3 Whole genome sequencing from individual cells**

Genomic sequences provide the most absolute indication of genetic variation and virulence potential for a bacterial strain. The documentation of the complete nucleic acid sequences of high priority beneficial and detrimental microorganisms in public databases are efforts that can greatly aid the identification of unknown strains. In studies involving closely related bacterial strains, shotgun library sequences can be assembled by mapping the reads to a reference genome.

Direct single bacterial cell genome sequencing can be carried out by multiple displacement amplification, using individually lysed bacteria and the few femtograms of DNA present in bacterial cells in order to generate template for shotgun sequencing. This reaction involves the use of 29 DNA polymerase and random primers to amplify DNA templates under isothermal conditions (Dean et al., 2001).

Genomic sequencing from individual uncultured bacterial cells was first shown by Raghunathan et al., using *E. coli* cells that had been isolated by flow cytometry (Raghunathan et al., 2005). This report illustrated contamination as a technical challenge

PCR Advances Towards the Identification

**7. References** 

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## **5. Conclusions and future challenges**

The safe use of biotechnology microbes for public health and in the environment requires knowledge of the identity and genetic potential of these organisms. In the first decade of the 21st century, amongst the genetic tools available for genetic characterization, PCR remains a cornerstone. Advances in miniaturization and parallelism of PCR have enhanced throughput and enabled second generation sequencing platforms. These technological advancements have been linked to progress in single cell microbial genomics, whole genome sequencing and the characterization of microbial mixtures. Collectively, these developments have direct implications for the safety assessments that are carried out by industry and governments.

These recent technological advances will allow new human and environmental surveys. As an example, movements of genes amongst microbes by horizontal gene transfer mechanisms may be tracked. Environmental surveys of the movements of particular nucleotide sequences are now possible by metagenomic methods. Culture-independent methodology for genetic analysis will allow greater throughput. However, at present, computational hurdles remain for the wide-spread implementation of such technology.

Miniaturization has been a hallmark of progress in electronics and computing. By this measure, PCR miniaturization that has taken place to date is of relatively low order. At the same time, the complexity of biotechnology microbes developed for commercial applications is increasing. The advances in PCR and genomic technologies must be considered in parallel with the technical advancements that have been made towards the *de novo* construction of synthetic microbes. High throughput, high efficiency microfluidic devices can enable the encapsulation of novel genetic material in abiotic chassis (Szita et al., 2010). PCR and sequencing advancements will remain important for microbial genetic characterization.

## **6. Acknowledgements**

Drs. Guillaume Pelletier and Azam Tayabali are thanked for constructive criticism of the manuscript. Open access charges are supported by the Canadian Regulatory System for Biotechnology Fund.

## **7. References**

450 Polymerase Chain Reaction

when working with individual microbial cells. The reaction involves random primers in order to initiate polymerization and this can result in amplification of contaminating DNA. In the case of poorly characterized or novel biotechnology microbes, the non-target DNA could confound conclusions about the target organism. In addition, there are biases introduced by multiple displacement amplification, particularly with the use of small input quantity of DNA. Segments of the chromosomes have been observed to be preferentially amplified. As well, chimeric rearrangements of DNA result from the linking of non-

Despite these challenges, there have been recent reports that are more encouraging about the acquisition of finished genomic sequence derived from a single bacterial cell (Woyke et al., 2010). Multiple displacement amplification artifacts have been overcome with new computational algorithms, that can compensate for amplification bias and chimeric

The safe use of biotechnology microbes for public health and in the environment requires knowledge of the identity and genetic potential of these organisms. In the first decade of the 21st century, amongst the genetic tools available for genetic characterization, PCR remains a cornerstone. Advances in miniaturization and parallelism of PCR have enhanced throughput and enabled second generation sequencing platforms. These technological advancements have been linked to progress in single cell microbial genomics, whole genome sequencing and the characterization of microbial mixtures. Collectively, these developments have direct implications for the safety assessments that are carried out by

These recent technological advances will allow new human and environmental surveys. As an example, movements of genes amongst microbes by horizontal gene transfer mechanisms may be tracked. Environmental surveys of the movements of particular nucleotide sequences are now possible by metagenomic methods. Culture-independent methodology for genetic analysis will allow greater throughput. However, at present, computational

Miniaturization has been a hallmark of progress in electronics and computing. By this measure, PCR miniaturization that has taken place to date is of relatively low order. At the same time, the complexity of biotechnology microbes developed for commercial applications is increasing. The advances in PCR and genomic technologies must be considered in parallel with the technical advancements that have been made towards the *de novo* construction of synthetic microbes. High throughput, high efficiency microfluidic devices can enable the encapsulation of novel genetic material in abiotic chassis (Szita et al., 2010). PCR and sequencing advancements will remain important for microbial genetic characterization.

Drs. Guillaume Pelletier and Azam Tayabali are thanked for constructive criticism of the manuscript. Open access charges are supported by the Canadian Regulatory System for

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**5. Conclusions and future challenges** 

industry and governments.

**6. Acknowledgements** 

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macrophage interactions: intracellular survival survival and escape. *Cell Microbiol.* 2

nature of chronic diabetic foot ulcer biofilm infections determined using bacterial tag encoded FLX amplicon pyrosequencing (bTEFAP). *PLoS One.* 3 (10): e3326. Dressman D, Yan H, Traverso G, Kinzler KW, and Vogelstein B. 2003. Transforming single

DNA molecules into fluorescent magnetic particles for detection and enumeration

for DNA attachment on glass and efficient generation of solid-phase amplified

Vandamme P, Thompson FL, and Swings J. 2005. Opinion: Re-evaluating

primers for pyrosequencing hundreds of samples in multiplex. *Nat Methods* 5 (3):

Maiden MC. 2006. Multilocus sequence typing of bacteria. *Annu. Rev. Microbiol.* 60: 561-588.


**21** 

Tammam Sipahi

 *Turkey* 

**Lack of Evidence for Contribution** 

**with Development of Ischemic Stroke in Turkish Subjects in Trakya Region** 

*Department of Biophysics, Medical Faculty, Trakya University, Edirne* 

**of eNOS, ACE and AT1R Gene Polymorphisms** 

Nitric oxide (NO) is produced in the endothelial cells, neurons, glia, and macrophages by the nitric oxide synthase (NOS) isoenzymes. Endothelial nitric oxide synthase (eNOS) is a subgroup of this family of enzymes that catalyze the production of nitric oxide (NO) from Larginine and oxygen, which causes vascular relaxation (1) by activates guanylate cyclase,

L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+

NO can also promote vasorelaxation indirectly by inhibiting the release of renin which converts angiotensinogen to angiotensin I. This is in turn cleaved to form active angiotensin II by Angiotensin-converting enzyme (ACE), the key component of the physiological control of blood pressure in human. Angiotensin II exerts its effects by binding to angiotensin II type 1, 2, 3, and 4 receptors (AT1R, AT2R, AT3R, AT4R). AT1R is the major mediator of physiological effects of angiotensin II. AT1R mediates its action by association with G proteins and followed by vasoconstriction. The activated receptor in turn couples to G proteins and thus activates phospholipases, increases the cytosolic

**1. Introduction** 

which induces smooth muscle relaxation.

The reaction catalyzed by eNOS is:


## **Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms with Development of Ischemic Stroke in Turkish Subjects in Trakya Region**

Tammam Sipahi

*Department of Biophysics, Medical Faculty, Trakya University, Edirne Turkey* 

#### **1. Introduction**

454 Polymerase Chain Reaction

Shendure JA, Porreca GJ, Church GM, Gardner AF, Hendrickson CL, Kieleczawa J, and

Shwed P.S., Crosthwait J, and Seligy VL. 2007. Comparative genomic and DNA microarray

Stahl DA, Lane DJ, Olsen GJ, and Pace NR. 1984. Analysis of hydrothermal vent-associated symbionts by ribosomal RNA sequences. *Science* 224 (4647): 409-411. Stahl DA, Lane DJ, Olsen GJ, and Pace NR. 1985. Characterization of a Yellowstone hot

Sykes PJ, Neoh SH, Brisco MJ, Hughes E, Condon J, and Morley AA. 1992. Quantitation of targets for PCR by use of limiting dilution. *Biotechniques* 13 (3): 444-449. Szita N, Polizzi K, Jaccard N, and Baganz F. 2010. Microfluidic approaches for systems and

Tadmor AD, Ottesen EA, Leadbetter JR, and Phillips R. 2011. Probing individual

Tewhey R, Warner JB, Nakano M, Libby B, Medkova M, David PH, Kotsopoulos SK,

Vandamme P, Pot B, Gillis M, De VP, Kersters K, and Swings J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. *Microbiol. Rev.* 60 (2): 407-438. von Mering C, Hugenholtz P, Raes J, Tringe SG, Doerks T, Jensen LJ, Ward N, and Bork P.

Woyke T, Tighe D, Mavromatis K, Clum A, Copeland A, Schackwitz W, Lapidus A, Wu D,

Yang B, Peng Y, Leung HC, Yiu SM, Chen JC, and Chin FY. 2010. Unsupervised binning of

Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW, and Church GM.

P Kumarathasan and R Vincent, 137-156. Research Signpost.

synthetic biology. *Curr. Opin. Biotechnol.* 21 (4): 517-523.

scale targeted sequencing. *Nat Biotechnol.* 27 (11): 1025-1031.

bacterial cell, one complete genome. *PLoS One.* 5 (4): e10314.

environments. *Science* 315 (5815): 1126-1130. Woese CR. 1987. Bacterial evolution. *Microbiol. Rev.* 51 (2): 221-271.

*BMC. Bioinformatics.* 11 Suppl 2: S5.

*Molecular Biology*, John Wiley & Sons, Inc.

1379-1384.

(6038): 58-62.

24 (6): 680-686.

Slatko BE. 2011. Overview of DNA Sequencing Strategies. In *Current Protocols in* 

applications in risk assessment of biotechnology microorganisms. In *Recent Advancements in Analytical Biochemistry: Applications in Environmental Toxicology*, eds

spring microbial community by 5S rRNA sequences. *Appl. Environ. Microbiol.* 49 (6):

environmental bacteria for viruses by using microfluidic digital PCR. *Science* 333

Samuels ML, Hutchison JB, Larson JW, Topol EJ, Weiner MP, Harismendy O, Olson J, Link DR, and Frazer KA. 2009. Microdroplet-based PCR enrichment for large-

2007. Quantitative phylogenetic assessment of microbial communities in diverse

McCutcheon JP, McDonald BR, Moran NA, Bristow J, and Cheng JF. 2010. One

environmental genomic fragments based on an error robust selection of l-mers.

2006. Sequencing genomes from single cells by polymerase cloning. *Nat Biotechnol.*

Nitric oxide (NO) is produced in the endothelial cells, neurons, glia, and macrophages by the nitric oxide synthase (NOS) isoenzymes. Endothelial nitric oxide synthase (eNOS) is a subgroup of this family of enzymes that catalyze the production of nitric oxide (NO) from Larginine and oxygen, which causes vascular relaxation (1) by activates guanylate cyclase, which induces smooth muscle relaxation.

The reaction catalyzed by eNOS is:

L-arginine + 3/2 NADPH + H+ + 2 O2 = citrulline + nitric oxide + 3/2 NADP+

NO can also promote vasorelaxation indirectly by inhibiting the release of renin which converts angiotensinogen to angiotensin I. This is in turn cleaved to form active angiotensin II by Angiotensin-converting enzyme (ACE), the key component of the physiological control of blood pressure in human. Angiotensin II exerts its effects by binding to angiotensin II type 1, 2, 3, and 4 receptors (AT1R, AT2R, AT3R, AT4R). AT1R is the major mediator of physiological effects of angiotensin II. AT1R mediates its action by association with G proteins and followed by vasoconstriction. The activated receptor in turn couples to G proteins and thus activates phospholipases, increases the cytosolic

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

prevalence, and mortality of stroke in the next decades.

fragment length polymorphism (RFLP) assay (5, 19).

**2. Material and methods** 

sequence (20).

with Development of Ischemic Stroke in Turkish Subjects in Trakya Region 457

In view of the aging population stroke is becoming a major problem, it is the most frequent disease leading to disability (3) and estimates forecast a continuing increase in the incidence,

The study included 341 subjects; 197 stroke patients and 144 controls **(Table 1)**. All participants gave informed consent that was approved by the local ethics committee. DNA was isolated from peripheral blood, collected into tubes containing ethylenediaminetetraacetic acid (EDTA) by eZNA (EaZy Nucleic Acid Isolation) blood DNA kits (Omega Bio-tek, Doraville, USA). eNOS (4 a/b) and ACE (I/D) gene polymorphisms were identified using a polymerase chain reaction (PCR) technique (5, 18). The AT1R (A1166C) and eNOS (Glu298Asp) gene polymorphisms were identified using PCR technique and restriction

**Hypertension (%) 61.3 83.1 <0.001** 

**Current smoker (%) 3.6 28.3 <0.001** 

**Diabetes mellitus (%) 17.6 33.8 0.001** 

**Family history of stroke (%) 17.6 33.0 0.002** 

**Age (years) 63.0 (17.0) 69.0 (14.0) <0.001** 

**SBP (mmHg) 120.0 (20.0) 140.0 (40.0) <0.001** 

**DBP (mmHg) 70.0 (10.0) 80.0 (20.0) <0.001** 

**FBG (mg/dl) 89.5 (18.3) 105.5 (41.0) <0.001** 

**TG (mg/dl) 117.5 (93.5) 145.0 (105.0) 0.008** 

**TC (mg/dl) 189.0 (44.0) 190.0 (52.0) NS** 

**HDL-C (mg/dl) 39.0 (19.5) 38.5 (14.0) NS** 

**LDL-C (mg/dl) 120.5 (35.0) 124.0 (41.5) NS** 

cholesterol, HDL-C/LDL-C; High/Low density lipoprotein cholesterol, NS: Non-significant. Table 1. Demographic and clinical characteristics of the control and stroke groups

SBP/DBP; Systolic/Diastolic blood pressure, FBG; Fasting blood glucose, TG; Triglycerides, TC; Total

PCR technique, developed in 1983 by Kary Mullis, is an in vitro indispensable scientific technique used in medical genetics and hereditary disorders researches to amplify a single (or a few copies) of a piece of DNA to generating millions of copies of a particular DNA

**Control Group Stroke Group p** 

Ca2+ concentrations, which triggers cellular responses such as stimulation of protein kinases. Activated receptor also inhibits adenylyl cyclases and activates various tyrosine kinases (2).

Ischemic stroke, caused either by thrombosis or embolism, is the most frequent disease leading to disability and/or to death (3). The genetic differentiations varying with ethnic properties may be related to the arrangement of the classic and non-classic risk factors for ischemic stroke (4).

During the last two decades, there has been an increasing interest in the study of the different polymorphisms of genes of the renin-angiotensin system (RAS) and its association with the pathogenesis of stroke disease (5, 6). The RAS gene system comprises the angiotensinogen (AGT), renin, angiotensin I, angiotensin I-converting enzyme (ACE), angiotensin II, and angiotensin II receptors (7).

The ACE is a key component of both the RAS and the kinin-kallikrein system. ACE cleaves the carboxy-terminal dipeptide of angiotensin I, releasing the physiologically active octapeptide angiotensin II (8). Angiotensin II is a potent vasoconstrictive molecule that plays a key role in modulating vascular tone. Angiotensin II exerts its effects by binding to the major mediator AT1R. Human AT1R is present predominantly in vascular cells and in both kidney and adrenal gland mediating physiological actions of angiotensin II. AT1R mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system, followed by vasoconstriction, hypertrophy, or catecholamine liberation at sympathetic nerve endings (9).

Our study aimed to assess the distribution of gene polymorphisms of ACE, AT1R and eNOS gene polymorphisms in ischemic stroke patients compared to healthy controls in the subjects from Trakya region.

The ACE gene maps to chromosome 17 (17q23.3), spans 21 kb, and comprises 26 exons and 25 introns, and is characterized by a polymorphism resulting from the presence (insertion) or absence (deletion) of a 287 base pairs fragment of a repeated Alu sequence at intron 16 hence, the corresponding designation of insertion (I) or deletion (D) of the two resulting alleles (10, 11).

The AT1R gene maps to chromosome 3 (3q21q25), spans 45.123 kb, and comprises 5 exons and 4 introns (12). AT1R entire coding region harbored only on exon 5, and is characterized by a polymorphism resulting from an A/C (adenine/cytosine) transversion located at position 1166 (A1166C polymorphism) in 3' untranslated region (13).

The eNOS gene is located on chromosome 7q35-36 and comprises 26 exons spanning 21 kb (14). Three classes of genetic polymorphisms in eNOS have been identified: those in intron regions, those in the promoter, and those in exon regions (15).

The variable number of tandem repeat (27 VNTR) polymorphism in intron 4 of the eNOS gene (eNOS 4 a/b), and Guanine (G) to Thymine (T) conversion at nucleotide position 894 in exon 7 causing Glutamic acid (Glu) to Aspartic acid (Asp) change at 298 are two of the most encountered polymorphisms. This polymorphism was shown to affect the response of vascular endothelium and the NO levels of plasma (16, 17).

In view of the aging population stroke is becoming a major problem, it is the most frequent disease leading to disability (3) and estimates forecast a continuing increase in the incidence, prevalence, and mortality of stroke in the next decades.

## **2. Material and methods**

456 Polymerase Chain Reaction

Ca2+ concentrations, which triggers cellular responses such as stimulation of protein kinases. Activated receptor also inhibits adenylyl cyclases and activates various tyrosine

Ischemic stroke, caused either by thrombosis or embolism, is the most frequent disease leading to disability and/or to death (3). The genetic differentiations varying with ethnic properties may be related to the arrangement of the classic and non-classic risk factors for

During the last two decades, there has been an increasing interest in the study of the different polymorphisms of genes of the renin-angiotensin system (RAS) and its association with the pathogenesis of stroke disease (5, 6). The RAS gene system comprises the angiotensinogen (AGT), renin, angiotensin I, angiotensin I-converting enzyme (ACE),

The ACE is a key component of both the RAS and the kinin-kallikrein system. ACE cleaves the carboxy-terminal dipeptide of angiotensin I, releasing the physiologically active octapeptide angiotensin II (8). Angiotensin II is a potent vasoconstrictive molecule that plays a key role in modulating vascular tone. Angiotensin II exerts its effects by binding to the major mediator AT1R. Human AT1R is present predominantly in vascular cells and in both kidney and adrenal gland mediating physiological actions of angiotensin II. AT1R mediates its action by association with G proteins that activate a phosphatidylinositol-calcium second messenger system, followed by vasoconstriction, hypertrophy, or catecholamine liberation

Our study aimed to assess the distribution of gene polymorphisms of ACE, AT1R and eNOS gene polymorphisms in ischemic stroke patients compared to healthy controls in the

The ACE gene maps to chromosome 17 (17q23.3), spans 21 kb, and comprises 26 exons and 25 introns, and is characterized by a polymorphism resulting from the presence (insertion) or absence (deletion) of a 287 base pairs fragment of a repeated Alu sequence at intron 16 hence, the corresponding designation of insertion (I) or deletion (D) of the two resulting

The AT1R gene maps to chromosome 3 (3q21q25), spans 45.123 kb, and comprises 5 exons and 4 introns (12). AT1R entire coding region harbored only on exon 5, and is characterized by a polymorphism resulting from an A/C (adenine/cytosine) transversion located at

The eNOS gene is located on chromosome 7q35-36 and comprises 26 exons spanning 21 kb (14). Three classes of genetic polymorphisms in eNOS have been identified: those in intron

The variable number of tandem repeat (27 VNTR) polymorphism in intron 4 of the eNOS gene (eNOS 4 a/b), and Guanine (G) to Thymine (T) conversion at nucleotide position 894 in exon 7 causing Glutamic acid (Glu) to Aspartic acid (Asp) change at 298 are two of the most encountered polymorphisms. This polymorphism was shown to affect the response of

position 1166 (A1166C polymorphism) in 3' untranslated region (13).

regions, those in the promoter, and those in exon regions (15).

vascular endothelium and the NO levels of plasma (16, 17).

kinases (2).

ischemic stroke (4).

angiotensin II, and angiotensin II receptors (7).

at sympathetic nerve endings (9).

subjects from Trakya region.

alleles (10, 11).

The study included 341 subjects; 197 stroke patients and 144 controls **(Table 1)**. All participants gave informed consent that was approved by the local ethics committee. DNA was isolated from peripheral blood, collected into tubes containing ethylenediaminetetraacetic acid (EDTA) by eZNA (EaZy Nucleic Acid Isolation) blood DNA kits (Omega Bio-tek, Doraville, USA). eNOS (4 a/b) and ACE (I/D) gene polymorphisms were identified using a polymerase chain reaction (PCR) technique (5, 18). The AT1R (A1166C) and eNOS (Glu298Asp) gene polymorphisms were identified using PCR technique and restriction fragment length polymorphism (RFLP) assay (5, 19).


SBP/DBP; Systolic/Diastolic blood pressure, FBG; Fasting blood glucose, TG; Triglycerides, TC; Total cholesterol, HDL-C/LDL-C; High/Low density lipoprotein cholesterol, NS: Non-significant.

Table 1. Demographic and clinical characteristics of the control and stroke groups

PCR technique, developed in 1983 by Kary Mullis, is an in vitro indispensable scientific technique used in medical genetics and hereditary disorders researches to amplify a single (or a few copies) of a piece of DNA to generating millions of copies of a particular DNA sequence (20).

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

and 0.1 mg/mL BSA.

DNA Thermal Cycler.

contains ACE (D) polymorphism.

letters were used for the primer sequences.

**PCR Conditions** 

**3. ACE I/D gene polymorphism (rs 4646994)** 

with Development of Ischemic Stroke in Turkish Subjects in Trakya Region 459

to 0.5 μg of PCR products and 10 mM Tris-HCl (pH 7.5 to 8.5), 10 mM MgCl2, 100 mM KCl

A genomic DNA were amplified by PCR technique in a total 25 μL PCR mixture containing 200 ng of DNA, deoxynucleotide triphosphates (0.2 mM of each), 0.5 nmol of sense and anti-sense oligonucleotide primers, 1X Taq buffer and 1.25 U of Taq DNA polymerase. eNOS (4a/b), ACE (I/D) and eNOS (Glu298Asp) gene polymorphism reactions were contained 2.5 mM MgCl2 whereas AT1R (A1166C) gene polymorphism reaction were contained 1.5 mM MgCl2. All reagents for PCR amplification and gel electrophoresis were purchased from Fermentas Life Sciences (ELİPS, Istanbul, Turkey). All other chemicals were from Sigma and Merck (BO&GA, Istanbul, Turkey) and of the highest purity available. DNA amplifications were performed with a Techne (TechGene)

The PCR primers with the sequences reported by Rigat B. et al. (27) were used. Sense and anti-sense primers were; 5'-CTGGAGACCACTCCCATCCTTTCT-3' and 5'-GATGTGGCCATCACATTCGTCAGAT-3', respectively. Normally the sense primer in Rigat et al. didn't contain (G), so our PCR products also didn't contain (G), and the antisense primer in Rigat et al. didn't contain (G); which is normally must be included, but it was instead of (G) contained (A). Figure 1 shows the sequencing of the region which

The expected insertion (I) and deletion (D) alleles were visualized after electrophoresis on a 2% agarose gel and ethidium bromide staining under UV light transillumination **(Fig. 2)**. Preferential amplification of the D allele in the heterozygotes has led to their mistyping as DD homozygotes (28). To exclude this possibility, all DD homozygotes were retyped using I

Fig. 1. The sequencing of the region which contains ACE (D) polymorphism. Italic and bold

The method relies on thermal cycling, consisting of steps of thermal cycling which can be accomplished automatically with the DNA thermal cycler. First step is DNA denaturation. DNA denaturation is necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). A base on one strand normally binds only to T on the other strand, and C base on one strand normally binds only to G on the other strand. The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds. To separate the tow strands of DNA, typical strand separation temperatures (Tss) are 95°C for 30 seconds, or 97°C for 15 seconds (21). For G and C rich region higher temperature may be appropriate (21). The second step is primer annealing. Primers contain sequences complementary to the target region of the DNA template. Primer annealing is required for initiation of DNA synthesis at a lower temperature. A temperature of 55°C is a starting degree for 20 base primers with equal GC/AT content (22). Annealing temperatures in the range of 55°C to 72°C generally yield the best results and occurs in a few seconds (21). The third step is primer extension. Primer extension depends upon the length of the target sequence. Extension at 72°C for fragments shorter than 500 base takes only 20 seconds, and fragments up to 1.2 kilo base 40 seconds is sufficient (23).

In the PCR a thermostable Taq DNA polymerase, an enzyme originally isolated from the bacterium *Thermus aquaticus,* are used. The half life of Taq DNA polymerase activity is larger than 2 hours at 92.5°C, 40 minutes at 95°C, and 5 minutes at 97.5°C (21). This DNA polymerase enzymatically assembles a new DNA strand from deoxynucleotide triphosphates (dNTPs), by using separated single-stranded DNA as a template and DNA primers. Because the primer extension products synthesized in one cycle can serve as a template in the next, the DNA template is exponentially amplified. Thus, 20 cycles of PCR yields about a million - fold (220) amplification (22). Since strand dissociation temperatures, primer annealing, product specificity, and Taq DNA polymerase activity affected by magnesium concentration, the magnesium ion concentration was optimized for all gene amplifications in the study. Also, a recommended buffer for PCR is 10 to 50 mM Tris-HCl pH 8.3, up to 50 mM KCl, and up to 0.1% detergents such as Tween 20 must be included. The PCR products of a particular segment of DNA in an ethidium bromide stained agarose gel visualized by UV transillumination. The minimum amount which can be detected by UV transillumination is larger than 10 ng DNA.

Restriction endonucleases are a set of enzymes expressed in bacteria against foreign DNA. Restriction enzymes cut or cleave double stranded DNA at specific recognition base sequences. In 1970 Smith H. et al identified the first restriction enzyme Hind II. Over 3000 of restriction enzymes have been isolated from different bacterial species (24, 25). Restriction enzymes can be used to distinguish single base changes in DNA (26). This method can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. The choice of a restriction enzyme for PCR product is dictated by the product itself. All restriction enzymes require Mg2+ ions as a cofactor and 37°C is optimal for most of them to works. The recommended units and digestion buffer for 100% digestion with restriction enzymes is 10-20 units for 0.1 to 0.5 μg of PCR products and 10 mM Tris-HCl (pH 7.5 to 8.5), 10 mM MgCl2, 100 mM KCl and 0.1 mg/mL BSA.

A genomic DNA were amplified by PCR technique in a total 25 μL PCR mixture containing 200 ng of DNA, deoxynucleotide triphosphates (0.2 mM of each), 0.5 nmol of sense and anti-sense oligonucleotide primers, 1X Taq buffer and 1.25 U of Taq DNA polymerase. eNOS (4a/b), ACE (I/D) and eNOS (Glu298Asp) gene polymorphism reactions were contained 2.5 mM MgCl2 whereas AT1R (A1166C) gene polymorphism reaction were contained 1.5 mM MgCl2. All reagents for PCR amplification and gel electrophoresis were purchased from Fermentas Life Sciences (ELİPS, Istanbul, Turkey). All other chemicals were from Sigma and Merck (BO&GA, Istanbul, Turkey) and of the highest purity available. DNA amplifications were performed with a Techne (TechGene) DNA Thermal Cycler.

## **3. ACE I/D gene polymorphism (rs 4646994)**

The PCR primers with the sequences reported by Rigat B. et al. (27) were used. Sense and anti-sense primers were; 5'-CTGGAGACCACTCCCATCCTTTCT-3' and 5'-GATGTGGCCATCACATTCGTCAGAT-3', respectively. Normally the sense primer in Rigat et al. didn't contain (G), so our PCR products also didn't contain (G), and the antisense primer in Rigat et al. didn't contain (G); which is normally must be included, but it was instead of (G) contained (A). Figure 1 shows the sequencing of the region which contains ACE (D) polymorphism.

#### **PCR Conditions**

458 Polymerase Chain Reaction

The method relies on thermal cycling, consisting of steps of thermal cycling which can be accomplished automatically with the DNA thermal cycler. First step is DNA denaturation. DNA denaturation is necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. The four bases found in DNA are adenine (A), cytosine (C), guanine (G) and thymine (T). A base on one strand normally binds only to T on the other strand, and C base on one strand normally binds only to G on the other strand. The two types of base pairs form different numbers of hydrogen bonds, AT forming two hydrogen bonds, and GC forming three hydrogen bonds. To separate the tow strands of DNA, typical strand separation temperatures (Tss) are 95°C for 30 seconds, or 97°C for 15 seconds (21). For G and C rich region higher temperature may be appropriate (21). The second step is primer annealing. Primers contain sequences complementary to the target region of the DNA template. Primer annealing is required for initiation of DNA synthesis at a lower temperature. A temperature of 55°C is a starting degree for 20 base primers with equal GC/AT content (22). Annealing temperatures in the range of 55°C to 72°C generally yield the best results and occurs in a few seconds (21). The third step is primer extension. Primer extension depends upon the length of the target sequence. Extension at 72°C for fragments shorter than 500 base takes only 20 seconds, and fragments

In the PCR a thermostable Taq DNA polymerase, an enzyme originally isolated from the bacterium *Thermus aquaticus,* are used. The half life of Taq DNA polymerase activity is larger than 2 hours at 92.5°C, 40 minutes at 95°C, and 5 minutes at 97.5°C (21). This DNA polymerase enzymatically assembles a new DNA strand from deoxynucleotide triphosphates (dNTPs), by using separated single-stranded DNA as a template and DNA primers. Because the primer extension products synthesized in one cycle can serve as a template in the next, the DNA template is exponentially amplified. Thus, 20 cycles of PCR yields about a million - fold (220) amplification (22). Since strand dissociation temperatures, primer annealing, product specificity, and Taq DNA polymerase activity affected by magnesium concentration, the magnesium ion concentration was optimized for all gene amplifications in the study. Also, a recommended buffer for PCR is 10 to 50 mM Tris-HCl pH 8.3, up to 50 mM KCl, and up to 0.1% detergents such as Tween 20 must be included. The PCR products of a particular segment of DNA in an ethidium bromide stained agarose gel visualized by UV transillumination. The minimum amount which can be detected by UV

Restriction endonucleases are a set of enzymes expressed in bacteria against foreign DNA. Restriction enzymes cut or cleave double stranded DNA at specific recognition base sequences. In 1970 Smith H. et al identified the first restriction enzyme Hind II. Over 3000 of restriction enzymes have been isolated from different bacterial species (24, 25). Restriction enzymes can be used to distinguish single base changes in DNA (26). This method can be used to genotype a DNA sample without the need for expensive gene sequencing. The sample is first digested with the restriction enzyme to generate DNA fragments, and then the different sized fragments separated by gel electrophoresis. The choice of a restriction enzyme for PCR product is dictated by the product itself. All restriction enzymes require Mg2+ ions as a cofactor and 37°C is optimal for most of them to works. The recommended units and digestion buffer for 100% digestion with restriction enzymes is 10-20 units for 0.1

up to 1.2 kilo base 40 seconds is sufficient (23).

transillumination is larger than 10 ng DNA.


The expected insertion (I) and deletion (D) alleles were visualized after electrophoresis on a 2% agarose gel and ethidium bromide staining under UV light transillumination **(Fig. 2)**. Preferential amplification of the D allele in the heterozygotes has led to their mistyping as DD homozygotes (28). To exclude this possibility, all DD homozygotes were retyped using I

Fig. 1. The sequencing of the region which contains ACE (D) polymorphism. Italic and bold letters were used for the primer sequences.

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

which are deleted in the VNTR 4a polymorphism.

**5. AT1R A1166C gene polymorphism (rs 5186)** 

with the restriction enzyme HaeIII (30).

restriction site.

with Development of Ischemic Stroke in Turkish Subjects in Trakya Region 461

Fig. 3. The sequencing of the region which contains eNOS 4 a/b (27 VNTRs) polymorphism. Italic letters were used for the primer sequences and bold letters were used for 27-bp repeats

Fig. 4. PCR products of eNOS VNTR gene polymorphism. The aa genotype (394 bp; lane 1), the ab genotype (394 bp and 421 bp; lane 2), and the bb genotype (421 bp; samples 3, 4, and 5). Lane (-) is a negative control, and 6 is a size marker (O'RangeRuler 100bp DNA Ladder).

AT1R A1166C gene polymorphism was identified with PCR technique followed by RFLP

PCR primers were generated to amplify the 255 bp fragment encompassing the A1166C variant (sense and anti-sense primers were 5'-GCAGCACTTCACTACCAAATGGGC-3' and 5'-CAGGACAAAAGCAGGCTAGGGAGA -3', respectively) in a 25 µL PCR mixture. Figure 5 shows the sequencing of the region which contains AT1R A1166C gene polymorphism. The sense primer contains one mismatch (A→G) which was required for

allele specific sense primer 5'-TTTGAGACGGAGTCTCGCTC-3' and anti-sense primer, also reported by Rigat B et al. (27) were used. Amplification was performed with a DNA Thermal Cycler with 3 min of denaturation at 93°C, followed by 30 cycles with 1 min of denaturation at 93°C, annealing for 1.5 min at 68°C, and extension for 2 min at 72°C, followed by 3 min of extension at 72°C. When a DD sample amplified using the I-specific primer, it was retyped ID.

Fig. 2. PCR products of ACE gene I/D polymorphism. The DD (190 bp; lane 2, 5, 7, and 8), the ID (190 bp, and 490 bp, lane 3, 4, 6, 9, and 10) and the II (490 bp, lane 1), 50 bp is a size marker, (-) is a negatif control.

## **4. eNOS 4 a/b (27 VNTRs) gene polymorphism**

The PCR primers with the sequences reported by Wang et al. (29) were used. Sense and antisense primers were; 5'-AGGCCCTATGGTAGTGCCTT-3' and 5'-TCTCTTAGTGCTGTGGTCAC-3', respectively. Figure 3 shows the sequencing of the region which contains eNOS 4 a/b (27 VNTRs) polymorphism.

#### **PCR Conditions**


The PCR products were electrophorized on 2.5% agarose gels, stained with ethidium bromide, and checked under UV light transillumination **(Fig. 4)**.

allele specific sense primer 5'-TTTGAGACGGAGTCTCGCTC-3' and anti-sense primer, also reported by Rigat B et al. (27) were used. Amplification was performed with a DNA Thermal Cycler with 3 min of denaturation at 93°C, followed by 30 cycles with 1 min of denaturation at 93°C, annealing for 1.5 min at 68°C, and extension for 2 min at 72°C, followed by 3 min of extension at 72°C. When a DD sample amplified using the I-specific

Fig. 2. PCR products of ACE gene I/D polymorphism. The DD (190 bp; lane 2, 5, 7, and 8), the ID (190 bp, and 490 bp, lane 3, 4, 6, 9, and 10) and the II (490 bp, lane 1), 50 bp is a size

The PCR primers with the sequences reported by Wang et al. (29) were used. Sense and antisense primers were; 5'-AGGCCCTATGGTAGTGCCTT-3' and 5'-TCTCTTAGTGCTGTGGTCAC-3', respectively. Figure 3 shows the sequencing of the

The PCR products were electrophorized on 2.5% agarose gels, stained with ethidium

primer, it was retyped ID.

marker, (-) is a negatif control.

**PCR Conditions** 

**4. eNOS 4 a/b (27 VNTRs) gene polymorphism** 

region which contains eNOS 4 a/b (27 VNTRs) polymorphism.

bromide, and checked under UV light transillumination **(Fig. 4)**.

Fig. 3. The sequencing of the region which contains eNOS 4 a/b (27 VNTRs) polymorphism. Italic letters were used for the primer sequences and bold letters were used for 27-bp repeats which are deleted in the VNTR 4a polymorphism.

Fig. 4. PCR products of eNOS VNTR gene polymorphism. The aa genotype (394 bp; lane 1), the ab genotype (394 bp and 421 bp; lane 2), and the bb genotype (421 bp; samples 3, 4, and 5). Lane (-) is a negative control, and 6 is a size marker (O'RangeRuler 100bp DNA Ladder).

## **5. AT1R A1166C gene polymorphism (rs 5186)**

AT1R A1166C gene polymorphism was identified with PCR technique followed by RFLP with the restriction enzyme HaeIII (30).

PCR primers were generated to amplify the 255 bp fragment encompassing the A1166C variant (sense and anti-sense primers were 5'-GCAGCACTTCACTACCAAATGGGC-3' and 5'-CAGGACAAAAGCAGGCTAGGGAGA -3', respectively) in a 25 µL PCR mixture. Figure 5 shows the sequencing of the region which contains AT1R A1166C gene polymorphism. The sense primer contains one mismatch (A→G) which was required for restriction site.

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

agarose gel and ethidium bromide staining (Fig. 6).

with the restriction enzyme BanII (19, 31).

and checked under UV light transillumination.

**PCR conditions** 

**6. eNOS Glu298Asp (rs 1799983) gene polymorphism** 

region which contains eNOS Glu298Asp gene polymorphism.

with Development of Ischemic Stroke in Turkish Subjects in Trakya Region 463

Ten microliters of PCR product were digested with 5 unite of the restriction enzyme HaeIII (Takara Bio Inc, Japan) in 1 X M buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM Dithiothreitol and 50 mM NaCl) for 2 hours at 37°C. When mutant allele (cytosine), digested with HaeIII that yield two fragments, whereas a wild allele (adenine) at nucleotide position 1166, had no cutting site for HaeIII, so that the PCR product was not cleaved into two fragments. The restriction digest products were visualized after electrophoresis on a 2.5%

Glu298Asp polymorphism of eNOS was identified with PCR technique followed by RFLP

PCR primers were generated to amplify the 248 bp fragment encompassing the eNOS Glu298Asp variant primers 5'-AAGGCAGGAGACAGTGGATGGA-3' (sense) and 5'-CCC AGTCAATCCCTTTGGTGCTCA-3' (anti-sense). Figure 7 shows the sequencing of the

The PCR products were electrophorized on 2% agarose gels, stained with ethidium bromide,

Fig. 7. The sequencing of the region which contains eNOS Glu298Asp polymorphism. Italic and bold letters were used for the primer sequences. The underlined and bold letters

Ten microliters of PCR product were digested with the restriction enzyme BanII to digest wild allele (guanine). When a guanine is at nucleotide position 894, resulting in a glutamic acid at amino acid position 298, BanII restriction enzyme produces two fragments of 163 and

represent the restriction site for Ban II (5'-G(A/G)GC(T/C)↓C-3').

#### **PCR Conditions**


The PCR products were electrophorized on 2% agarose gels, stained with ethidium bromide, and checked under UV light transillumination.

Fig. 5. The sequencing of the region which contains AT1R A1166C polymorphism. Italic and bold letters were used for the primer sequences. The underlined and bold letters represent the restriction site for HaeIII (5'-GG↓CC-3').

Fig. 6. EtBr stained gel of HaeIII digested PCR products of AT1R A1166C shows the AA genotype (255 bp; lane 2, 3, 7, and 8), the AC genotype (255 bp, 231 bp, and 24 bp; lane 5, 6, 9, and 11), the CC genotype (231 bp, and 24bp; lane 1, 4, and 10), lane O'GR is a size marker (100bp DNA Ladder).

Ten microliters of PCR product were digested with 5 unite of the restriction enzyme HaeIII (Takara Bio Inc, Japan) in 1 X M buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM Dithiothreitol and 50 mM NaCl) for 2 hours at 37°C. When mutant allele (cytosine), digested with HaeIII that yield two fragments, whereas a wild allele (adenine) at nucleotide position 1166, had no cutting site for HaeIII, so that the PCR product was not cleaved into two fragments. The restriction digest products were visualized after electrophoresis on a 2.5% agarose gel and ethidium bromide staining (Fig. 6).

## **6. eNOS Glu298Asp (rs 1799983) gene polymorphism**

Glu298Asp polymorphism of eNOS was identified with PCR technique followed by RFLP with the restriction enzyme BanII (19, 31).

PCR primers were generated to amplify the 248 bp fragment encompassing the eNOS Glu298Asp variant primers 5'-AAGGCAGGAGACAGTGGATGGA-3' (sense) and 5'-CCC AGTCAATCCCTTTGGTGCTCA-3' (anti-sense). Figure 7 shows the sequencing of the region which contains eNOS Glu298Asp gene polymorphism.

## **PCR conditions**

462 Polymerase Chain Reaction

The PCR products were electrophorized on 2% agarose gels, stained with ethidium bromide,

Fig. 5. The sequencing of the region which contains AT1R A1166C polymorphism. Italic and bold letters were used for the primer sequences. The underlined and bold letters represent

Fig. 6. EtBr stained gel of HaeIII digested PCR products of AT1R A1166C shows the AA genotype (255 bp; lane 2, 3, 7, and 8), the AC genotype (255 bp, 231 bp, and 24 bp; lane 5, 6, 9, and 11), the CC genotype (231 bp, and 24bp; lane 1, 4, and 10), lane O'GR is a size marker

**PCR Conditions** 

and checked under UV light transillumination.

the restriction site for HaeIII (5'-GG↓CC-3').

(100bp DNA Ladder).


The PCR products were electrophorized on 2% agarose gels, stained with ethidium bromide, and checked under UV light transillumination.

Fig. 7. The sequencing of the region which contains eNOS Glu298Asp polymorphism. Italic and bold letters were used for the primer sequences. The underlined and bold letters represent the restriction site for Ban II (5'-G(A/G)GC(T/C)↓C-3').

Ten microliters of PCR product were digested with the restriction enzyme BanII to digest wild allele (guanine). When a guanine is at nucleotide position 894, resulting in a glutamic acid at amino acid position 298, BanII restriction enzyme produces two fragments of 163 and

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

with Development of Ischemic Stroke in Turkish Subjects in Trakya Region 465

aa ab bb

Non-Significant Non-Significant Non-Significant

Non-Significant Non-Significant Non-Significant

GG GT TT

Non-Significant Non-Significant Non-Significant

Controls (%) 49.3 45.8 4.9

Table 5. Distribution of eNOS (Glu298Asp) genotype frequency in the controls and stroke

In our previous study about potential angiotensinogen (AGT) gene that predispose to hypertension, we failed to detect any relation between T174M and M235T gene polymorphisms of the AGT gene in the RAS and the development of hypertension (32).

Now, we are working on the AGT gene to clarify the role of T174M and M235T gene

In addition to demographic and clinical characteristics, which are important in the developing of ischemic stroke, our data does not suggest that ACE (I/D), AT1R (A1166C), eNOS (4 a/b) and eNOS (Glu298Asp) gene polymorphisms, in contrast to other studies which shows a positive association between this gene polymorphisms and ischemic stroke,

polymorphisms of the AGT gene in the stroke Turkish patients from Trakya region.

are a common cause of ischemic stroke in Turkish patients from Trakya region.

Stroke Patients (%) 56.3 40.6 3.1

Controls (%) 2.8 29.8 67.4

Table 3. Distribution of eNOS (4 a/b) genotype frequency in the controls and stroke patients

Controls (%) 60.1 35.7 4.2

Stroke Patients (%) 58.0 34.6 7.4

Table 4. Distribution of AT1R (A1166C) genotype frequency in the controls and stroke

patients

patients

**8. Conclusions** 

AA AC CC

Stroke Patients (%) 2.0 35.0 63.0

85 bp. In contrast, when a thymine is at nucleotide position 894 (mutant allele), resulting in an aspartic acid in the amino acid sequence, the Asp298 variant had no cutting site for BanII, so that the 248 bp PCR product was not cleaved into 163 and 85 bp fragments. The restriction digest products were analyzed through electrophoresis on 2.5% agarose gel and ethidium bromide staining (Fig. 8).

Fig. 8. EtBr stained gel of BanII digested products of eNOS gene Glu298Asp polymorphism. Line 1 and 4; GT alleles (85, 163, and 248 bp), line 2; GG alleles (85 and 163 bp), line 3; TT alleles (248 bp) and line 100bp; Gene Ruler 100 bp DNA Ladder.

## **7. Results and discussion**

Table 2, 3, 4, and 5 shows the distributions of ACE I/D, eNOS (4 a/b), AT1R (A1166C), and eNOS Glu298Asp genotypes, respectively.

Statistical analyses were performed with the SPSS 15.0 software and STATA program. Genotypic distributions were in accordance with Hardy-Weinberg equilibrium in the stroke group as well as in the control group. Several studies have shown differences in the genotypic distributions of these genes while, others have shown no differences between the controls and patients. Our results didn't show any significant difference between the ischemic stroke patients and the controls (p>0.05) and suggested the lack of an association between the 4 gene polymorphisms and ischemic stroke (Table 2, 3, 4, and 5). So the 4 gene polymorphisms did not enhance the predictability of stroke.


Table 2. Distribution of ACE (I/D) genotype frequency in the controls and stroke patients


Table 3. Distribution of eNOS (4 a/b) genotype frequency in the controls and stroke patients


Table 4. Distribution of AT1R (A1166C) genotype frequency in the controls and stroke patients


Table 5. Distribution of eNOS (Glu298Asp) genotype frequency in the controls and stroke patients

In our previous study about potential angiotensinogen (AGT) gene that predispose to hypertension, we failed to detect any relation between T174M and M235T gene polymorphisms of the AGT gene in the RAS and the development of hypertension (32).

Now, we are working on the AGT gene to clarify the role of T174M and M235T gene polymorphisms of the AGT gene in the stroke Turkish patients from Trakya region.

## **8. Conclusions**

464 Polymerase Chain Reaction

85 bp. In contrast, when a thymine is at nucleotide position 894 (mutant allele), resulting in an aspartic acid in the amino acid sequence, the Asp298 variant had no cutting site for BanII, so that the 248 bp PCR product was not cleaved into 163 and 85 bp fragments. The restriction digest products were analyzed through electrophoresis on 2.5% agarose gel and

Fig. 8. EtBr stained gel of BanII digested products of eNOS gene Glu298Asp polymorphism. Line 1 and 4; GT alleles (85, 163, and 248 bp), line 2; GG alleles (85 and 163 bp), line 3; TT

Table 2, 3, 4, and 5 shows the distributions of ACE I/D, eNOS (4 a/b), AT1R (A1166C), and

Statistical analyses were performed with the SPSS 15.0 software and STATA program. Genotypic distributions were in accordance with Hardy-Weinberg equilibrium in the stroke group as well as in the control group. Several studies have shown differences in the genotypic distributions of these genes while, others have shown no differences between the controls and patients. Our results didn't show any significant difference between the ischemic stroke patients and the controls (p>0.05) and suggested the lack of an association between the 4 gene polymorphisms and ischemic stroke (Table 2, 3, 4, and 5). So the 4 gene

DD ID II

Non-Significant Non-Significant Non-Significant

Controls (%) 34.3 49.7 16.1

Stroke Patients (%) 34.0 50.0 16.0

Table 2. Distribution of ACE (I/D) genotype frequency in the controls and stroke patients

alleles (248 bp) and line 100bp; Gene Ruler 100 bp DNA Ladder.

polymorphisms did not enhance the predictability of stroke.

ethidium bromide staining (Fig. 8).

**7. Results and discussion** 

eNOS Glu298Asp genotypes, respectively.

In addition to demographic and clinical characteristics, which are important in the developing of ischemic stroke, our data does not suggest that ACE (I/D), AT1R (A1166C), eNOS (4 a/b) and eNOS (Glu298Asp) gene polymorphisms, in contrast to other studies which shows a positive association between this gene polymorphisms and ischemic stroke, are a common cause of ischemic stroke in Turkish patients from Trakya region.

Lack of Evidence for Contribution of eNOS, ACE and AT1R Gene Polymorphisms

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[30] Berge K. E., Berg K. Polymorphisms at the angiotensinogen (AGT) and angiotensin II

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Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke. Mol. Biol. Rep. (ISI), 539-1543 pp., DOI:

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**22** 

*Republic of Serbia* 

**Analysis of Genomic Instability and** 

Nikola Tanic1, Jasna Bankovic1 and Nasta Tanic2

**Arbitrarily Primed PCR** 

*2Institute of nuclear Sciences "Vinca", Belgrade* 

**Tumor-Specific Genetic Alterations by** 

*1Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade* 

It is now widely accepted that cancer development is a multistage process that results from an accumulation of mutations (Lengauer et al., 1998). Since spontaneous mutation rates in human cells are considerably lower then the large number of mutations observed in cancer cells, cancer cells must be a manifestation of the mutator phenotype. The mutator phenotype, also referred to as genomic instability, designates the increased mutation rate that occurs in neoplastic cells (Loeb, 1991). The induction of the genomic instability phenotype is emerging to be a crucial early event in carcinogenesis that enables an initiated cell to evolve into a cancer cell by achieving a greater proliferative capacity and genetic plasticity, which can overcome host immunological resistance, localized toxic environments and a suboptimal supply of micronutrients (Loeb, 1991; Cahill et al., 1999; Fenech 2002). Two distinct forms of genomic instability have been identified, microsatellite instability (MIN) and chromosomal instability (CIN). They probably encompass most characterized malignances (Lengauer et al., 1998; Breivik & Gaudernack, 1999). Genomic instability is present in all stages of cancer, from precancerous lesions to advanced cancers (Negrini et al.,

Measurements of instability have been performed by a variety of techniques, including flow cytometry, comparative genomic hybridization (CGH), allelotyping, and analysis of gene amplification rates (Vogelstein et al., 1989; Kallioniemi et al., 1994; Jass et al., 1994). These approaches, although informative, are generally cumbersome and somewhat impractical for widespread clinical use. Unlike these techniques, DNA fingerprinting methods, RAPD (Random Amplified Polymorphic DNA) and AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) are rapid and simple procedures that examine the whole genome and detect the propensity of a tumor to undergo genomic rearrangements (Peinado et al., 1992;

AP-PCR is a PCR-based DNA fingerprinting method that utilizes arbitrarily chosen primers to co-amplify multiple and independent sequences under low stringency conditions during the first cycles. It was first described by Welsh and McClelland (1990), who designed it to amplify multiple DNA fragments from anonymous regions of the genome. Initial cycles of

**1. Introduction** 

2010; Markovic et al., 2008)

Perucho et al., 1996).

Association of the Missense Glu298Asp Variant of the Endothelial Nitric Oxide Synthase Gene With Myocardial Infarction. JACC. 31: 1506-10, (1998).

[32] Basak, A. A., Sipahi T., Ustundag S., Ozgen Z., Budak M., Sen S., Sener S. Association of Angiotensinogen T174M and M235T Gene Variants with Development of Hypertension in Turkish Subjects of Trakya Region. Biotechnol. & Biotechnol. Eq., 22, 984-989, (2008).

## **Analysis of Genomic Instability and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR**

Nikola Tanic1, Jasna Bankovic1 and Nasta Tanic2 *1Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Belgrade 2Institute of nuclear Sciences "Vinca", Belgrade Republic of Serbia* 

## **1. Introduction**

468 Polymerase Chain Reaction

Synthase Gene With Myocardial Infarction. JACC. 31: 1506-10, (1998). [32] Basak, A. A., Sipahi T., Ustundag S., Ozgen Z., Budak M., Sen S., Sener S. Association of

22, 984-989, (2008).

Association of the Missense Glu298Asp Variant of the Endothelial Nitric Oxide

Angiotensinogen T174M and M235T Gene Variants with Development of Hypertension in Turkish Subjects of Trakya Region. Biotechnol. & Biotechnol. Eq.,

> It is now widely accepted that cancer development is a multistage process that results from an accumulation of mutations (Lengauer et al., 1998). Since spontaneous mutation rates in human cells are considerably lower then the large number of mutations observed in cancer cells, cancer cells must be a manifestation of the mutator phenotype. The mutator phenotype, also referred to as genomic instability, designates the increased mutation rate that occurs in neoplastic cells (Loeb, 1991). The induction of the genomic instability phenotype is emerging to be a crucial early event in carcinogenesis that enables an initiated cell to evolve into a cancer cell by achieving a greater proliferative capacity and genetic plasticity, which can overcome host immunological resistance, localized toxic environments and a suboptimal supply of micronutrients (Loeb, 1991; Cahill et al., 1999; Fenech 2002). Two distinct forms of genomic instability have been identified, microsatellite instability (MIN) and chromosomal instability (CIN). They probably encompass most characterized malignances (Lengauer et al., 1998; Breivik & Gaudernack, 1999). Genomic instability is present in all stages of cancer, from precancerous lesions to advanced cancers (Negrini et al., 2010; Markovic et al., 2008)

> Measurements of instability have been performed by a variety of techniques, including flow cytometry, comparative genomic hybridization (CGH), allelotyping, and analysis of gene amplification rates (Vogelstein et al., 1989; Kallioniemi et al., 1994; Jass et al., 1994). These approaches, although informative, are generally cumbersome and somewhat impractical for widespread clinical use. Unlike these techniques, DNA fingerprinting methods, RAPD (Random Amplified Polymorphic DNA) and AP-PCR (Arbitrarily Primed Polymerase Chain Reaction) are rapid and simple procedures that examine the whole genome and detect the propensity of a tumor to undergo genomic rearrangements (Peinado et al., 1992; Perucho et al., 1996).

> AP-PCR is a PCR-based DNA fingerprinting method that utilizes arbitrarily chosen primers to co-amplify multiple and independent sequences under low stringency conditions during the first cycles. It was first described by Welsh and McClelland (1990), who designed it to amplify multiple DNA fragments from anonymous regions of the genome. Initial cycles of

Analysis of Genomic Instability

**2. Materials and methods** 

**2.1 Tissue samples and DNA extraction** 

down in the 1964 Declaration of Helsinki.

**2.2 AP-PCR DNA fingerprinting** 

impurities in the DNA preparations.

Table 1.

DNA concentration was assessed spectrophotometrically.

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 471

Paired tumor and normal tissue samples (adjacent normal lung tissue and blood for malignant gliomas, HNSCC and leukoplakias) were analyzed. Specifically, 30 malignant glioma patients who underwent surgical resection at Clinic for Neurosurgery, Clinical Center of Serbia, 30 NSCLC patients who underwent surgery at the Institute for Lung Diseases and Tuberculosis, Clinical Centre of Serbia, 32 leukoplakia patients and 30 HNSCC patients who underwent surgery at the Clinic of Maxillofacial Surgery, School of Dentistry, University of Belgrade. Freshly excised tissue samples were partitioned for histopathology and DNA analyses. The specimens for DNA analyses were frozen in liquid nitrogen until DNA extraction. The samples were collected and used after obtaining informed consents and approval from the Ethics Committee, in accordance with the ethical standards laid

DNA was extracted using the phenol/chloroform/isoamyl alcohol method (Sambrook et al., 1989). The quality of the DNA was verified by electrophoresis on 0.8% agarose gel. The

Genomic instability was determined by comparing the AP-PCR profiles of paired tumor and normal DNA samples of the same patient. Altogether, twenty primers were tested for the ability to generate informative fingerprints that distinguish tumor from normal tissue. Optimization of AP-PCR reactions was done for each primer according to Cobb (1997) and included the search for conditions that would yield profiles of moderate complexity in order to simplify the analysis (McClelland & Welsh, 1994). Normally, optimization of AP-PCR DNA fingerprinting would require each variable to be tested independently. An experiment investigating the effects and interactions of four critical reaction components (dNTPs, MgCl2, primer and DNA), each at three concentrations, would require 81 (i.e., 34) separate reactions. However, using modified Taguchi method (Taguchi & Wu, 1980, as cited in Cobb, 1997) only nine reactions are required to perform the same optimization. Here an estimate of the effect of individual components is achieved by looking at the effects that component interactions have on the fingerprint. These interactions are determined by arranging those components that are likely to affect the reactions into an orthogonal array. The product yield for each reaction is used to estimate the effects of individual components on amplification. We varied the PCR components in the following final concentrations: dNTPs (0.2 mM, 0.4 mM, 0.6 mM), MgCl2 (1.5 mM, 2.5 mM, 3.5 mM), primer (1.5 *μ*M, 3.0 *μ*M, 5.0 *μ*M) and DNA (50 ng, 100 ng, 150 ng). DNA concentration did not affect AP-PCR fingerprints and it was used to validate the method. Namely, after optimal reaction conditions were established, each experiment included the analysis of two template concentrations (25 ng and 50 ng in a final volume of 25 µL) for each individual in order to exclude artifacts arising from

Twelve out of twenty primers produced informative profiles differentiating normal from tumor tissue. Primer sequences, AP-PCR conditions and reaction mixtures are given in

the reaction are performed under low stringency conditions which are achieved with low temperatures during the annealing step of PCR and/or high magnesium concentration in the reaction. Under these conditions the arbitrary primer anneals to the best matches in the template. The priming events during the initial low stringency cycles are arbitrary since they depend on the nucleotide sequence of the PCR primer, which is arbitrarily chosen. Competition between these annealing events results in reproducible and quantitative amplification of many discrete bands. Further amplification of these sequences (discrete bands) under high stringency conditions produces a complex fingerprint which can be visualized by gel electrophoresis. The obtained band pattern is characteristic and representative of the genome used as template.

The large number of bands amplified with a single arbitrary primer generates a complex fingerprint that can be used to detect differences in the arbitrary amplified DNA sequences from two different but closely related genomes, like DNA from normal and cancer cells. Such differences correspond to somatic genetic alterations. In addition, AP-PCR method permits direct cloning and identification of altered variant bands i.e. altered DNA sequences. Therefore, this unbiased methodology allows for molecular karyotyping of somatically acquired genomic abnormalities, comparing related genomes, whereby one is a derivative of the other emerging via undefined and abnormal genomic events. Indeed, AP-PCR has been successfully used as a molecular alternative for cancer cytogenetics since it has proved to be capable of detecting chromosomal gains and losses as well as point mutations associated with carcinogenesis (Perucho et al., 1996; Chariyalertsak et al., 2005). This is based on the following favorable properties of the method: (i) the amplified bands usually originate from single copy sequences rather then from repetitive elements; (ii) there is no apparent bias for the chromosomal origins of the amplified bands, and therefore, fingerprints representative of the full chromosomal complement can be obtained by using a few arbitrary primers; (iii) the amplification is semi-quantitative, that is, the intensities of the amplified bands are almost proportional to the concentration of the corresponding template sequences.

Taking into account the potential and advantages of AP-PCR method, it seems as a reasonable approach to use this method to detect and quantify the level of genomic instability in various cancer samples. Therefore, we applied AP-PCR to measure genomic instability in samples of patients with Non Small Cell Lung Carcinoma (NSCLC) of various stages and grades, samples of patients with Malignant Gliomas of various grades (Anaplastic Astrocytomas and Glioblastomas) and samples of patients with Head and Neck Squamous Cell Carcinoma (HNSCC) and their premalignant lesions leukoplakias. Moreover, we aimed to identify some of these genomic alterations associated with the process of carcinogenesis in these types of tumors.

Here we describe the procedure for analyzing the level of genomic instability and identifying specific genetic alterations that occur during the tumorigenic process by Arbitrarily Primed PCR. This procedure involves the following steps: (i) comparative AP-PCR analysis of matching normal and tumor tissue and determination of the frequency of DNA alterations, a measurement of genomic instability; (ii) correlation between the level of genomic instability and histological grade and stage of each tumor; (iii) isolation and identification of altered amplified bands. Obtained results are presented and discussed in terms of the evolution of these types of tumors.

## **2. Materials and methods**

470 Polymerase Chain Reaction

the reaction are performed under low stringency conditions which are achieved with low temperatures during the annealing step of PCR and/or high magnesium concentration in the reaction. Under these conditions the arbitrary primer anneals to the best matches in the template. The priming events during the initial low stringency cycles are arbitrary since they depend on the nucleotide sequence of the PCR primer, which is arbitrarily chosen. Competition between these annealing events results in reproducible and quantitative amplification of many discrete bands. Further amplification of these sequences (discrete bands) under high stringency conditions produces a complex fingerprint which can be visualized by gel electrophoresis. The obtained band pattern is characteristic and

The large number of bands amplified with a single arbitrary primer generates a complex fingerprint that can be used to detect differences in the arbitrary amplified DNA sequences from two different but closely related genomes, like DNA from normal and cancer cells. Such differences correspond to somatic genetic alterations. In addition, AP-PCR method permits direct cloning and identification of altered variant bands i.e. altered DNA sequences. Therefore, this unbiased methodology allows for molecular karyotyping of somatically acquired genomic abnormalities, comparing related genomes, whereby one is a derivative of the other emerging via undefined and abnormal genomic events. Indeed, AP-PCR has been successfully used as a molecular alternative for cancer cytogenetics since it has proved to be capable of detecting chromosomal gains and losses as well as point mutations associated with carcinogenesis (Perucho et al., 1996; Chariyalertsak et al., 2005). This is based on the following favorable properties of the method: (i) the amplified bands usually originate from single copy sequences rather then from repetitive elements; (ii) there is no apparent bias for the chromosomal origins of the amplified bands, and therefore, fingerprints representative of the full chromosomal complement can be obtained by using a few arbitrary primers; (iii) the amplification is semi-quantitative, that is, the intensities of the amplified bands are almost proportional to the concentration of the corresponding template

Taking into account the potential and advantages of AP-PCR method, it seems as a reasonable approach to use this method to detect and quantify the level of genomic instability in various cancer samples. Therefore, we applied AP-PCR to measure genomic instability in samples of patients with Non Small Cell Lung Carcinoma (NSCLC) of various stages and grades, samples of patients with Malignant Gliomas of various grades (Anaplastic Astrocytomas and Glioblastomas) and samples of patients with Head and Neck Squamous Cell Carcinoma (HNSCC) and their premalignant lesions leukoplakias. Moreover, we aimed to identify some of these genomic alterations associated with the

Here we describe the procedure for analyzing the level of genomic instability and identifying specific genetic alterations that occur during the tumorigenic process by Arbitrarily Primed PCR. This procedure involves the following steps: (i) comparative AP-PCR analysis of matching normal and tumor tissue and determination of the frequency of DNA alterations, a measurement of genomic instability; (ii) correlation between the level of genomic instability and histological grade and stage of each tumor; (iii) isolation and identification of altered amplified bands. Obtained results are presented and discussed in

representative of the genome used as template.

process of carcinogenesis in these types of tumors.

terms of the evolution of these types of tumors.

sequences.

## **2.1 Tissue samples and DNA extraction**

Paired tumor and normal tissue samples (adjacent normal lung tissue and blood for malignant gliomas, HNSCC and leukoplakias) were analyzed. Specifically, 30 malignant glioma patients who underwent surgical resection at Clinic for Neurosurgery, Clinical Center of Serbia, 30 NSCLC patients who underwent surgery at the Institute for Lung Diseases and Tuberculosis, Clinical Centre of Serbia, 32 leukoplakia patients and 30 HNSCC patients who underwent surgery at the Clinic of Maxillofacial Surgery, School of Dentistry, University of Belgrade. Freshly excised tissue samples were partitioned for histopathology and DNA analyses. The specimens for DNA analyses were frozen in liquid nitrogen until DNA extraction. The samples were collected and used after obtaining informed consents and approval from the Ethics Committee, in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki.

DNA was extracted using the phenol/chloroform/isoamyl alcohol method (Sambrook et al., 1989). The quality of the DNA was verified by electrophoresis on 0.8% agarose gel. The DNA concentration was assessed spectrophotometrically.

## **2.2 AP-PCR DNA fingerprinting**

Genomic instability was determined by comparing the AP-PCR profiles of paired tumor and normal DNA samples of the same patient. Altogether, twenty primers were tested for the ability to generate informative fingerprints that distinguish tumor from normal tissue. Optimization of AP-PCR reactions was done for each primer according to Cobb (1997) and included the search for conditions that would yield profiles of moderate complexity in order to simplify the analysis (McClelland & Welsh, 1994). Normally, optimization of AP-PCR DNA fingerprinting would require each variable to be tested independently. An experiment investigating the effects and interactions of four critical reaction components (dNTPs, MgCl2, primer and DNA), each at three concentrations, would require 81 (i.e., 34) separate reactions. However, using modified Taguchi method (Taguchi & Wu, 1980, as cited in Cobb, 1997) only nine reactions are required to perform the same optimization. Here an estimate of the effect of individual components is achieved by looking at the effects that component interactions have on the fingerprint. These interactions are determined by arranging those components that are likely to affect the reactions into an orthogonal array. The product yield for each reaction is used to estimate the effects of individual components on amplification. We varied the PCR components in the following final concentrations: dNTPs (0.2 mM, 0.4 mM, 0.6 mM), MgCl2 (1.5 mM, 2.5 mM, 3.5 mM), primer (1.5 *μ*M, 3.0 *μ*M, 5.0 *μ*M) and DNA (50 ng, 100 ng, 150 ng). DNA concentration did not affect AP-PCR fingerprints and it was used to validate the method. Namely, after optimal reaction conditions were established, each experiment included the analysis of two template concentrations (25 ng and 50 ng in a final volume of 25 µL) for each individual in order to exclude artifacts arising from impurities in the DNA preparations.

Twelve out of twenty primers produced informative profiles differentiating normal from tumor tissue. Primer sequences, AP-PCR conditions and reaction mixtures are given in Table 1.


Table 1. Primer sequences, AP-PCR conditions and reaction mixtures

Analysis of Genomic Instability

*Fixation Pretreatment* 

*Fixation* 

*Water Washing* 

*Water Washing* 

*Silver Impregnation* 

*Developing - Reduction*

*Stopping Reduction* 

9700 (Applied Biosystems, Foster City, California, USA).

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 473

The reactions consisted of an initial denaturation step (95ºC for 5 min), 4 cycles at lowstringency conditions (specific for each primer), 35 cycles at high-stringency conditions (specific for each primer), and a final extension (72ºC for 7 min) in a GeneAmp® PCR System

The AP-PCR products were separated on 6 – 8% non-denaturing polyacrylamide (PAA) gels and visualized by silver staining. Silver- staining procedure creates permanent record of the electrophoresis results and includes several steps: fixing, silver impregnation, development and stopping the reaction. In the fixing step, the gel is treated with 1% nitric acid solution to render the macromolecules in the gel insoluble and prevent diffusion during the subsequent staining steps. In the silver impregnation step, soluble silver ion (Ag+) derived from the silver nitrate, 12mM AgNO3 solution, binds to nucleic acid bases fixed in gel. Generally, DNA bases promotes the reduction of silver ion to metallic silver (Ag0), which is insoluble and visible, allowing nucleic acid-containing bands to be seen. In order to prevent reduction of silver ion to metallic silver before the end of silver impregnation, this step is often performed in mildly acidic acid conditions. During the development step, formaldehyde reduces silver ions to metallic silver in process that only proceed at high pH, approximately 12. For that reason, sodium carbonate is included as one of the main component that render development solution alkaline. Stopping reaction step imply prevention of any further silver ion reduction by soaking the gels in the 10 % acetic acid solution. Finally, it should be emphasized that water washes are also included between some of the above mentioned

steps in the silver staining procedure (detailed procedure is given in Table 2).

*Step Solution Time* 

10% Ethanol

10 minutes

3 minutes

2 x 1 minute

30 minutes

3 x 1 minutes

Until desired images appear

5 – 10 minutes

1% Nitric Acid Solution

Destiled H2O

12 mM Silver Nitrate Solution

Destiled H2O

0.28 M Sodium Carbonat with 0.019 % Formaldehyde

10 % Acetic Acid

Gel images were acquired with the Multi-Analyst/PC Software Image Analysis System (Bio Rad Gel Doc 1000). Digitized images were loaded into the specialized public software Image J (National Institute of Health, USA, www.rsb.info.nih.gov/ij) and analyzed by the image enhancement function 'adapthisteq'. This function performs contrast-limited adaptive

Table 2. In-house procedure for silver- staining of PAA gels.

*μ*M primer; 1 U

Taq DNA

*μ*M primer; 1 U

Taq DNA

*μ*M primer; 1 U

Taq DNA

*μ*M primer; 1

U Taq DNA

E5A p53 5'–CAG CCC TGT CGT CTC TCC AG–3' 950C 30"; 400C 2'; 720C 1' 950C 30"; 550C 1'; 720C 1' 0,6 mM each dNTP; 3,5 mM MgCl2; 3

p53 A 5'–TTG GGC AGT GCT CGC TTA GT–3' 950C 30"; 400C 2'; 720C 1' 950C 30"; 600C 1'; 720C 1' 0,2 mM each dNTP; 3,5 mM MgCl2; 5

H61-5' 5'-AGG TGG TCA TTG ATG GGG AG-3' 940C 1'; 450C 2", 720C 2' 940C 1'; 620C 1'; 720C 2' 0.4 mM each dNTPs, 2.5 mM MgCl2 5

Table 1. Primer sequences, AP-PCR conditions and reaction mixtures

Primer Primer sequence AP-PCR low-stringency conditions AP-PCR high-stringency conditions AP-PCR reaction mixture CCNA1 5'-AAG AGG ACC AGG AGA ATA TCA-3' 95oC 30″ 45oC 2′ 72oC 1′ 95oC 30″ 60oC 1′ 72oC 1′ 0,2mM each dNTP; 3,5mM MgCl2; 5μM primer; 1U Taq DNA LRP-A 5'-GCT TCC GAG GTC TCA AAG C-3' 95oC 30″ 40oC 2′ 72oC 1′ 95oC 30″ 58oC 1′ 72oC 1′ 0,2mM each dNTP; 3,5mM MgCl2; 5μM primer; 1U Taq DNA MDR-A 5'-GTT CAA ACT TCT GCT CCT GA-3' 95oC 30″ 40oC 2′ 72oC 1′ 95oC 30″ 58oC 1′ 72oC 1′ 0,4mM each dNTP; 2,5mM MgCl2; 5μM primer; 1U Taq DNA E8S p53 5'-TAA ATG GGA CAG GTA GGA CC-3' 95oC 30″ 40oC 2′ 72oC 1′ 95oC 30″ 58oC 1′ 72oC 1′ 0,4mM each dNTP; 2,5mM MgCl2; 5μM primer; 1U Taq DNA GAPDH-S 5'– CGG AGT CAA CGG ATT TGG TCG TAT– 3' 95ºC 30''; 50ºC 2'; 72ºC 1' 95ºC 30''; 70ºC 1'; 72ºC 1' 0,4mM each dNTP; 2,5mM MgCl2; 5μM primer; 1 U Taq DNA GAPDH-A 5'–AGC CTT CTC CAT GGTGGT GAA GAC–3' 950C 30"; 500C 2'; 720C 1' 950C 30"; 720C 1'; 720C 1' 0,2 mM each dNTP; 2,5 mM MgCl2; 3

The reactions consisted of an initial denaturation step (95ºC for 5 min), 4 cycles at lowstringency conditions (specific for each primer), 35 cycles at high-stringency conditions (specific for each primer), and a final extension (72ºC for 7 min) in a GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, California, USA).

The AP-PCR products were separated on 6 – 8% non-denaturing polyacrylamide (PAA) gels and visualized by silver staining. Silver- staining procedure creates permanent record of the electrophoresis results and includes several steps: fixing, silver impregnation, development and stopping the reaction. In the fixing step, the gel is treated with 1% nitric acid solution to render the macromolecules in the gel insoluble and prevent diffusion during the subsequent staining steps. In the silver impregnation step, soluble silver ion (Ag+) derived from the silver nitrate, 12mM AgNO3 solution, binds to nucleic acid bases fixed in gel. Generally, DNA bases promotes the reduction of silver ion to metallic silver (Ag0), which is insoluble and visible, allowing nucleic acid-containing bands to be seen. In order to prevent reduction of silver ion to metallic silver before the end of silver impregnation, this step is often performed in mildly acidic acid conditions. During the development step, formaldehyde reduces silver ions to metallic silver in process that only proceed at high pH, approximately 12. For that reason, sodium carbonate is included as one of the main component that render development solution alkaline. Stopping reaction step imply prevention of any further silver ion reduction by soaking the gels in the 10 % acetic acid solution. Finally, it should be emphasized that water washes are also included between some of the above mentioned steps in the silver staining procedure (detailed procedure is given in Table 2).


Table 2. In-house procedure for silver- staining of PAA gels.

Gel images were acquired with the Multi-Analyst/PC Software Image Analysis System (Bio Rad Gel Doc 1000). Digitized images were loaded into the specialized public software Image J (National Institute of Health, USA, www.rsb.info.nih.gov/ij) and analyzed by the image enhancement function 'adapthisteq'. This function performs contrast-limited adaptive

Analysis of Genomic Instability

plasmid with DNA ladder.

NCBI GenBank and EBI (Sanger Institute) database.

**3. Results and discussion** 

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 475

Before the transformation procedure, the preparation of competent bacteria of *E. coli* GM2163 strain was performed using TransformAidTM Bacterial Transformation Kit

The next step was to recover plasmid DNA from recombinant *E.coli* cultures using GeneJETTM Plasmid Miniprep Kit (Fermentas Life Sciences, Lithuania). A single colony was picked from a freshly streaked selective plate for inoculation of 5 mL of LB liquid medium (Fermentas Life Sciences, Lithuania) supplemented with the ampicillin. A bacterial culture is harvested and lysed. The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration. The adsorbed DNA is washed to remove contaminants, and the pure plasmid DNA is eluted in a small volume of elution buffer or water. The purified DNA is ready for immediate use in all molecular biology procedures such as automated sequencing. Before sequencing, the ligation of DNA fragment into the plasmid was verified using restriction enzymes HindIII and EcoRI (Sigma-Aldrich Chemie GmbH, Germany). The fragments obtained after restriction were analyzed on 1% agarose gels. The sequencing was performed only after the presence of the DNA fragment in plasmid was confirmed by comparing the molecular weight of recombinant

Sequences were determined on ABI Prism 3130 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing was performed in both directions on several clones for each selected DNA band. The obtained sequences were analyzed using BLAST software in the NCBI GenBank and EBI (Sanger Institute) database. The sequencing procedure itself involved: 1) two independent cycle sequencing PCRs, each with one primer only (5' and 3'), for the sequencing in both directions; 2) precipitation of the amplicons; 3) their denaturation and 4) automatic electrophoresis. Cycle sequencing PCRs were performed on the GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the final concentration of 100-300 ng of the plasmid DNA and 4pmol of the primer under the following conditions: initial denaturation at 96°C for 1 min, 25 cycles at 96°C for 10 s, 50°C for 5 s, 60°C for 4 min and at 4°C indefinitely. The obtained PCR products were precipitated and EDTA (25 mM final) and EtOH (70-75% final) added.The mixture was incubated for 15 min at RT and then centrifuged 30-45 min at 6000 rpm and +4°C. The supernatant was removed, a new quantity of 70% EtOH added, followed by centrifugation for 25 min at 5000 rpm and +4°C. Supernatant was removed again and the obtained pellet dried at 90°C. Then, 15 μl of Hi-DI™ Formamide (Applied Biosystems, Foster City, CA, USA) was added for the denaturation at 95°C. 10 μl of the amplicons dissolved in formamide were subjected to the automatic electrophoresis and sequence reading on ABI Prism 3130 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were analyzed using BLAST software in the

Genomic instability was determined by comparing the AP-PCR profiles of DNA isolated from paired normal and tumor tissues of patients with non small cell lung cancer (NSCLC),

(Fermentas Life Sciences, Lithuania) according to the manufacturer instruction.

histogram equalization on small regions of the image, called tiles. Contrast of each tile is enhanced so that the histogram of the output region approximately matches a specified histogram. After equalization, adapthisteq combines neighboring tiles using bilinear interpolation to eliminate artificially induced boundaries.

### **2.2.1 Reproducibility**

The problem of reproducibility of AP-PCR has been a matter of concern for quite some time (Meunier and Grimont, 1993; McClelland and Welsh, 1994). In our study, reproducibility was verified by at least three independent reactions and a reaction with a two-fold higher template concentration. Occasional irreproducibility was found to be due to template quality, where additional round of purification solved the problem. Template carry-over was routinely monitored by systematic incorporation of "no-template reaction" in each set of experiments. Day to day variation was found only in respect of band intensities. This variability was in the range of less than 10% (± 5%) as estimated by integration of densitometric scans. Interlab variation was not assessed but we presume that it does not affect the interpretation of data from this report.

#### **2.3 Isolation, cloning and DNA sequencing of variant bands obtained by AP-PCR**

Selected variant DNA bands, bands with altered mobility, were further characterized. The PCR amplicons resolved on the silver stained gels were gently removed with a hypodermic 22-gauge needle pre-wetted with the PCR master mix solution. The needle was dipped in the PCR master mix for 2 min and then discarded. The PCR products were reamplified with the same primers used for AP-PCR reactions at high-stringency conditions specific for each particular primer. The reamplified material was administrated on 1.5% agarose gels, purified using DNA Extraction Kit (Fermentas Life Sciences, Lithuania) and cloned with GeneJetTM PCR Cloning Kit (Fermentas Life Sciences, Lithuania) according to manufacturers' instructions. Plasmids were purified using GeneJetTM Plasmid Miniprep Kit (Fermentas Life Sciences, Lithuania).

Cloning process consisted of setting up the blunting and ligation reactions. Blunting reaction allows the conversion of PCR products generated with non-proofreading Taq DNA polymerase to DNA fragments with blunt ends using thermostable DNA Blunting Enzyme provided with the kit. The reaction consists of 10 μL of 2x Reaction Buffer, 2 μL of nonpurified PCR product, 5 μL of nuclease free water and 1 μL of DNA Blunting Enzyme in 18 μL reaction mixture. The resulting blunt-ended DNA can be ligated efficiently into a vector, pJET1.2/blunt, using the included DNA Ligation Kit Solutions: 1 μL of pJET1.2/blunt Cloning Vector (50ng/ μl) and 1 μL of T4 DNA Ligase (5u/μl). The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Recircularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme which kills the host *E.coli* cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection. The reactions can be used directly for bacterial transformation and in vitro packaging procedures without further purification. All common laboratory *E.coli* strains can be directly transformed with the ligation product.

histogram equalization on small regions of the image, called tiles. Contrast of each tile is enhanced so that the histogram of the output region approximately matches a specified histogram. After equalization, adapthisteq combines neighboring tiles using bilinear

The problem of reproducibility of AP-PCR has been a matter of concern for quite some time (Meunier and Grimont, 1993; McClelland and Welsh, 1994). In our study, reproducibility was verified by at least three independent reactions and a reaction with a two-fold higher template concentration. Occasional irreproducibility was found to be due to template quality, where additional round of purification solved the problem. Template carry-over was routinely monitored by systematic incorporation of "no-template reaction" in each set of experiments. Day to day variation was found only in respect of band intensities. This variability was in the range of less than 10% (± 5%) as estimated by integration of densitometric scans. Interlab variation was not assessed but we presume that it does not

**2.3 Isolation, cloning and DNA sequencing of variant bands obtained by AP-PCR** 

Selected variant DNA bands, bands with altered mobility, were further characterized. The PCR amplicons resolved on the silver stained gels were gently removed with a hypodermic 22-gauge needle pre-wetted with the PCR master mix solution. The needle was dipped in the PCR master mix for 2 min and then discarded. The PCR products were reamplified with the same primers used for AP-PCR reactions at high-stringency conditions specific for each particular primer. The reamplified material was administrated on 1.5% agarose gels, purified using DNA Extraction Kit (Fermentas Life Sciences, Lithuania) and cloned with GeneJetTM PCR Cloning Kit (Fermentas Life Sciences, Lithuania) according to manufacturers' instructions. Plasmids were purified using GeneJetTM Plasmid Miniprep Kit

Cloning process consisted of setting up the blunting and ligation reactions. Blunting reaction allows the conversion of PCR products generated with non-proofreading Taq DNA polymerase to DNA fragments with blunt ends using thermostable DNA Blunting Enzyme provided with the kit. The reaction consists of 10 μL of 2x Reaction Buffer, 2 μL of nonpurified PCR product, 5 μL of nuclease free water and 1 μL of DNA Blunting Enzyme in 18 μL reaction mixture. The resulting blunt-ended DNA can be ligated efficiently into a vector, pJET1.2/blunt, using the included DNA Ligation Kit Solutions: 1 μL of pJET1.2/blunt Cloning Vector (50ng/ μl) and 1 μL of T4 DNA Ligase (5u/μl). The vector contains a lethal restriction enzyme gene that is disrupted by ligation of a DNA insert into the cloning site. As a result, only bacterial cells with recombinant plasmids are able to form colonies. Recircularized pJET1.2/blunt vector molecules lacking an insert express a lethal restriction enzyme which kills the host *E.coli* cell after transformation. This positive selection drastically accelerates the process of colony screening and eliminates additional costs required for blue/white selection. The reactions can be used directly for bacterial transformation and in vitro packaging procedures without further purification. All common laboratory *E.coli*

interpolation to eliminate artificially induced boundaries.

affect the interpretation of data from this report.

(Fermentas Life Sciences, Lithuania).

strains can be directly transformed with the ligation product.

**2.2.1 Reproducibility** 

Before the transformation procedure, the preparation of competent bacteria of *E. coli* GM2163 strain was performed using TransformAidTM Bacterial Transformation Kit (Fermentas Life Sciences, Lithuania) according to the manufacturer instruction.

The next step was to recover plasmid DNA from recombinant *E.coli* cultures using GeneJETTM Plasmid Miniprep Kit (Fermentas Life Sciences, Lithuania). A single colony was picked from a freshly streaked selective plate for inoculation of 5 mL of LB liquid medium (Fermentas Life Sciences, Lithuania) supplemented with the ampicillin. A bacterial culture is harvested and lysed. The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration. The adsorbed DNA is washed to remove contaminants, and the pure plasmid DNA is eluted in a small volume of elution buffer or water. The purified DNA is ready for immediate use in all molecular biology procedures such as automated sequencing. Before sequencing, the ligation of DNA fragment into the plasmid was verified using restriction enzymes HindIII and EcoRI (Sigma-Aldrich Chemie GmbH, Germany). The fragments obtained after restriction were analyzed on 1% agarose gels. The sequencing was performed only after the presence of the DNA fragment in plasmid was confirmed by comparing the molecular weight of recombinant plasmid with DNA ladder.

Sequences were determined on ABI Prism 3130 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequencing was performed in both directions on several clones for each selected DNA band. The obtained sequences were analyzed using BLAST software in the NCBI GenBank and EBI (Sanger Institute) database.

The sequencing procedure itself involved: 1) two independent cycle sequencing PCRs, each with one primer only (5' and 3'), for the sequencing in both directions; 2) precipitation of the amplicons; 3) their denaturation and 4) automatic electrophoresis. Cycle sequencing PCRs were performed on the GeneAmp® PCR System 9700 (Applied Biosystems, Foster City, CA, USA) using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the final concentration of 100-300 ng of the plasmid DNA and 4pmol of the primer under the following conditions: initial denaturation at 96°C for 1 min, 25 cycles at 96°C for 10 s, 50°C for 5 s, 60°C for 4 min and at 4°C indefinitely. The obtained PCR products were precipitated and EDTA (25 mM final) and EtOH (70-75% final) added.The mixture was incubated for 15 min at RT and then centrifuged 30-45 min at 6000 rpm and +4°C. The supernatant was removed, a new quantity of 70% EtOH added, followed by centrifugation for 25 min at 5000 rpm and +4°C. Supernatant was removed again and the obtained pellet dried at 90°C. Then, 15 μl of Hi-DI™ Formamide (Applied Biosystems, Foster City, CA, USA) was added for the denaturation at 95°C. 10 μl of the amplicons dissolved in formamide were subjected to the automatic electrophoresis and sequence reading on ABI Prism 3130 Genetic Analyzer automated sequencer (Applied Biosystems, Foster City, CA, USA). The obtained sequences were analyzed using BLAST software in the NCBI GenBank and EBI (Sanger Institute) database.

## **3. Results and discussion**

Genomic instability was determined by comparing the AP-PCR profiles of DNA isolated from paired normal and tumor tissues of patients with non small cell lung cancer (NSCLC),

Analysis of Genomic Instability

with increased intensity.

parameter.

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 477

This type of analysis differentiates individuals and, thus, displays the cardinal feature of the DNA profile analysis. Additionally, some bands are characteristic for the human genome, being common to all analyzed patients. Importantly, some electrophoretic bands were present in DNA profiles of tumor but not in normal tissue, and vice versa, indicating the mutational like events. The unbiased nature of AP-PCR profiling allows for the screening of anonymous regions of a genome without any prior knowledge of its structure (Welsh and McClelland, 1990; Williams et al., 1990) and provides information about two distinct types of DNA alterations: qualitative and quantitative. Qualitative differences, which represent microsatellite instability (MIN), are detected as mobility shifts in the banding pattern, i.e., the presence or absence of specific bands in tumor and control samples. Quantitative differences appear as altered band intensities and represent amplifications or deletions of existing chromosomal material as manifestations of chromosomal instability (CIN). Observed changes should be cautiously regarded as semiquantitative and semi-qualitative due to the competitive nature of AP-PCR where sequence context may play unpredictable role. This situation may present a serious problem for simple to moderate patterns but not for complex patterns. Unfortunately, the former are preferred due to simplicity of interpretation. Since the profile is the result of a competition between many PCR products, the problem may appear with very simple profiles in analysis of similar but non-identical genomes. For this reason, it had been suggested to use profile pattern with more than 10 prominent PCR products of moderate complexity (McClelland and Welsh, 1994). We followed this reasoning and the necessary precautions for reproducibility and reliability of DNA profiling analysis in comparing DNA fingerprints of paired normal – tumor samples. We identified significant genomic instability in most cases as qualitative and quantitative electrophoretic changes. The qualitative alterations represented as a loss or a gain of a band are the result of mutations at the primer-template interaction sites leading to a mobility shift of a band. Quantitative changes were observed as bands of either decreased or increased intensity. Allelic losses, which may occur as a result of their linkage to suppressor genes, produce bands with decreased intensity. Gene amplification or chromosomal aneuploidy appears as bands

For each type of DNA change, as well as for the total number of changes, the frequency of DNA alterations, a measurement of genomic instability, was calculated as the number of altered bands in the AP-PCR profile of tumor tissue divided by the total number of amplicons in the fingerprint of normal tissue from each patient. AP-PCR fingerprints were analyzed and qualitative and quantitative changes determined using image enhancement

DNA alterations were detected in all analyzed samples with the frequency varying among different types of tumors (Table 3). The largest variation of the frequency of total DNA alterations was in NSCLC patients ranging from 8% to even 68%. The contribution of qualitative changes to overall genomic instability was significantly greater than the contribution of quantitative changes. This large range of instability raised the question of its distribution among samples of NSCLC patients. In other words we were interested to see if there was association between the level of genomic instability and any clinicopathological

function 'adapthisteq' of the specialized public software Image J (Figure 2).

malignant glioma, head and neck squamous cell carcinoma (HNSCC) and leukoplakia (L). Twelve out of twenty tested primers produced informative amplification profiles differentiating normal from tumor tissue or normal from leukoplakia (Table 1). Specifically, five primers produced informative sequence alterations that distinguish NSCLC from normal tissue, a set of four primers produced informative fingerprints differentiating malignant gliomas from normal tissue and another set of four primers produced informative sequence alterations that distinguish HNSCC and leukoplakias from their normal counterparts. The AP-PCR products were separated on 6-8% nondenaturing polyacrylamide (PAA) gels and visualized by silver staining. Typical fingerprints are shown in Figure 1.

Fig. 1. AP-PCR fingerprint profiles of tumor (T) and normal (N) tissues from patients with NSCLC obtained with GAPDH AS primer. Reactions were performed in duplicate with 25 ng and 50 ng of DNA. Numbers 1–5 represent the patients; M–the DNA ladder; NTC–no template control. Arrows and arrowheads indicate examples of quantitative and qualitative changes, respectively.

malignant glioma, head and neck squamous cell carcinoma (HNSCC) and leukoplakia (L). Twelve out of twenty tested primers produced informative amplification profiles differentiating normal from tumor tissue or normal from leukoplakia (Table 1). Specifically, five primers produced informative sequence alterations that distinguish NSCLC from normal tissue, a set of four primers produced informative fingerprints differentiating malignant gliomas from normal tissue and another set of four primers produced informative sequence alterations that distinguish HNSCC and leukoplakias from their normal counterparts. The AP-PCR products were separated on 6-8% nondenaturing polyacrylamide (PAA) gels and visualized by silver staining. Typical fingerprints are

Fig. 1. AP-PCR fingerprint profiles of tumor (T) and normal (N) tissues from patients with NSCLC obtained with GAPDH AS primer. Reactions were performed in duplicate with 25 ng and 50 ng of DNA. Numbers 1–5 represent the patients; M–the DNA ladder; NTC–no template control. Arrows and arrowheads indicate examples of quantitative and qualitative

shown in Figure 1.

changes, respectively.

This type of analysis differentiates individuals and, thus, displays the cardinal feature of the DNA profile analysis. Additionally, some bands are characteristic for the human genome, being common to all analyzed patients. Importantly, some electrophoretic bands were present in DNA profiles of tumor but not in normal tissue, and vice versa, indicating the mutational like events. The unbiased nature of AP-PCR profiling allows for the screening of anonymous regions of a genome without any prior knowledge of its structure (Welsh and McClelland, 1990; Williams et al., 1990) and provides information about two distinct types of DNA alterations: qualitative and quantitative. Qualitative differences, which represent microsatellite instability (MIN), are detected as mobility shifts in the banding pattern, i.e., the presence or absence of specific bands in tumor and control samples. Quantitative differences appear as altered band intensities and represent amplifications or deletions of existing chromosomal material as manifestations of chromosomal instability (CIN). Observed changes should be cautiously regarded as semiquantitative and semi-qualitative due to the competitive nature of AP-PCR where sequence context may play unpredictable role. This situation may present a serious problem for simple to moderate patterns but not for complex patterns. Unfortunately, the former are preferred due to simplicity of interpretation. Since the profile is the result of a competition between many PCR products, the problem may appear with very simple profiles in analysis of similar but non-identical genomes. For this reason, it had been suggested to use profile pattern with more than 10 prominent PCR products of moderate complexity (McClelland and Welsh, 1994). We followed this reasoning and the necessary precautions for reproducibility and reliability of DNA profiling analysis in comparing DNA fingerprints of paired normal – tumor samples. We identified significant genomic instability in most cases as qualitative and quantitative electrophoretic changes. The qualitative alterations represented as a loss or a gain of a band are the result of mutations at the primer-template interaction sites leading to a mobility shift of a band. Quantitative changes were observed as bands of either decreased or increased intensity. Allelic losses, which may occur as a result of their linkage to suppressor genes, produce bands with decreased intensity. Gene amplification or chromosomal aneuploidy appears as bands with increased intensity.

For each type of DNA change, as well as for the total number of changes, the frequency of DNA alterations, a measurement of genomic instability, was calculated as the number of altered bands in the AP-PCR profile of tumor tissue divided by the total number of amplicons in the fingerprint of normal tissue from each patient. AP-PCR fingerprints were analyzed and qualitative and quantitative changes determined using image enhancement function 'adapthisteq' of the specialized public software Image J (Figure 2).

DNA alterations were detected in all analyzed samples with the frequency varying among different types of tumors (Table 3). The largest variation of the frequency of total DNA alterations was in NSCLC patients ranging from 8% to even 68%. The contribution of qualitative changes to overall genomic instability was significantly greater than the contribution of quantitative changes. This large range of instability raised the question of its distribution among samples of NSCLC patients. In other words we were interested to see if there was association between the level of genomic instability and any clinicopathological parameter.

Analysis of Genomic Instability

type of DNA alteration

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 479

decreased with the increase of the histological grade (Figure 3). These results support the idea that mutational alterations conferring genomic instability and the mutator phenotype occur early during tumor formation. The mutator phenotype hypothesis proposes that such phenotypes result from mutations in genes that maintain genomic stability in normal cells. Instability promotes mutations in other genes, oncogenes and tumor suppressor genes, providing the tumor cell with a selective growth advantage. These findings strongly support the increasingly popular explanation of neoplastic transformation in terms of Darwinian evolutionary mechanisms (Breivik, 2001; Breivik & Gaudernack, 1999; Cahill et al., 1999). Evolution through natural selection depends on two essential elements, the availability of

qualitative 0.07 – 0.53 0.06 – 0.27 0.18 – 0.41 0.05 – 0.21 quantitative 0.01 – 0.16 0.05 – 0.27 0.07 – 0.16 0.07 – 0.21 TOTAL 0.08 – 0.68 0.14 – 0.49 0.30 – 0.48 0.12 – 0.31

Fig. 3. The relationship between the total frequency of DNA alterations and the histological grades of the lung tumors. All values are presented as means ± SEM. # *p <* 0.05 when grade 1 was compared to grade 2; \*\*\* *p <* 0.005 when grade 1 was compared to grade 3; & *p <* 0.05

**g1 g2 g3**

when grade 2 was compared to grade 3.

**0**

**0.05**

**0.1**

**0.15**

**0.2**

**0.25**

**0.3**

**0.35**

Table 3. Measurement of genomic instability in various types of tumors.

**#\*\*\***

frequency of DNA alterations

NSCLC glioma leukoplakia HNSCC

**#&**

**\*\*\*&**

Fig. 2. AP-PCR fingerprinting analysis of genomic instability in glioma samples. AP-PCR profiles of tumor (T) and blood (N) tissues from the same patient obtained using MDRa primer, separated on 6% non-denaturing polyacrylamide (PAA) gel and corresponding contrast-limited adaptive histograms obtained using image enhancement function 'adapthisteq' of the specialized public software Image J (**A**). Arrows and arrowheads indicate examples of qualitative and quantitative electrophoretic changes respectively, clearly seen on the overlap of tumor and blood histograms (**B**).

The most noteworthy finding of this study was the association between the level of genomic instability and histological grades of NSCLC. Namely, we found the significant decrease of the total number of DNA alterations with increasing histological grade of the NSCLC. The same pattern was found for quantitative changes alone – the frequency of alterations

Fig. 2. AP-PCR fingerprinting analysis of genomic instability in glioma samples. AP-PCR profiles of tumor (T) and blood (N) tissues from the same patient obtained using MDRa primer, separated on 6% non-denaturing polyacrylamide (PAA) gel and corresponding contrast-limited adaptive histograms obtained using image enhancement function 'adapthisteq' of the specialized public software Image J (**A**). Arrows and arrowheads indicate examples of qualitative and quantitative electrophoretic changes respectively,

The most noteworthy finding of this study was the association between the level of genomic instability and histological grades of NSCLC. Namely, we found the significant decrease of the total number of DNA alterations with increasing histological grade of the NSCLC. The same pattern was found for quantitative changes alone – the frequency of alterations

clearly seen on the overlap of tumor and blood histograms (**B**).

decreased with the increase of the histological grade (Figure 3). These results support the idea that mutational alterations conferring genomic instability and the mutator phenotype occur early during tumor formation. The mutator phenotype hypothesis proposes that such phenotypes result from mutations in genes that maintain genomic stability in normal cells. Instability promotes mutations in other genes, oncogenes and tumor suppressor genes, providing the tumor cell with a selective growth advantage. These findings strongly support the increasingly popular explanation of neoplastic transformation in terms of Darwinian evolutionary mechanisms (Breivik, 2001; Breivik & Gaudernack, 1999; Cahill et al., 1999). Evolution through natural selection depends on two essential elements, the availability of


Table 3. Measurement of genomic instability in various types of tumors.

Fig. 3. The relationship between the total frequency of DNA alterations and the histological grades of the lung tumors. All values are presented as means ± SEM. # *p <* 0.05 when grade 1 was compared to grade 2; \*\*\* *p <* 0.005 when grade 1 was compared to grade 3; & *p <* 0.05 when grade 2 was compared to grade 3.

Analysis of Genomic Instability

leukoplakia samples.

(Tanic et al., 2009).

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 481

clearly distinguished two groups of leukoplakias: a group of six leukoplakias had a frequency of DNA alterations of 0.3–0.34 and was denoted as leukoplakias with a moderate degree of instability while the other group of 26 leukoplakias had a frequency of DNA alterations of > 0.4 and was denoted as leukoplakias with a high degree of instability (Tanic et al., 2009). However, such high levels of genomic instability in leukoplakia samples were a surprise mainly because they are defined as white patches or plaques of oral mucosa that cannot be rubbed off and cannot be diagnosed clinically or pathologically as other specific diseases and have been considered premalignant lesions only since recently (Neville & Day, 2002; Hunter et al., 2005). It is impossible to state, with precision, the proportion of leukoplakias that undergo malignant transformation. For oral mucosa, in general, up to 20% of leukoplakias exhibit dysplasia. Dysplastic leukoplakias have a greater probability of developing into cancer, although leukoplakias without evidence of dysplastic changes may also progress to highly aggressive squamous cell carcinoma. Still, the majority of leukoplakias fail to undergo malignant transformation. The frequency of malignant alterations in oral leukoplakia varies from study to study and ranges from 8.9 to 17.5% (summarized in Neville & Day, 2002). These facts and our finding were the reasons to include samples of Head and Neck Squamous Cell Carcinoma patients, identify and quantify genomic instability in these samples and compare obtained results with those of

Obtained frequency of DNA alterations in HNSCC samples was significantly lower than that of leukoplakia samples, as shown in Table 3. When comparing mean frequencies of DNA alterations the result is even more convincing. Namely, mean frequency of total DNA changes was 0.42 for leukoplakia samples vs. 0.28 for HNSCC samples. Interestingly, contribution of quantitative changes to the total instability in HNSCC samples is significantly higher (0.21) than the contribution of qualitative changes (0.16) which is quite opposite in leukoplakia samples. In other words, the level of genomic instability decreased during HNSCC promotion from premalignant lesions but more serious alterations, quantitative changes as manifestations of chromosomal instability, were selected. These results fit nicely into Darwinian evolutionary theory of neoplastic transformation. High instability is present at the very beginning of HNSCC genesis, providing genetic variability in the population of premalignant cells, which is absolutely necessary for the evolution by natural selection. During tumor progression the level of instability decreases due to selection of genotypes that are better adapted to the micro-environment in which natural selection took place. However, the question remains: why the majority of leukoplakias with such a huge instability fail to undergo malignant transformation? The answer may be in exceeding the error threshold for cell replication and viability (Eigen, 1993) with so many mutations. In other words, it seems that leukoplakias with a high degree of genomic instability have less chance to develop into HNSCC, whereas leukoplakias with a lower (moderate) degree of genomic instability have a better chance of transforming, probably because they carry a certain number of mutations that have favorable effects on cell growth

Following the same reasoning as in the case of NSCLC we attempted to identify some of detected DNA changes in leukoplakias, with the aim of identifying tumor-specific alterations (Peinado et al., 1992) that could lead to the development of potential diagnostic

genetic variation and selection pressure (Dawkins, 1989.). In general evolutionary terms, it could be said that genomic instability accelerates the somatic evolutionary process by promoting genetic variation in an organism. Extensive genomic instability is thus expected in early phases of cancer progression (histological grade 1 in this study). At the same time, an increased mutation rate is expected to cause mutations that are deleterious or lethal at higher frequencies rather than mutations that have favorable effects on cellular proliferation. Consequently, elevated mutation rates must generally be regarded as disadvantageous to cellular growth (Tomlinson et al.,1996). Theoretical arguments suggest that the accumulation of large numbers of mutations can exceed the error threshold for cell replication and viability (Eigen, 1993). Only cells carrying reasonable number of mutations with favorable effects on cell growth would survive. Therefore, it seems probable that the expression of the mutator phenotype could be decreased and lost in the late phases of tumor progression. As a result, tumors may no longer exhibit a mutator phenotype but will nevertheless reveal its history, i.e. random mutations, throughout their genome (Loeb, 2001). In other words, the result showing the lower degree of genomic instability in advanced NSCLCs (grades 2 and 3) is not unexpected in the light of these arguments and could be considered as a marker of poor prognosis.

Following the study of genomic instability in NSCLC tissue samples, we made an attempt to identify some of detected DNA changes in order to identify genes that alter during NSCLC promotion and progression (Bankovic et al., 2010). Selected DNA bands with altered mobility were further characterized. Twenty one unique bands present only in tumor but not in normal tissue were retrieved from the gels and cloned. Variant bands that appeared in more than one sample (new bands with the same mobility), were chosen in order to identify DNA alterations common to as many NSCLC patients as possible. Bands (amplicons) with the same electrophoretic mobility were isolated and characterized from at least two patients in order to confirm that they represent the same DNA sequence. Three clones of each band were sequenced. Obtained sequences were submitted to homology or identity search in NCBI GenBank and EBI (Sanger Institute) databases. Following genes were identified: tetraspanin 14 (TSPAN14*)*, cadherin 12 (CDH12), retinol dehydrogenase 10 (RDH10), cytochrome P450, family 4, subfamily Z, polypeptide 1 (CYP4Z1), killer cell immunoglobulin-like receptor (KIR), E2F transcription factor 4 (E2F4), phosphatase and actin regulator 3 (PHACTR3), PHD finger protein 20 (PHF20), PRAME (preferentially expressed antigen in melanoma) family member and solute carrier family 2 (facilitated glucose transporter), member 13 (SLC2A13). Moreover, we were able to identify types of mutations in revealed genes according to sequence data and BLAST search results and to examine their presence in relation to NSCLC subtype, histological grade and stage of the tumor, lymph node invasion and patients' survival. Examining their relation to the patients' clinicopathological parameters and survival we concluded that TSPAN14, SLC2A13 and PHF20 could have a role in NSCLC promotion, CYP4Z1, KIR and RDH10 would possibly play a role in NSCLC progression, while E2F4, PHACTR3, CDH12 and PRAME family member probably play important role in NSCLC geneses. Patients with altered E2F4 and PHACTR3 lived significantly shorter.

Unlike NSCLC samples, all leukoplakias demonstrated extensive instability in a relatively small range (Table 3). The frequency of total DNA alterations ranged from 0.30 to 0.48 and

genetic variation and selection pressure (Dawkins, 1989.). In general evolutionary terms, it could be said that genomic instability accelerates the somatic evolutionary process by promoting genetic variation in an organism. Extensive genomic instability is thus expected in early phases of cancer progression (histological grade 1 in this study). At the same time, an increased mutation rate is expected to cause mutations that are deleterious or lethal at higher frequencies rather than mutations that have favorable effects on cellular proliferation. Consequently, elevated mutation rates must generally be regarded as disadvantageous to cellular growth (Tomlinson et al.,1996). Theoretical arguments suggest that the accumulation of large numbers of mutations can exceed the error threshold for cell replication and viability (Eigen, 1993). Only cells carrying reasonable number of mutations with favorable effects on cell growth would survive. Therefore, it seems probable that the expression of the mutator phenotype could be decreased and lost in the late phases of tumor progression. As a result, tumors may no longer exhibit a mutator phenotype but will nevertheless reveal its history, i.e. random mutations, throughout their genome (Loeb, 2001). In other words, the result showing the lower degree of genomic instability in advanced NSCLCs (grades 2 and 3) is not unexpected in the light of these arguments and could be

Following the study of genomic instability in NSCLC tissue samples, we made an attempt to identify some of detected DNA changes in order to identify genes that alter during NSCLC promotion and progression (Bankovic et al., 2010). Selected DNA bands with altered mobility were further characterized. Twenty one unique bands present only in tumor but not in normal tissue were retrieved from the gels and cloned. Variant bands that appeared in more than one sample (new bands with the same mobility), were chosen in order to identify DNA alterations common to as many NSCLC patients as possible. Bands (amplicons) with the same electrophoretic mobility were isolated and characterized from at least two patients in order to confirm that they represent the same DNA sequence. Three clones of each band were sequenced. Obtained sequences were submitted to homology or identity search in NCBI GenBank and EBI (Sanger Institute) databases. Following genes were identified: tetraspanin 14 (TSPAN14*)*, cadherin 12 (CDH12), retinol dehydrogenase 10 (RDH10), cytochrome P450, family 4, subfamily Z, polypeptide 1 (CYP4Z1), killer cell immunoglobulin-like receptor (KIR), E2F transcription factor 4 (E2F4), phosphatase and actin regulator 3 (PHACTR3), PHD finger protein 20 (PHF20), PRAME (preferentially expressed antigen in melanoma) family member and solute carrier family 2 (facilitated glucose transporter), member 13 (SLC2A13). Moreover, we were able to identify types of mutations in revealed genes according to sequence data and BLAST search results and to examine their presence in relation to NSCLC subtype, histological grade and stage of the tumor, lymph node invasion and patients' survival. Examining their relation to the patients' clinicopathological parameters and survival we concluded that TSPAN14, SLC2A13 and PHF20 could have a role in NSCLC promotion, CYP4Z1, KIR and RDH10 would possibly play a role in NSCLC progression, while E2F4, PHACTR3, CDH12 and PRAME family member probably play important role in NSCLC geneses. Patients with altered E2F4 and

Unlike NSCLC samples, all leukoplakias demonstrated extensive instability in a relatively small range (Table 3). The frequency of total DNA alterations ranged from 0.30 to 0.48 and

considered as a marker of poor prognosis.

PHACTR3 lived significantly shorter.

clearly distinguished two groups of leukoplakias: a group of six leukoplakias had a frequency of DNA alterations of 0.3–0.34 and was denoted as leukoplakias with a moderate degree of instability while the other group of 26 leukoplakias had a frequency of DNA alterations of > 0.4 and was denoted as leukoplakias with a high degree of instability (Tanic et al., 2009). However, such high levels of genomic instability in leukoplakia samples were a surprise mainly because they are defined as white patches or plaques of oral mucosa that cannot be rubbed off and cannot be diagnosed clinically or pathologically as other specific diseases and have been considered premalignant lesions only since recently (Neville & Day, 2002; Hunter et al., 2005). It is impossible to state, with precision, the proportion of leukoplakias that undergo malignant transformation. For oral mucosa, in general, up to 20% of leukoplakias exhibit dysplasia. Dysplastic leukoplakias have a greater probability of developing into cancer, although leukoplakias without evidence of dysplastic changes may also progress to highly aggressive squamous cell carcinoma. Still, the majority of leukoplakias fail to undergo malignant transformation. The frequency of malignant alterations in oral leukoplakia varies from study to study and ranges from 8.9 to 17.5% (summarized in Neville & Day, 2002). These facts and our finding were the reasons to include samples of Head and Neck Squamous Cell Carcinoma patients, identify and quantify genomic instability in these samples and compare obtained results with those of leukoplakia samples.

Obtained frequency of DNA alterations in HNSCC samples was significantly lower than that of leukoplakia samples, as shown in Table 3. When comparing mean frequencies of DNA alterations the result is even more convincing. Namely, mean frequency of total DNA changes was 0.42 for leukoplakia samples vs. 0.28 for HNSCC samples. Interestingly, contribution of quantitative changes to the total instability in HNSCC samples is significantly higher (0.21) than the contribution of qualitative changes (0.16) which is quite opposite in leukoplakia samples. In other words, the level of genomic instability decreased during HNSCC promotion from premalignant lesions but more serious alterations, quantitative changes as manifestations of chromosomal instability, were selected. These results fit nicely into Darwinian evolutionary theory of neoplastic transformation. High instability is present at the very beginning of HNSCC genesis, providing genetic variability in the population of premalignant cells, which is absolutely necessary for the evolution by natural selection. During tumor progression the level of instability decreases due to selection of genotypes that are better adapted to the micro-environment in which natural selection took place. However, the question remains: why the majority of leukoplakias with such a huge instability fail to undergo malignant transformation? The answer may be in exceeding the error threshold for cell replication and viability (Eigen, 1993) with so many mutations. In other words, it seems that leukoplakias with a high degree of genomic instability have less chance to develop into HNSCC, whereas leukoplakias with a lower (moderate) degree of genomic instability have a better chance of transforming, probably because they carry a certain number of mutations that have favorable effects on cell growth (Tanic et al., 2009).

Following the same reasoning as in the case of NSCLC we attempted to identify some of detected DNA changes in leukoplakias, with the aim of identifying tumor-specific alterations (Peinado et al., 1992) that could lead to the development of potential diagnostic

Analysis of Genomic Instability

tissues.

**4. Conclusions** 

and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 483

Finally, it is worth mentioning that measurements of genomic instability could be performed by another DNA fingerprinting technique, RAPD (Random Amplified Polymorphic DNA). Wang et al. (2002) measured genomic instability in various cancer types using RAPD and the instability they detected was in average higher than 40% for lung cancer tissues. In another study (Ong et al., 1998), DNAs from 20 lung cancer (18 non-small cell lung cancers and two small cell lung cancers) and their corresponding normal tissues were amplified individually by RAPD with seven different 10-base arbitrary primers. PCR products from RAPD were electrophoretically separated in agarose gels and banding profiles were visualized by ethidium bromide staining. The ability to detect genomic instability in 20 cancer tissues by each single primer ranged from 15 to 75%. DNA changes were detected by at least one primer in 19 (95%) cancer tissues. They concluded that these results seem to indicate that genomic rearrangement is associated with lung carcinogenesis and that RAPD analysis is useful for the detection of genomic instability in lung cancer

Misra A. et al. (2007) used RAPD to attempt to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin . They targeted genomic loci of a different nature and estimated that the number of overall alterations in tumor genome seemed to be greater than expected. They also observed a higher number of genetic changes in tumors of lower grade and suggested that it could be a consequence of an increased mutation rate in early tumorigenesis due to acquisition of a mutator phenotype. The increased extent of alterations occurring in tumors of a lower grade is consistent with our study. The results of Misra et al. showed the acquisition of a mutator phenotype early in tumorigenesis and

AP-PCR DNA fingerprinting is an efficient tool to quickly and easily screen a very large number of loci for possible DNA alterations in cancer cells. It has several advantages: first, minor amounts of template DNA are sufficient for analysis; second, it allows for the screening of anonymous regions of a genome without any prior knowledge of its structure; third, two types of DNA alterations could be detected in single reaction, chromosomal rearrangements and random mutations dispersed over the genome; and forth, possibility of reamplification, cloning and sequencing of variant bands enables the rapid identification of the genes probably linked to tumor progression. Here, we demonstrated the use of AP-PCR DNA fingerprinting in detection and quantification of genomic instability (microsatellite, chromosomal and total) in three types of tumors as well as in search for molecular biomarkers for cancer promotion and progression. Therefore, we conclude that AP-PCR

support the mutator hypothesis proposed by Loeb (1991, 2001).

Anaplastic Astrocytoma vs. Glioblastoma Multiforme

instability Mean frequency

microsatellite 0.15 vs. 0.16 chromosomal 0.19 vs. 0.16 TOTAL 0.34 vs. 0.33

Table 4. Mean frequency of DNA alterations in malignant glioma samples.

markers involved in the genesis of HNSCC. To that end, nine variant bands present in leukoplakias but not in normal tissue, were selected. Unexpectedly, two different amplicons, originating from distinct leukoplakias, were identified as altered part of the TIMP-3 gene (tissue inhibitors of metalloproteinases 3), two were identified as mutated DNMT 3A gene (DNA (cytosine-5)-methyltransferase 3 alpha) and two represented copies of the Ty1-copialike retrotransposon.

Further investigations of the detected genes in both, leukoplakia and NSCLC samples, on larger sample size, with special emphases on tumor promoting genes, are underway. We expect more detailed profile of their involvement in NSCLC and HNSCC after extensive analyses of their mutational status and detailed analyses of their expression profile at RNA and protein level in a larger sample. We expect that some of them might prove to be a good prognostic biomarkers for NSCLC or HNSCC patients.

Finally, we analyzed malignant gliomas, tumors that originate from glia, the most common and deadly brain tumors. All patients had histologically confirmed diagnosis of anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM) according to the new World Health Organisation (WHO) classification. Anaplastic astrocytomas (WHO grade III) and glioblastomas (WHO grade IV) are two major groups of malignant gliomas. Glioblastomas are further classified as primary and secondary. Distinction between them is based on different genetic pathways leading to their development (Ohgaki & Kleihues, 2007; Van Meir et al., 2010). Primary glioblastoma develop rapidly *de novo*, without clinical or histological evidence of a less malignant precursor lesion. Secondary glioblastoma develop slowly progressing from low-grade diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III).

Examination of the extent of genomic instability revealed that samples of patients with anaplastic astrocytoma had similar level of total, microsatellite and chromosomal genomic instability as patients with glioblastoma multiforme, with very high values in both histological subtypes (Table 4). It was unexpected and, at first sight, looked like these results contradicted the expectation and results obtained from NSCLC and HNSCC samples. However, all analyzed grade IV glioblastomas were classified as primary glioblastomas (because glioblastoma diagnosis was made at the first biopsy, without clinical or histopathologic evidence of a less malignant precursor lesion), which are considered to be *de novo* tumors and not the progressive form of grade III astrocytomas. Therefore, obtained results are still consistent with the evolutionary theory of neoplastic transformation and the decrease of the level of genomic instability could be expected in secondary glioblastomas. In other words, extensive genomic instability might be used as diagnostic character where pathology cannot provide unambiguous distinction between primary and secondary GBM. Similar results were obtained by Nishizaki et al. (2002) who demonstrated that there was no significant difference in FISH heterogeneity between malignant gliomas of WHO grades III and IV. We expect that further research involving secondary glioblastomas will confirm our hypothesis and will provide additional confirmation for the evolutionary theory of tumor progression. Moreover, we hope that cloning and sequencing of amplified DNA bands showing genetic alterations specific for glioma genome, will allow the detection of new genes implicated in glioma pathogenesis and progression.

markers involved in the genesis of HNSCC. To that end, nine variant bands present in leukoplakias but not in normal tissue, were selected. Unexpectedly, two different amplicons, originating from distinct leukoplakias, were identified as altered part of the TIMP-3 gene (tissue inhibitors of metalloproteinases 3), two were identified as mutated DNMT 3A gene (DNA (cytosine-5)-methyltransferase 3 alpha) and two represented copies of the Ty1-copia-

Further investigations of the detected genes in both, leukoplakia and NSCLC samples, on larger sample size, with special emphases on tumor promoting genes, are underway. We expect more detailed profile of their involvement in NSCLC and HNSCC after extensive analyses of their mutational status and detailed analyses of their expression profile at RNA and protein level in a larger sample. We expect that some of them might prove to be a good

Finally, we analyzed malignant gliomas, tumors that originate from glia, the most common and deadly brain tumors. All patients had histologically confirmed diagnosis of anaplastic astrocytoma (AA) or glioblastoma multiforme (GBM) according to the new World Health Organisation (WHO) classification. Anaplastic astrocytomas (WHO grade III) and glioblastomas (WHO grade IV) are two major groups of malignant gliomas. Glioblastomas are further classified as primary and secondary. Distinction between them is based on different genetic pathways leading to their development (Ohgaki & Kleihues, 2007; Van Meir et al., 2010). Primary glioblastoma develop rapidly *de novo*, without clinical or histological evidence of a less malignant precursor lesion. Secondary glioblastoma develop slowly progressing from low-grade diffuse astrocytoma (WHO grade II) or anaplastic

Examination of the extent of genomic instability revealed that samples of patients with anaplastic astrocytoma had similar level of total, microsatellite and chromosomal genomic instability as patients with glioblastoma multiforme, with very high values in both histological subtypes (Table 4). It was unexpected and, at first sight, looked like these results contradicted the expectation and results obtained from NSCLC and HNSCC samples. However, all analyzed grade IV glioblastomas were classified as primary glioblastomas (because glioblastoma diagnosis was made at the first biopsy, without clinical or histopathologic evidence of a less malignant precursor lesion), which are considered to be *de novo* tumors and not the progressive form of grade III astrocytomas. Therefore, obtained results are still consistent with the evolutionary theory of neoplastic transformation and the decrease of the level of genomic instability could be expected in secondary glioblastomas. In other words, extensive genomic instability might be used as diagnostic character where pathology cannot provide unambiguous distinction between primary and secondary GBM. Similar results were obtained by Nishizaki et al. (2002) who demonstrated that there was no significant difference in FISH heterogeneity between malignant gliomas of WHO grades III and IV. We expect that further research involving secondary glioblastomas will confirm our hypothesis and will provide additional confirmation for the evolutionary theory of tumor progression. Moreover, we hope that cloning and sequencing of amplified DNA bands showing genetic alterations specific for glioma genome, will allow the detection of new genes implicated in glioma pathogenesis

like retrotransposon.

astrocytoma (WHO grade III).

and progression.

prognostic biomarkers for NSCLC or HNSCC patients.


Table 4. Mean frequency of DNA alterations in malignant glioma samples.

Finally, it is worth mentioning that measurements of genomic instability could be performed by another DNA fingerprinting technique, RAPD (Random Amplified Polymorphic DNA). Wang et al. (2002) measured genomic instability in various cancer types using RAPD and the instability they detected was in average higher than 40% for lung cancer tissues. In another study (Ong et al., 1998), DNAs from 20 lung cancer (18 non-small cell lung cancers and two small cell lung cancers) and their corresponding normal tissues were amplified individually by RAPD with seven different 10-base arbitrary primers. PCR products from RAPD were electrophoretically separated in agarose gels and banding profiles were visualized by ethidium bromide staining. The ability to detect genomic instability in 20 cancer tissues by each single primer ranged from 15 to 75%. DNA changes were detected by at least one primer in 19 (95%) cancer tissues. They concluded that these results seem to indicate that genomic rearrangement is associated with lung carcinogenesis and that RAPD analysis is useful for the detection of genomic instability in lung cancer tissues.

Misra A. et al. (2007) used RAPD to attempt to quantify the number of clonal mutations in primary human gliomas of astrocytic cell origin . They targeted genomic loci of a different nature and estimated that the number of overall alterations in tumor genome seemed to be greater than expected. They also observed a higher number of genetic changes in tumors of lower grade and suggested that it could be a consequence of an increased mutation rate in early tumorigenesis due to acquisition of a mutator phenotype. The increased extent of alterations occurring in tumors of a lower grade is consistent with our study. The results of Misra et al. showed the acquisition of a mutator phenotype early in tumorigenesis and support the mutator hypothesis proposed by Loeb (1991, 2001).

## **4. Conclusions**

AP-PCR DNA fingerprinting is an efficient tool to quickly and easily screen a very large number of loci for possible DNA alterations in cancer cells. It has several advantages: first, minor amounts of template DNA are sufficient for analysis; second, it allows for the screening of anonymous regions of a genome without any prior knowledge of its structure; third, two types of DNA alterations could be detected in single reaction, chromosomal rearrangements and random mutations dispersed over the genome; and forth, possibility of reamplification, cloning and sequencing of variant bands enables the rapid identification of the genes probably linked to tumor progression. Here, we demonstrated the use of AP-PCR DNA fingerprinting in detection and quantification of genomic instability (microsatellite, chromosomal and total) in three types of tumors as well as in search for molecular biomarkers for cancer promotion and progression. Therefore, we conclude that AP-PCR

Analysis of Genomic Instability

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and Tumor-Specific Genetic Alterations by Arbitrarily Primed PCR 485

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DNA fingerprinting is important and practically feasible technique for elucidating the genetic background of various tumors. Accordingly, we believe that this technique is rather neglected in contemporary research and should make a comeback because it still has a particularly promising future in experimental oncology.

## **5. Acknowledgments**

This study was supported by Grant # III41031 from the Ministry of Education and Science, Republic of Serbia.

#### **6. References**


DNA fingerprinting is important and practically feasible technique for elucidating the genetic background of various tumors. Accordingly, we believe that this technique is rather neglected in contemporary research and should make a comeback because it still has a

This study was supported by Grant # III41031 from the Ministry of Education and Science,

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particularly promising future in experimental oncology.

**5. Acknowledgments** 

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Republic of Serbia.

**6. References** 


**1. Introduction**

2007).

using PCR.

al., 2007).

**2. Background information**

or exclusion of an exon, the exon skipping.

In 1977 it was discovered that the one-gene-one-enzyme hypothesis was not true (Chow et al.,1977; Berget at al., 1977). The primary transcription product can be spliced in different ways and give rise to several proteins depending on the exons being present in the final mRNA. This phenomenon is called alternative splicing and indeed is common to many genes. Several possible modes of alternative splicing are known and the most common one is the inclusion

**Analysis of Alternatively Spliced Domains in** 

**Matrix Glycoprotein Tenascin C** 

Ursula Theocharidis and Andreas Faissner

*Ruhr-University Bochum* 

*Germany* 

**23**

**Multimodular Gene Products - The Extracellular** 

Based on polymerase chain reaction (PCR) techniques we developed a method to analyse combinations of alternatively spliced domains in multimodular gene products. This method was used to determine the combinatorial variability of tenascin C isoforms in the mouse central nervous system (Joester & Faissner, 1999) and in neural stem cells (von Holst et al.,

Here, we present the method of amplifying different sized isoforms of a gene product with several alternatively spliced domains via PCR and the isolation and subcloning of the PCR products. Clones are analyzed for alternatively spliced domains contained therein by a dot blot *in vitro* hybridization method with domain-specific DNA probes which were generated

Tenascin C is a multimodular glycoprotein of the extracellular matrix which is mainly expressed during central nervous system development and in pathological states such as brain tumours or lesions. We have studied the expression pattern of this molecule and its function *in vivo* and *in vitro* and collected evidence concerning its structural diversity. We and others determined its functions during neural development, in the adult neural stem cell niche and in lesions and tumours (Czopka et al., 2009, 2010; Dobbertin et al., 2010; Garcion et al., 2001, 2004; Garwood et al., 2011; Gates et al., 1995; Orend & Chiquet-Ehrismann, 2006; von Holst et

Tenascin C contains a constant part including eight contitutive fibronectin type III (fnIII) domains and a variable part of six alternatively spliced fnIII domains in the mouse which

can be included independently into the gene product (figure 1).


## **Analysis of Alternatively Spliced Domains in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C**

Ursula Theocharidis and Andreas Faissner *Ruhr-University Bochum Germany* 

#### **1. Introduction**

486 Polymerase Chain Reaction

Wang, J.; Wang, Q. & Ye, F. (2002). Genetic instability in cancer tissues analyzed by random

Welsh, J. & McClelland, M. (1990). Fingerprinting genomes using PCR with arbitrary primers. *Nucleic Acid Research*, Vol.18, No.24, pp. 7213–7218, ISSN 0305-1048. Williams, J.G.K.; Kubelik, A.R.; Livak, K.J.; Rafalski, J.A. & Tingey, S.V. (1990). DNA

*Nucleic Asids Research*, Vol.18, No.22, pp. 6531-6535, ISSN 0305-1048.

ISSN 0366-6999.

amplified polymorphic DNA PCR. *Chin Med J (Engl)*, Vol.115, No.3, pp. 430-432,

polymorphisms amplified by arbitrary primers are useful as genetic markers.

In 1977 it was discovered that the one-gene-one-enzyme hypothesis was not true (Chow et al.,1977; Berget at al., 1977). The primary transcription product can be spliced in different ways and give rise to several proteins depending on the exons being present in the final mRNA. This phenomenon is called alternative splicing and indeed is common to many genes. Several possible modes of alternative splicing are known and the most common one is the inclusion or exclusion of an exon, the exon skipping.

Based on polymerase chain reaction (PCR) techniques we developed a method to analyse combinations of alternatively spliced domains in multimodular gene products. This method was used to determine the combinatorial variability of tenascin C isoforms in the mouse central nervous system (Joester & Faissner, 1999) and in neural stem cells (von Holst et al., 2007).

Here, we present the method of amplifying different sized isoforms of a gene product with several alternatively spliced domains via PCR and the isolation and subcloning of the PCR products. Clones are analyzed for alternatively spliced domains contained therein by a dot blot *in vitro* hybridization method with domain-specific DNA probes which were generated using PCR.

## **2. Background information**

Tenascin C is a multimodular glycoprotein of the extracellular matrix which is mainly expressed during central nervous system development and in pathological states such as brain tumours or lesions. We have studied the expression pattern of this molecule and its function *in vivo* and *in vitro* and collected evidence concerning its structural diversity. We and others determined its functions during neural development, in the adult neural stem cell niche and in lesions and tumours (Czopka et al., 2009, 2010; Dobbertin et al., 2010; Garcion et al., 2001, 2004; Garwood et al., 2011; Gates et al., 1995; Orend & Chiquet-Ehrismann, 2006; von Holst et al., 2007).

Tenascin C contains a constant part including eight contitutive fibronectin type III (fnIII) domains and a variable part of six alternatively spliced fnIII domains in the mouse which can be included independently into the gene product (figure 1).

Matrix Glycoprotein Tenascin C 3

<sup>489</sup> Analysis of Alternatively Spliced Domains

Tenascin C has its variable region between the constantly expressed fnIII domains 5 and 6. We used two different primer combinations to determine the isoform pattern of the molecule in

containing only these two constant domains. Another primer pair we used was called 5for

6. The smallest amplicons are then represented by forms with one alternatively spliced fnIII domain. The further analysis was carried out with PCR products obtained with this primer

end of the sixth domain and result in PCR products with the smallest form

end of domain number 5 and the 5

end of the sixth domain. The resulting amplicons therefore contain the minimum

end of fnIII domain 6. Only PCR products with the minimum of one

of two fnIII domains, namely 5 and 6. The further insertion of alternatively spliced cassettes increases the size of the PCR product. The primers 5for and 6rev bind to the 3 end of fnIII

alternatively spliced fnIII domain can be generated. Every additional domain increases the

The expression analysis can be performed on RNA isolated from tissue or cell culture material which was processed by reverse transcription. Several commercially available kits help to isolate total RNA or mRNA from tissue or cell cultures. The resulting RNA can then be used to generate cDNA by reverse transcription which can also be carried out using kits from different suppliers. If oligo-dT primers or random primers are used for the reverse transcription makes no difference in our experience. The generated cDNA is the template for the PCR which possibly needs some optimization steps to generate all bands of interest. According to our experience it is of outstanding importance to test the performance of different Taq polymerases in advance because not every enzyme from each supplier will work equally efficiently. Different polymerases in their respective buffer system show variable results and

The PCR conditions with regard to annealing temperature and time, elongation time as well as concentration of cDNA, primers and Magnesium must be worked out in advance. Addition

the desired results. To be able to generate all the expected amplicons the longest product determines the elongation time. The rule of thumb to calculate 1 minute elongation time per

The resulting PCR products can be processed on an agarose gel and the DNA bands made visible with ethidium bromide or a substitute. The concentration of the agarose must be high enough to discriminate between contiguous bands but sufficiently low that the longest products can enter the analysis area. A long gel chamber increases the migration way

of DMSO or betain may be needed and checked when the standard conditions don

end of the

end of domain number

end of the fifth

t lead to

various tissues and cell cultures (figure 2). The primers 5s and 6as bind to the 5

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

Fig. 2. Primer binding sites. Two different primer pairs were used to amplify the alternatively spliced region of tenascin C and analyse the expression profile of different isoforms. The primers 5s / 6as and 5for / 6rev bind to the constant fnIII domains 5 and 6 at

their outer or inner tails, respectively. The primers 5s and 6as bind to the 5

size of the amplicons by 273 bp, the size of the single domains.

fifth and the 3

pair.

and the 3

domain 5 and the 5

**3.1 Expression analysis by RT-PCR**

should be adapted to the reaction requirements.

1000 base pairs gives a good estimation here.

and 6rev and these bind to the 3

The alternatively spliced fnIII domains of the tenascin C molecule have different functions, e.g. affecting the axon outgrowth of developing nerve cells or the migration potential of brain tumour cells (Rigato et al., 2002; Michele & Faissner, 2009; Broesicke & Faissner, personal communication). Therefore it is important to have a method to determine the isoform composition of the molecule in the tissue or cell cultures used.

Fig. 1. Schematic representation of mouse tenascin C. The monomer consists of several distinct protein domains. At the N-terminal tenascin C assembly domain six monomers can be assembled to the so called hexabrachion (Erickson & Inglesias, 1984). 14,5 epidermal growth factor (EGF) like domains and eight constitutive fibronectin type III (fnIII) domains follow before the C-terminal globular lobe homologous to the beta- and gamma-chains of fibrinogen. Between the fifth and sixth constant fnIII domain up to six alternatively spliced domains can be inserted and an independent alternative splicing at each position could lead to the generation of 64 (=26) possible isoforms of the molecule. All possible numbers of domains can be inserted in the final splicing product, but the combination of cassettes is unclear in most cases. Only the largest variant necessarily contains all six alternatively spliced domains.

Gene products with different exons being alternatively spliced and inserted into the sequence can be distinguished by PCR when the sizes of the resulting mRNAs are different. A PCR analysis uses primers flanking the alternatively spliced region and results in amplicons with different sizes. These can be analysed by agarose gel electrophoresis and show bands in distinct positions. Tenascin C has six domains that can be alternatively spliced and independently inserted into the sequence. The analysis of these domains on an agarose gel shows the size of the resulting amplicons and therefore the number of inserted domains but leaves the question open which of the possible domains are included. A further analysis is therefore needed. We have shown that it can be performed using an *in vitro* dot blot hybridization technique to verify the exact domain combinations.

#### **3. Analysis of isoform sizes in multimodular gene products by RT-PCR**

When the sequence of the gene of interest is known primers can be generated which allow the amplification of the relevant region. The primers can either bind in the alternatively spliced region itself and therefore generate PCR products only when the target sequence is expressed. When the primers bind outside of the alternatively spliced part of the sequence the products can contain every possible insert additionally to the constant parts of the sequence which are defined by the primer binding sites. Additionally, isoforms without any insert can be amplified with these primer combinations.

Tenascin C has its variable region between the constantly expressed fnIII domains 5 and 6. We used two different primer combinations to determine the isoform pattern of the molecule in various tissues and cell cultures (figure 2). The primers 5s and 6as bind to the 5 end of the fifth and the 3 end of the sixth domain and result in PCR products with the smallest form containing only these two constant domains. Another primer pair we used was called 5for and 6rev and these bind to the 3 end of domain number 5 and the 5 end of domain number 6. The smallest amplicons are then represented by forms with one alternatively spliced fnIII domain. The further analysis was carried out with PCR products obtained with this primer pair.

Fig. 2. Primer binding sites. Two different primer pairs were used to amplify the alternatively spliced region of tenascin C and analyse the expression profile of different isoforms. The primers 5s / 6as and 5for / 6rev bind to the constant fnIII domains 5 and 6 at their outer or inner tails, respectively. The primers 5s and 6as bind to the 5 end of the fifth and the 3 end of the sixth domain. The resulting amplicons therefore contain the minimum of two fnIII domains, namely 5 and 6. The further insertion of alternatively spliced cassettes increases the size of the PCR product. The primers 5for and 6rev bind to the 3 end of fnIII domain 5 and the 5 end of fnIII domain 6. Only PCR products with the minimum of one alternatively spliced fnIII domain can be generated. Every additional domain increases the size of the amplicons by 273 bp, the size of the single domains.

## **3.1 Expression analysis by RT-PCR**

2 Will-be-set-by-IN-TECH

The alternatively spliced fnIII domains of the tenascin C molecule have different functions, e.g. affecting the axon outgrowth of developing nerve cells or the migration potential of brain tumour cells (Rigato et al., 2002; Michele & Faissner, 2009; Broesicke & Faissner, personal communication). Therefore it is important to have a method to determine the isoform

Fig. 1. Schematic representation of mouse tenascin C. The monomer consists of several distinct protein domains. At the N-terminal tenascin C assembly domain six monomers can be assembled to the so called hexabrachion (Erickson & Inglesias, 1984). 14,5 epidermal growth factor (EGF) like domains and eight constitutive fibronectin type III (fnIII) domains follow before the C-terminal globular lobe homologous to the beta- and gamma-chains of fibrinogen. Between the fifth and sixth constant fnIII domain up to six alternatively spliced domains can be inserted and an independent alternative splicing at each position could lead to the generation of 64 (=26) possible isoforms of the molecule. All possible numbers of domains can be inserted in the final splicing product, but the combination of cassettes is unclear in most cases. Only the largest variant necessarily contains all six alternatively

Gene products with different exons being alternatively spliced and inserted into the sequence can be distinguished by PCR when the sizes of the resulting mRNAs are different. A PCR analysis uses primers flanking the alternatively spliced region and results in amplicons with different sizes. These can be analysed by agarose gel electrophoresis and show bands in distinct positions. Tenascin C has six domains that can be alternatively spliced and independently inserted into the sequence. The analysis of these domains on an agarose gel shows the size of the resulting amplicons and therefore the number of inserted domains but leaves the question open which of the possible domains are included. A further analysis is therefore needed. We have shown that it can be performed using an *in vitro* dot blot

When the sequence of the gene of interest is known primers can be generated which allow the amplification of the relevant region. The primers can either bind in the alternatively spliced region itself and therefore generate PCR products only when the target sequence is expressed. When the primers bind outside of the alternatively spliced part of the sequence the products can contain every possible insert additionally to the constant parts of the sequence which are defined by the primer binding sites. Additionally, isoforms without any insert can be

hybridization technique to verify the exact domain combinations.

amplified with these primer combinations.

**3. Analysis of isoform sizes in multimodular gene products by RT-PCR**

composition of the molecule in the tissue or cell cultures used.

spliced domains.

The expression analysis can be performed on RNA isolated from tissue or cell culture material which was processed by reverse transcription. Several commercially available kits help to isolate total RNA or mRNA from tissue or cell cultures. The resulting RNA can then be used to generate cDNA by reverse transcription which can also be carried out using kits from different suppliers. If oligo-dT primers or random primers are used for the reverse transcription makes no difference in our experience. The generated cDNA is the template for the PCR which possibly needs some optimization steps to generate all bands of interest. According to our experience it is of outstanding importance to test the performance of different Taq polymerases in advance because not every enzyme from each supplier will work equally efficiently. Different polymerases in their respective buffer system show variable results and should be adapted to the reaction requirements.

The PCR conditions with regard to annealing temperature and time, elongation time as well as concentration of cDNA, primers and Magnesium must be worked out in advance. Addition of DMSO or betain may be needed and checked when the standard conditions don t lead to the desired results. To be able to generate all the expected amplicons the longest product determines the elongation time. The rule of thumb to calculate 1 minute elongation time per 1000 base pairs gives a good estimation here.

The resulting PCR products can be processed on an agarose gel and the DNA bands made visible with ethidium bromide or a substitute. The concentration of the agarose must be high enough to discriminate between contiguous bands but sufficiently low that the longest products can enter the analysis area. A long gel chamber increases the migration way

Matrix Glycoprotein Tenascin C 5

<sup>491</sup> Analysis of Alternatively Spliced Domains

different forms expressed in parallel? Are all possible product sizes present? What is the ratio

The analysis of the PCR products on an agarose gel answers the question for size and ratio of the isoforms expressed but leaves open which of the possible domains are contained in the bands. Some further experimental steps are necessary to determine the domains being expressed. Because several domain combinations can migrate in the same position they must be separated from each other. This can be achieved by subcloning the different PCR amplicons

The PCR bands are cut out of the gel under visual control at an UV desk and the gel slices collected in separate tubes. It is important to use different knives for each band because otherwise DNA from other bands might be carried over and contaminate the samples. Isolation of the DNA from the gel can be performed using classic methods or commercially available kits. The elution should be done with the minimal amount of water to avoid problems with following reaction steps. For the subcloning of the PCR products we used the TOPO-TA cloning kit from invitrogen but any other similar kit will do. In our experience it is important to handle the bacteria quite carefully and leave them grow in antibiotic-free medium for 30 minutes after the transformation. Spread the bacteria to LB agar plates with

Fig. 4. Check for positive clones after direct colony lysis. The colonies grown after the cloning and transformation of the PCR products are checked for their content of fnIII domains. The primer pair 5for / 6rev was used to generate amplicons of the expected size when the clones have taken up the plasmids containing the fnIII domains. This example shows seven clones from a DNA band containing 2 fnIII domains. Two of the clones shown here do not contain any fnIII domains and are therefore not selected for the following screen. The other clones show PCR bands of the expected size and are analysed in the subsequent

The content of the resulting clones can quickly and easily be checked by direct lysis of the bacteria and a subsequent PCR with the primers used before. The colonies grown on the agar plate are picked with a pipette tip and transferred to another (the "master�� plate) into numbered fields. The tip is then shaken in 25*μ*L 70% ethanol in PCR tubes to lyse the bacteria. The master plate can be placed in the incubator while in the meantime the colonies are checked for their content. In an incubation step the ethanol is evaporated at 80◦C for approx. 15

between different forms? Does the expression profile change with the conditions?

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

**3.2 Cloning of resulting PCR products**

and analysis of the resulting clones.

dot blot hybridizations.

appropriate antibiotics then and let them grow over night.

and a lower voltage over a longer time period narrows the single bands and makes the discrimination easier.

We used brain tissue from postnatal mice or cultures of neural stem cells to isolate total RNA and analysed the expression pattern of the alternatively spliced forms of Tenascin C in the respective system (Joester & Faissner, 1999; von Holst et al., 2007). In these cases we found isoforms of all possible sizes to be present and performed the further analysis for isoforms containing between one and six additional cassettes. The use of the primer pair 5s and 6as leads to DNA bands on the agarose gel where the smallest one is 546bp, representing only the two constant fnIII domains 5 and 6 of 273bp each. Every additional cassette increases the amplicon size by 273bp. Therefore, we can see a "ladder" structure of up to seven DNA bands on the agarose gel when using this primer pair (figure 3B). When the primers 5for and 6rev are present in the PCR mix instead we get products where the smallest isoform contains the minimum of one alternatively spliced fnIII domain. The larger bands represent the larger forms with up to six fnIII domains. Here, we get the maximum of 6 DNA bands on the gel (figure 3A).

Fig. 3. Examples of tenascin C isoform PCRs. The primer pairs 5for / 6rev and 5s / 6as were used to amplify the alternatively spliced region of tenascin C. The PCR products were separated on an 1,2 % agarose gel. (A) The smallest amplicon generated using the primers 5for and 6rev contains only one of the alternatively spliced cassettes. The insertion of additional domains increases the product size by 273 bp. Up to six bands appear representing the different possible amplicon sizes. PCR products amplified with this primer pair were used for the further analysis of the domain expression profile after separation on an agarose gel. (B) The use of the alternative primer pair 5s and 6as leads to the generation of up to seven DNA bands on the agarose gel because the smallest band represents only the constant fnIII domains 5 and 6 without any insert. When alternatively spliced domains are included in the sequence the product size increases by 273 bp for each domain. Up to six domains can be added and therefore the largest DNA band on the gel represents the total of eight fnIII domains. This primer pair was mainly used for the analysis of expression profiles.

The agarose gel shows the expression profile of the alternatively spliced gene products in the analysed tissue or cells. The resulting amplicons answer the first questions in this respect: Are different forms expressed in parallel? Are all possible product sizes present? What is the ratio between different forms? Does the expression profile change with the conditions?

#### **3.2 Cloning of resulting PCR products**

4 Will-be-set-by-IN-TECH

and a lower voltage over a longer time period narrows the single bands and makes the

We used brain tissue from postnatal mice or cultures of neural stem cells to isolate total RNA and analysed the expression pattern of the alternatively spliced forms of Tenascin C in the respective system (Joester & Faissner, 1999; von Holst et al., 2007). In these cases we found isoforms of all possible sizes to be present and performed the further analysis for isoforms containing between one and six additional cassettes. The use of the primer pair 5s and 6as leads to DNA bands on the agarose gel where the smallest one is 546bp, representing only the two constant fnIII domains 5 and 6 of 273bp each. Every additional cassette increases the amplicon size by 273bp. Therefore, we can see a "ladder" structure of up to seven DNA bands on the agarose gel when using this primer pair (figure 3B). When the primers 5for and 6rev are present in the PCR mix instead we get products where the smallest isoform contains the minimum of one alternatively spliced fnIII domain. The larger bands represent the larger forms with up to six fnIII domains. Here, we get the maximum of 6 DNA bands on the gel

Fig. 3. Examples of tenascin C isoform PCRs. The primer pairs 5for / 6rev and 5s / 6as were used to amplify the alternatively spliced region of tenascin C. The PCR products were separated on an 1,2 % agarose gel. (A) The smallest amplicon generated using the primers 5for and 6rev contains only one of the alternatively spliced cassettes. The insertion of additional domains increases the product size by 273 bp. Up to six bands appear

representing the different possible amplicon sizes. PCR products amplified with this primer pair were used for the further analysis of the domain expression profile after separation on an agarose gel. (B) The use of the alternative primer pair 5s and 6as leads to the generation of up to seven DNA bands on the agarose gel because the smallest band represents only the constant fnIII domains 5 and 6 without any insert. When alternatively spliced domains are included in the sequence the product size increases by 273 bp for each domain. Up to six domains can be added and therefore the largest DNA band on the gel represents the total of eight fnIII domains. This primer pair was mainly used for the analysis of expression profiles. The agarose gel shows the expression profile of the alternatively spliced gene products in the analysed tissue or cells. The resulting amplicons answer the first questions in this respect: Are

discrimination easier.

(figure 3A).

The analysis of the PCR products on an agarose gel answers the question for size and ratio of the isoforms expressed but leaves open which of the possible domains are contained in the bands. Some further experimental steps are necessary to determine the domains being expressed. Because several domain combinations can migrate in the same position they must be separated from each other. This can be achieved by subcloning the different PCR amplicons and analysis of the resulting clones.

The PCR bands are cut out of the gel under visual control at an UV desk and the gel slices collected in separate tubes. It is important to use different knives for each band because otherwise DNA from other bands might be carried over and contaminate the samples. Isolation of the DNA from the gel can be performed using classic methods or commercially available kits. The elution should be done with the minimal amount of water to avoid problems with following reaction steps. For the subcloning of the PCR products we used the TOPO-TA cloning kit from invitrogen but any other similar kit will do. In our experience it is important to handle the bacteria quite carefully and leave them grow in antibiotic-free medium for 30 minutes after the transformation. Spread the bacteria to LB agar plates with appropriate antibiotics then and let them grow over night.

Fig. 4. Check for positive clones after direct colony lysis. The colonies grown after the cloning and transformation of the PCR products are checked for their content of fnIII domains. The primer pair 5for / 6rev was used to generate amplicons of the expected size when the clones have taken up the plasmids containing the fnIII domains. This example shows seven clones from a DNA band containing 2 fnIII domains. Two of the clones shown here do not contain any fnIII domains and are therefore not selected for the following screen. The other clones show PCR bands of the expected size and are analysed in the subsequent dot blot hybridizations.

The content of the resulting clones can quickly and easily be checked by direct lysis of the bacteria and a subsequent PCR with the primers used before. The colonies grown on the agar plate are picked with a pipette tip and transferred to another (the "master�� plate) into numbered fields. The tip is then shaken in 25*μ*L 70% ethanol in PCR tubes to lyse the bacteria. The master plate can be placed in the incubator while in the meantime the colonies are checked for their content. In an incubation step the ethanol is evaporated at 80◦C for approx. 15

Matrix Glycoprotein Tenascin C 7

<sup>493</sup> Analysis of Alternatively Spliced Domains

These inserts are labelled to use them in expression studies. The labelling with fluorescein has several advantages. When using non-radioactive probes no special safety regulations must be obeyed. Additionally, the probes can be used for a longer time period. This is of

fluorescein-labelled probes are stable for 6 months without decreasing activity. Radioactively labelled probes would lose sensitivity after a few days because the isotopes disintegrate continuously. Indeed, the probes generated in our lab could be used for several years (Joester

The labelling was performed with fluorescein-coupled dUTP (Amersham). The

labelling method. This uses the hybridization of 8 to 10 bases long random primers to single DNA strands. In a polymerization mix with the labelled nucleotide the Klenow fragment of the DNA polymerase I generates the complementary probe. But the tenascin C domains are less than 300 base pairs long and therefore have only a few potential binding sites for random primers which may lead to only very short probes. The labelling efficiency is too

labelling frequency. Therefore we developed a labelling protocol which uses a PCR method to

The Taq polymerase incorporates dUTP with less efficiency than unlabelled dTTP. Therefore the exclusive use of dUTP would show the optimum of labelled probe but only low yield of amplification product. A low amount of fluorescein-dUTP and higher amount of dTTP reverses this effect and leads to a higher product yield but low labelling efficiency. We adjusted

The optimal reaction conditions required only 10 pg of plasmid DNA and a low amount of dNTPs (20 *μ*M). The reaction mix contained 60 mM Tris/HCl, pH 8,5; 15 mM (NH4)2SO4; 2 mM MgCl2; 0,2 *μ*M sense primer; 0,2 *μ*M antisense primer and 1 Unit Taq polymerase in 25 *μ*L volume. The cycling conditions are dependent on the hybridization temperature of the

In the first labelling reactions with domain C different amounts of dTTP were replaced with FI-dUTP (3 to 50% equivalent to 0,6 to 10 *μ*M FI-dUTP). The amount of the products increases with decreasing amounts of the labelled nucleotide. The labelling efficiency was also tested on dot blots with different concentrations of the plasmid containing the C domain. 1*μ*L of the PCR products were used in the hybridization solution. After an over-night incubation the blots were developed with an alkaline phosphatase-coupled antibody detecting fluorescein. The detection sensitivity was proportional to the concentration of the FI-dUTP used in the labelling reaction. The subsequent labelling reactions were performed using 17,5 *μ*M dTTP and 2,5 *μ*M fluorescein-11-dUTP. All probes detecting the fnIII domains A1, A2, A4, B, C and D of tenascin C were labelled with this method and called FI-A1, FI-A2,... Figure 5 shows the

The fluorescein-labelled probes were tested for their detection capability of different dilutions of the respective plasmids. The senstivity was different for the probes and therefore their concentration was adjusted in the hybridization solution. The hybridization results show that the sensitivity is equal between 3 pg up to 1 ng of the target sequence (figure 6A). This sensitivity is much higher than that seen for agarose gels stained with ethidium bromide which is in the range of 1 ng DNA. The sensitivity was tested regularly to adjust the stability or labelling efficiency of the different probes but none showed a significant reduction in detection

s labelling kit could not be used because it is based on a random primer

s data state that

s data) to achieve an appropriate

special advantage when several probes are used in parallel. Manufacturer

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

& Faissner, 1999; von Holst et al., 2007).

generate dUTP-labelled DNA probes.

resulting PCR amplicons.

efficiency over time.

low (1 labelled base in 50 bases, according to manufacturer

respective primers and the length of the expected product.

the PCR conditions to the optimal yield of labelled amplification product.

manufacturer

minutes before the PCR master mix containing buffer, primers and polymerase is added. The reaction conditions can be the same as before. The products can be analysed on an agarose gel and should show single bands in the expected position for each positive clone (figure 4). These can subsequently be picked from the master plate and propagated in miniprep scale. The plasmid DNA from the miniprep cultures can be isolated by alcaline lysis or with appropriate kits.

#### **3.3 Analysis of clones - dot blot**

Of course, these plasmids could be sequenced and their composition clarified by this method at this point. Because sequencing is not cheap when analysing hundreds of clones a method was developed that renders the identification of many samples in one step possible and is cheaper. The basis is a dot blot of the isolated plasmids to nylon membranes which subsequently can be used in hybridizations with domain specific probes.

The plasmid solutions should be adjusted to similar concentrations with water to have equal amounts of target DNA in the spots. The easiest way to apply the plasmids to the membranes is the use of a dot blot apparatus with a vacuum manifold, but it is also possible to spot the liquid using a master plate onto the membrane which is placed on filter paper. For our analysis we used Hybond N+ membranes from Amersham which were pre-wetted with 10x SSC (1,5 M NaCl; 150 mM Na3Citrate, pH 7,0) buffer. Because there are six possible domains to detect (A1, A2, A4, B, C and D) and we used a negative control we prepared seven membranes with identical spot patterns. The plasmid solutions were diluted in 10x SSC in a volume of 100*μ*L when we used the dot blot apparatus and 6*μ*L when a pattern was used.

After application of the plasmid solutions the membranes are incubated for 10 minutes in denaturing buffer (500 mM NaOH; 1,5 M NaCl) and 10 minutes in renaturing buffer (500 mM Tris/HCl, pH 7,5; 1,5 M NaCl) to prepare the DNA for the hybridization. The nylon membranes are dried and baked at 80◦C for two hours to have the DNA bound covalently to them. These dot blots can be stored for some time at room temperature.

#### **3.4 Positive and negative controls**

To determine the specificity of the method and to be sure that no false-positive or false-negative results appear the use of positive and negative controls is important. For every application appropriate controls must be defined. In our case we could exploit the fact that the fnIII domain number 6 is not included in the alternatively spliced region which we amplify with the primer pair 5for / 6rev in the initial PCRs. Therefore a probe detecting the fnIII domain 6 serves as negative control. Another control we use is a plasmid, called pJT1# which contains the constant part of tenascin C between the fnIII domains 2 and 8, but none of the alternatively spliced domains. The positive control is a plasmid containing all six alternatively spliced domains. On the dot blot it is applied in addition to the clones under investigation.

#### **3.5 Generation of domain specific DNA probes by PCR**

The hybridization of membrane-bound DNA with probes detecting defined DNA fragments identifies specific sequences in the bound nucleic acids. Probes detecting the desired target sequences are generated based on the cDNA of these fragments which are cloned into common plasmid vectors. We used the sequences of the tenascin C fnIII domains A1, A2, A4, B, C, D and 6 as negative control which were inserted in pBluescript II KS+ vectors.

6 Will-be-set-by-IN-TECH

minutes before the PCR master mix containing buffer, primers and polymerase is added. The reaction conditions can be the same as before. The products can be analysed on an agarose gel and should show single bands in the expected position for each positive clone (figure 4). These can subsequently be picked from the master plate and propagated in miniprep scale. The plasmid DNA from the miniprep cultures can be isolated by alcaline lysis or with appropriate

Of course, these plasmids could be sequenced and their composition clarified by this method at this point. Because sequencing is not cheap when analysing hundreds of clones a method was developed that renders the identification of many samples in one step possible and is cheaper. The basis is a dot blot of the isolated plasmids to nylon membranes which

The plasmid solutions should be adjusted to similar concentrations with water to have equal amounts of target DNA in the spots. The easiest way to apply the plasmids to the membranes is the use of a dot blot apparatus with a vacuum manifold, but it is also possible to spot the liquid using a master plate onto the membrane which is placed on filter paper. For our analysis we used Hybond N+ membranes from Amersham which were pre-wetted with 10x SSC (1,5 M NaCl; 150 mM Na3Citrate, pH 7,0) buffer. Because there are six possible domains to detect (A1, A2, A4, B, C and D) and we used a negative control we prepared seven membranes with identical spot patterns. The plasmid solutions were diluted in 10x SSC in a volume of 100*μ*L when we used the dot blot apparatus and 6*μ*L when a pattern was used. After application of the plasmid solutions the membranes are incubated for 10 minutes in denaturing buffer (500 mM NaOH; 1,5 M NaCl) and 10 minutes in renaturing buffer (500 mM Tris/HCl, pH 7,5; 1,5 M NaCl) to prepare the DNA for the hybridization. The nylon membranes are dried and baked at 80◦C for two hours to have the DNA bound covalently to

To determine the specificity of the method and to be sure that no false-positive or false-negative results appear the use of positive and negative controls is important. For every application appropriate controls must be defined. In our case we could exploit the fact that the fnIII domain number 6 is not included in the alternatively spliced region which we amplify with the primer pair 5for / 6rev in the initial PCRs. Therefore a probe detecting the fnIII domain 6 serves as negative control. Another control we use is a plasmid, called pJT1# which contains the constant part of tenascin C between the fnIII domains 2 and 8, but none of the alternatively spliced domains. The positive control is a plasmid containing all six alternatively spliced domains. On the dot blot it is applied in addition to the clones under investigation.

The hybridization of membrane-bound DNA with probes detecting defined DNA fragments identifies specific sequences in the bound nucleic acids. Probes detecting the desired target sequences are generated based on the cDNA of these fragments which are cloned into common plasmid vectors. We used the sequences of the tenascin C fnIII domains A1, A2, A4, B, C, D and 6 as negative control which were inserted in pBluescript II KS+ vectors.

subsequently can be used in hybridizations with domain specific probes.

them. These dot blots can be stored for some time at room temperature.

**3.5 Generation of domain specific DNA probes by PCR**

kits.

**3.3 Analysis of clones - dot blot**

**3.4 Positive and negative controls**

These inserts are labelled to use them in expression studies. The labelling with fluorescein has several advantages. When using non-radioactive probes no special safety regulations must be obeyed. Additionally, the probes can be used for a longer time period. This is of special advantage when several probes are used in parallel. Manufacturer s data state that fluorescein-labelled probes are stable for 6 months without decreasing activity. Radioactively labelled probes would lose sensitivity after a few days because the isotopes disintegrate continuously. Indeed, the probes generated in our lab could be used for several years (Joester & Faissner, 1999; von Holst et al., 2007).

The labelling was performed with fluorescein-coupled dUTP (Amersham). The manufacturer s labelling kit could not be used because it is based on a random primer labelling method. This uses the hybridization of 8 to 10 bases long random primers to single DNA strands. In a polymerization mix with the labelled nucleotide the Klenow fragment of the DNA polymerase I generates the complementary probe. But the tenascin C domains are less than 300 base pairs long and therefore have only a few potential binding sites for random primers which may lead to only very short probes. The labelling efficiency is too low (1 labelled base in 50 bases, according to manufacturer s data) to achieve an appropriate labelling frequency. Therefore we developed a labelling protocol which uses a PCR method to generate dUTP-labelled DNA probes.

The Taq polymerase incorporates dUTP with less efficiency than unlabelled dTTP. Therefore the exclusive use of dUTP would show the optimum of labelled probe but only low yield of amplification product. A low amount of fluorescein-dUTP and higher amount of dTTP reverses this effect and leads to a higher product yield but low labelling efficiency. We adjusted the PCR conditions to the optimal yield of labelled amplification product.

The optimal reaction conditions required only 10 pg of plasmid DNA and a low amount of dNTPs (20 *μ*M). The reaction mix contained 60 mM Tris/HCl, pH 8,5; 15 mM (NH4)2SO4; 2 mM MgCl2; 0,2 *μ*M sense primer; 0,2 *μ*M antisense primer and 1 Unit Taq polymerase in 25 *μ*L volume. The cycling conditions are dependent on the hybridization temperature of the respective primers and the length of the expected product.

In the first labelling reactions with domain C different amounts of dTTP were replaced with FI-dUTP (3 to 50% equivalent to 0,6 to 10 *μ*M FI-dUTP). The amount of the products increases with decreasing amounts of the labelled nucleotide. The labelling efficiency was also tested on dot blots with different concentrations of the plasmid containing the C domain. 1*μ*L of the PCR products were used in the hybridization solution. After an over-night incubation the blots were developed with an alkaline phosphatase-coupled antibody detecting fluorescein. The detection sensitivity was proportional to the concentration of the FI-dUTP used in the labelling reaction. The subsequent labelling reactions were performed using 17,5 *μ*M dTTP and 2,5 *μ*M fluorescein-11-dUTP. All probes detecting the fnIII domains A1, A2, A4, B, C and D of tenascin C were labelled with this method and called FI-A1, FI-A2,... Figure 5 shows the resulting PCR amplicons.

The fluorescein-labelled probes were tested for their detection capability of different dilutions of the respective plasmids. The senstivity was different for the probes and therefore their concentration was adjusted in the hybridization solution. The hybridization results show that the sensitivity is equal between 3 pg up to 1 ng of the target sequence (figure 6A). This sensitivity is much higher than that seen for agarose gels stained with ethidium bromide which is in the range of 1 ng DNA. The sensitivity was tested regularly to adjust the stability or labelling efficiency of the different probes but none showed a significant reduction in detection efficiency over time.

Matrix Glycoprotein Tenascin C 9

<sup>495</sup> Analysis of Alternatively Spliced Domains

and the use of 0,5% SDS in the washing buffer. The highest probability for a cross-reactivity exists between the domains A1 and A4 because their nucleotide sequence is 80% identical. Only in a few cases a light background signal could be detected when using these probes.

The generated probes are used in an *in vitro* hybridization protocol and applied to the nylon membranes containing the plasmid DNA from the clones which shall be analysed. The nylon membranes with the bound plasmids are washed in 5xSSC and pre-incubated in hybridization solution (5x SSC; 0,1% SDS; 5% dextrane sulfate; 5% liquid block (Amersham)) for 30 minutes at 72◦C with gentle agitation. An appropriate amount of the probes which must be determined in preliminary experiments is added to 200*μ*L of hybridization solution. The probes are denatured at 96◦C for 5 minutes and applied to the membranes. The hybridization takes

We used two different methods for the detection of the hybridized probes. A detection protocol to obtain chemoluminescence signals uses the fluorescein gene images CDP-*Star* detection system (Amersham). The other option was the development of a colour reaction

When the DNA on the membranes was hybridized over night with the fluorescein-coupled probes the membranes can be washed for 2 x 15 minutes in wash buffer 1 (0,1x SSC; 0,5% SDS) at 72◦C. To block unspecific binding sites they are incubated for one hour in blocking buffer (10% liquid block (Amersham) in detection buffer (100 mM Tris/HCl, pH 7,5; 300 mM NaCl)) before the alkaline phosphatase-coupled anti-fluorescein antibody (1:5000 in detection buffer with 0,5% BSA) is applied for an hour. Unbound antibody is washed away with wash buffer 2 (0,3 % Tween-20 in detection buffer) 4x 8 minutes. For the chemoluminescence detection the blot is moistened with a dioxetane-based substrate solution (CDP-*Star* detection reagent (Amersham). After 3 minutes the excess substrate solution is removed and the blot placed between two sheets of foil and laid on an autoradiographic film. Depending on the DNA

For the alternative developing method the membranes are washed 3x 5 minutes in wash buffer A (100 mM Tris/HCl, pH 7,4; 150 mM NaCl; 0,3% Tween-20) after the antibody incubation, 2x 5 minutes in wash buffer B (100 mM Tris/HCl, pH 9,5; 100 mM NaCl) and 3x 10 minutes in TBS (50 mM Tris/HCl, pH 7,5; 150 mM NaCl). To develop the colour reaction the membranes are wetted with staining solution containing NBT and BCIP (Roche) and not shaken any more. The colour reaction will appear after five to 60 minutes. The reaction can be stopped with water and the membranes dried afterwards. Because the detection sensitivity is lower for the colour reaction a higher amount of plasmid DNA must be used in this case. 50 ng of target

The read-out of the results is straight forward. The membranes show positive signals whenever the respective domain is present in the clone. Every plasmid DNA shows a specific pattern of positive and negative signals and therefore stands for the presence or absence of a

place over night at 72◦C with constant agitation in a hybridization oven.

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

concentration the optimal detection time was between 10 and 60 minutes.

sequence in the spots lead to good results (data not shown).

**3.8 Analysis of domain combinations**

given single domain.

using NBT and BCIP as alkaline phosphatase substrates.

**3.6 Hybridization**

**3.7 Signal detection**

Fig. 5. Fluorescein-labelled probes on an agarose gel. The DNA probes labelled with FI-dUTP were applied to an agarose gel and show bands in the expected size of less than 300 bp. The asterisk designates the fluorescence signal of the non-incorporated nucleotides.

The specificity of the fluorescein-labelled probes was tested with seven dot blot stripes containing the plasmids pA1, pA2, pA4, pB, pC, pD, p6 and pJT1# in equal concentrations. The stripes were hybridized with the probes for the single fnIII domains (FI-A1, FI-A2,...) and the alkaline phosphatase reaction was developed (figure 6B). Highly stringent hybridization and washing conditions minimised the cross-reactivity of the probes with unspecific target sequences. These conditions included the hybridization and the first washing steps at 72◦C

Fig. 6. (A) Sensitivity of fluorescein-labelled probes. The plasmids containing the single fnIII domains A1, A2, A4, B, C, D and 6 (designated pA1, pA2, ...) were diluted and applied to the nylon membranes in dots. The membranes were incubated with the respective probes and the reaction developed with an anti-fluorescein antibody coupled to alkaline phosphatase. A minimum of about 10 pg of target sequence was detected by each of the probes. The probes were diluted so that all of them detected their targets in a comparable way. (B) Specificity of fluorescein-labelled probes. Seven identical dot blots containing 10 ng of the plasmids pA1, pA2, pA4, pB, pC, pD, p6 and 19 ng pJT1# (corresponding to 1 ng target sequence) were hybridized with the different fluorescein-labelled probes. The probes detect their target sequences with high specificity. Although domains A1 and A4 are highly identical the probes do not show a significant cross-reactivity. The probe FI-6 detects the plasmid pJT1# which contains domain number 6.

and the use of 0,5% SDS in the washing buffer. The highest probability for a cross-reactivity exists between the domains A1 and A4 because their nucleotide sequence is 80% identical. Only in a few cases a light background signal could be detected when using these probes.

#### **3.6 Hybridization**

8 Will-be-set-by-IN-TECH

Fig. 5. Fluorescein-labelled probes on an agarose gel. The DNA probes labelled with FI-dUTP were applied to an agarose gel and show bands in the expected size of less than 300 bp. The

The specificity of the fluorescein-labelled probes was tested with seven dot blot stripes containing the plasmids pA1, pA2, pA4, pB, pC, pD, p6 and pJT1# in equal concentrations. The stripes were hybridized with the probes for the single fnIII domains (FI-A1, FI-A2,...) and the alkaline phosphatase reaction was developed (figure 6B). Highly stringent hybridization and washing conditions minimised the cross-reactivity of the probes with unspecific target sequences. These conditions included the hybridization and the first washing steps at 72◦C

Fig. 6. (A) Sensitivity of fluorescein-labelled probes. The plasmids containing the single fnIII domains A1, A2, A4, B, C, D and 6 (designated pA1, pA2, ...) were diluted and applied to the nylon membranes in dots. The membranes were incubated with the respective probes and the reaction developed with an anti-fluorescein antibody coupled to alkaline phosphatase. A minimum of about 10 pg of target sequence was detected by each of the probes. The probes were diluted so that all of them detected their targets in a comparable way. (B) Specificity of fluorescein-labelled probes. Seven identical dot blots containing 10 ng of the plasmids pA1, pA2, pA4, pB, pC, pD, p6 and 19 ng pJT1# (corresponding to 1 ng target sequence) were hybridized with the different fluorescein-labelled probes. The probes detect their target sequences with high specificity. Although domains A1 and A4 are highly identical the probes do not show a significant cross-reactivity. The probe FI-6 detects the plasmid pJT1# which

contains domain number 6.

asterisk designates the fluorescence signal of the non-incorporated nucleotides.

The generated probes are used in an *in vitro* hybridization protocol and applied to the nylon membranes containing the plasmid DNA from the clones which shall be analysed. The nylon membranes with the bound plasmids are washed in 5xSSC and pre-incubated in hybridization solution (5x SSC; 0,1% SDS; 5% dextrane sulfate; 5% liquid block (Amersham)) for 30 minutes at 72◦C with gentle agitation. An appropriate amount of the probes which must be determined in preliminary experiments is added to 200*μ*L of hybridization solution. The probes are denatured at 96◦C for 5 minutes and applied to the membranes. The hybridization takes place over night at 72◦C with constant agitation in a hybridization oven.

### **3.7 Signal detection**

We used two different methods for the detection of the hybridized probes. A detection protocol to obtain chemoluminescence signals uses the fluorescein gene images CDP-*Star* detection system (Amersham). The other option was the development of a colour reaction using NBT and BCIP as alkaline phosphatase substrates.

When the DNA on the membranes was hybridized over night with the fluorescein-coupled probes the membranes can be washed for 2 x 15 minutes in wash buffer 1 (0,1x SSC; 0,5% SDS) at 72◦C. To block unspecific binding sites they are incubated for one hour in blocking buffer (10% liquid block (Amersham) in detection buffer (100 mM Tris/HCl, pH 7,5; 300 mM NaCl)) before the alkaline phosphatase-coupled anti-fluorescein antibody (1:5000 in detection buffer with 0,5% BSA) is applied for an hour. Unbound antibody is washed away with wash buffer 2 (0,3 % Tween-20 in detection buffer) 4x 8 minutes. For the chemoluminescence detection the blot is moistened with a dioxetane-based substrate solution (CDP-*Star* detection reagent (Amersham). After 3 minutes the excess substrate solution is removed and the blot placed between two sheets of foil and laid on an autoradiographic film. Depending on the DNA concentration the optimal detection time was between 10 and 60 minutes.

For the alternative developing method the membranes are washed 3x 5 minutes in wash buffer A (100 mM Tris/HCl, pH 7,4; 150 mM NaCl; 0,3% Tween-20) after the antibody incubation, 2x 5 minutes in wash buffer B (100 mM Tris/HCl, pH 9,5; 100 mM NaCl) and 3x 10 minutes in TBS (50 mM Tris/HCl, pH 7,5; 150 mM NaCl). To develop the colour reaction the membranes are wetted with staining solution containing NBT and BCIP (Roche) and not shaken any more. The colour reaction will appear after five to 60 minutes. The reaction can be stopped with water and the membranes dried afterwards. Because the detection sensitivity is lower for the colour reaction a higher amount of plasmid DNA must be used in this case. 50 ng of target sequence in the spots lead to good results (data not shown).

#### **3.8 Analysis of domain combinations**

The read-out of the results is straight forward. The membranes show positive signals whenever the respective domain is present in the clone. Every plasmid DNA shows a specific pattern of positive and negative signals and therefore stands for the presence or absence of a given single domain.

Matrix Glycoprotein Tenascin C 11

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

<sup>497</sup> Analysis of Alternatively Spliced Domains

usually the absence of fnIII domain C and only few miss A1. The extensive screens for the expression profiles of fnIII domains being expressed in postnatal mouse cerebellum or neural stem cells show that the possible variability among the clones is not utilised. 64 different isoforms of tenascin C would be theoretically possible but only 28 forms were found. Some combinations of domains were never seen like the direct link between the fnIII domains C and

To confirm the results of the hybridization and to clarify ambiguous signals we carried out PCRs for the single fnIII domains that were detected in the plasmid DNA. It is important to highly dilute the plasmid DNA and use only 10 to 20 pg plasmid DNA as template and to use highly specific PCR conditions. The specificity of the PCR conditions was confirmed before because the fnIII domains show high similarities and could therefore lead to false-positive signals when using standard PCR conditions on plasmid templates. We used a 2-step PCR with a high annealing temperature of the primers of 72◦C and combined the annealing step with the elongation step to a 40-seconds 72◦C incubation step. The cycling was therefore between 20 seconds 94◦C and 40 seconds 72◦C. We also skipped the final 5-minutes elongation step which we usually applied, especially for the addition of adenosines for cloning purposes. Figure 8 shows the high specificity of these PCR conditions when the different fnIII domain-containing plasmids were used in these reactions. Only for those plasmids amplicons were generated when the respective primer pair was used. Therefore, we had an additional

Fig. 8. PCRs for single fnIII domains. The primer pairs A1-s / A1-as, A2-s / A2-as,... were used in PCRs for the amplification of single fnIII domains. The domains have some similarities in their sequences. Therefore the PCR conditions must be highly specific. PCRs with stringent conditions amplify only products from plasmids containing the respective domain. Such conditions can be used to test plasmids with unclear domain composition.

6 or A4 and C.

**3.9 PCR of single domains**

tool to confirm the dot blot results.

Fig. 7. Example of screening results. Plasmids containing different numbers of alternatively spliced fnIII domains were applied to nylon membranes in dots. Seven identical blots were generated and hybridized with the fluorescein-labelled probes FI-A1, FI-A2, FI-A4, FI-B, FI-C, FI-D and FI-6 as negative control. After the development of the alkaline phosphatase reaction the dot blots show positive signals whenever the respective fnIII domain is present in the plasmid. Therefore the domain combination of every single clone can be directly read out from the blots.

In intensive studies of the expression pattern of Tenascin C isoforms in the developing brain and in neural stem cells (Joester & Faissner, 1999 and von Holst et al., 2007) we detected 28 different isoforms of Tenascin C out of 64 possible ones which could theoretically be generated with six independently spliced domains (=26). We had several hundred clones to identify which contained between one and six alternatively spliced fnIII domains. The membranes we prepared were handled separately depending on the expected number of domains to be present in the plasmids. Figure 7 shows an example of the analysis of several clones with different numbers of fnIII domains. Plasmids from the distinct subcloning reactions were spotted onto seven nylon membranes and hybridized with the probes FI-A1, FI-A2, FI-A4, FI-B, FI-C, FI-D and FI-6 as negative control. The signals show that different combinations of fnIII domains can be contained in the plasmids. The clones with only one alternatively spliced domain displayed here for example contain the domains A1 or D, respectively. Indeed, these were the most common domains among single-domain clones when a complete screen was performed (Joester & Faissner, 1999; von Holst et al., 2007). The variability of domain combinations is higher in the middle-size clones with two, three or four alternatively spliced fnIII domains. The plasmids containing five additional cassettes on the other hand show usually the absence of fnIII domain C and only few miss A1. The extensive screens for the expression profiles of fnIII domains being expressed in postnatal mouse cerebellum or neural stem cells show that the possible variability among the clones is not utilised. 64 different isoforms of tenascin C would be theoretically possible but only 28 forms were found. Some combinations of domains were never seen like the direct link between the fnIII domains C and 6 or A4 and C.

#### **3.9 PCR of single domains**

10 Will-be-set-by-IN-TECH

Fig. 7. Example of screening results. Plasmids containing different numbers of alternatively spliced fnIII domains were applied to nylon membranes in dots. Seven identical blots were generated and hybridized with the fluorescein-labelled probes FI-A1, FI-A2, FI-A4, FI-B, FI-C, FI-D and FI-6 as negative control. After the development of the alkaline phosphatase reaction the dot blots show positive signals whenever the respective fnIII domain is present in the plasmid. Therefore the domain combination of every single clone can be directly read

In intensive studies of the expression pattern of Tenascin C isoforms in the developing brain and in neural stem cells (Joester & Faissner, 1999 and von Holst et al., 2007) we detected 28 different isoforms of Tenascin C out of 64 possible ones which could theoretically be generated with six independently spliced domains (=26). We had several hundred clones to identify which contained between one and six alternatively spliced fnIII domains. The membranes we prepared were handled separately depending on the expected number of domains to be present in the plasmids. Figure 7 shows an example of the analysis of several clones with different numbers of fnIII domains. Plasmids from the distinct subcloning reactions were spotted onto seven nylon membranes and hybridized with the probes FI-A1, FI-A2, FI-A4, FI-B, FI-C, FI-D and FI-6 as negative control. The signals show that different combinations of fnIII domains can be contained in the plasmids. The clones with only one alternatively spliced domain displayed here for example contain the domains A1 or D, respectively. Indeed, these were the most common domains among single-domain clones when a complete screen was performed (Joester & Faissner, 1999; von Holst et al., 2007). The variability of domain combinations is higher in the middle-size clones with two, three or four alternatively spliced fnIII domains. The plasmids containing five additional cassettes on the other hand show

out from the blots.

To confirm the results of the hybridization and to clarify ambiguous signals we carried out PCRs for the single fnIII domains that were detected in the plasmid DNA. It is important to highly dilute the plasmid DNA and use only 10 to 20 pg plasmid DNA as template and to use highly specific PCR conditions. The specificity of the PCR conditions was confirmed before because the fnIII domains show high similarities and could therefore lead to false-positive signals when using standard PCR conditions on plasmid templates. We used a 2-step PCR with a high annealing temperature of the primers of 72◦C and combined the annealing step with the elongation step to a 40-seconds 72◦C incubation step. The cycling was therefore between 20 seconds 94◦C and 40 seconds 72◦C. We also skipped the final 5-minutes elongation step which we usually applied, especially for the addition of adenosines for cloning purposes.

Figure 8 shows the high specificity of these PCR conditions when the different fnIII domain-containing plasmids were used in these reactions. Only for those plasmids amplicons were generated when the respective primer pair was used. Therefore, we had an additional tool to confirm the dot blot results.

Fig. 8. PCRs for single fnIII domains. The primer pairs A1-s / A1-as, A2-s / A2-as,... were used in PCRs for the amplification of single fnIII domains. The domains have some similarities in their sequences. Therefore the PCR conditions must be highly specific. PCRs with stringent conditions amplify only products from plasmids containing the respective domain. Such conditions can be used to test plasmids with unclear domain composition.

Matrix Glycoprotein Tenascin C 13

<sup>499</sup> Analysis of Alternatively Spliced Domains

the domains can be analysed using the method presented here. Some preliminary steps are necessary before a screen for expressed domains can be started but when the system is set up

A screen includes the generation of the plasmids and the dot blot before the hybridization can

With the method presented here we developed a possibility to unravel unknown structures of splice products for alternatively spliced transcripts. The example we analysed was the extracellular matrix molecule tenascin C but any other multimodular protein can be examined in a similar way. With some preliminary preparations an operational tool is at hand which makes the screening of many clones and therefore the generation of an expression profile

The authors wish to thank very much Dr. Angret Joester for providing unpublished material concerning the dot blot assays. We also thank the German Research Foundation (DFG) (SPP

Berget, S.M., Moore, C. & Sharp, P.A. (1977) Spliced segments at the 5 terminus of adenovirus

Chow, L.T., Gelinas, R.E., Broker, T.R. & Roberts, R.J. (1977) An amazing sequence

arrangement at the 5 ends of adenovirus 2 messenger RNA. Cell. 1977 Sep;12(1):1-8.

2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171-5.

once it can be used for the screening of many PCR products over a long time.

in Multimodular Gene Products - The Extracellular Matrix Glycoprotein Tenascin C

4. Use these vectors as templates for PCRs generating dUTP-labelled DNA probes

3. Use this cDNA as template in PCRs for the alternatively spliced region of your gene

4. Separate the amplicons on an agarose gel and cut off the single bands

To start such a screen the following steps must be accomplished:

start. Therefore conduct the following steps to start a screen: 1. Isolate RNA from the tissue or cell type under investigation

6. Dilute the plasmid vectors to appropriate concentrations 7. Apply the plasmid solutions onto nylon membranes 8. Denature the DNA and bind it covalently to the membranes 9. Hybridize the DNA on the membranes with the probes

2. Generate primers detecting the single domains 3. Clone the single domains into plasmid vectors

5. Clone the PCR products into plasmid vectors

11. Apply an antibody to the labelling marker

Figure 9 shows the summary of the method:

1048, Fa 159/11-1, 2, 3) and the GRK 736 for support.

1. Clarify the domain sequence

2. Prepare cDNA based on this RNA

10. Wash under stringent conditions

12. Develop the enzyme reaction 13. Read out your domain structure

**5. Conclusion**

possible.

**7. References**

**6. Acknowledgements**

#### **4. Adaptation to general application**

Many genes are subject to alternative splicing and most of them show an exon skipping mode which implies the inclusion or exclusion of single exons. When the possible exon structure leading to the appearance or absence of single domains is known the expression profile of

Fig. 9. Schematic presentation of the method. Primers flanking the alternatively spliced region of the molecule are used to generate PCR products of different sizes which are separated on an agarose gel. The single bands are cut out off the gel and subcloned separately. The resulting clones can be analysed with a dot blot hybridization procedure with non-radioactively labelled probes. Positive and negative signals display the domain composition of every single clone.

the domains can be analysed using the method presented here. Some preliminary steps are necessary before a screen for expressed domains can be started but when the system is set up once it can be used for the screening of many PCR products over a long time.

To start such a screen the following steps must be accomplished:

1. Clarify the domain sequence

12 Will-be-set-by-IN-TECH

Many genes are subject to alternative splicing and most of them show an exon skipping mode which implies the inclusion or exclusion of single exons. When the possible exon structure leading to the appearance or absence of single domains is known the expression profile of

Fig. 9. Schematic presentation of the method. Primers flanking the alternatively spliced region of the molecule are used to generate PCR products of different sizes which are separated on an agarose gel. The single bands are cut out off the gel and subcloned

non-radioactively labelled probes. Positive and negative signals display the domain

composition of every single clone.

separately. The resulting clones can be analysed with a dot blot hybridization procedure with

**4. Adaptation to general application**


A screen includes the generation of the plasmids and the dot blot before the hybridization can start. Therefore conduct the following steps to start a screen:


Figure 9 shows the summary of the method:

#### **5. Conclusion**

With the method presented here we developed a possibility to unravel unknown structures of splice products for alternatively spliced transcripts. The example we analysed was the extracellular matrix molecule tenascin C but any other multimodular protein can be examined in a similar way. With some preliminary preparations an operational tool is at hand which makes the screening of many clones and therefore the generation of an expression profile possible.

## **6. Acknowledgements**

The authors wish to thank very much Dr. Angret Joester for providing unpublished material concerning the dot blot assays. We also thank the German Research Foundation (DFG) (SPP 1048, Fa 159/11-1, 2, 3) and the GRK 736 for support.

### **7. References**

Berget, S.M., Moore, C. & Sharp, P.A. (1977) Spliced segments at the 5 terminus of adenovirus 2 late mRNA. Proc Natl Acad Sci U S A. 1977 Aug;74(8):3171-5.

Chow, L.T., Gelinas, R.E., Broker, T.R. & Roberts, R.J. (1977) An amazing sequence arrangement at the 5 ends of adenovirus 2 messenger RNA. Cell. 1977 Sep;12(1):1-8.

**24** 

 *Gabon* 

Fousseyni S. Touré Ndouo *Medical Parasitology Unit,* 

**Submicroscopic Human Parasitic Infections** 

*Centre International de Recherches Médicales de Franceville (CIRMF), Franceville* 

Polymerase chain reaction (PCR) amplification provides a powerful tool for parasite detection. This chapter examines the use of PCR to diagnose malaria in patients with low parasite densities (submicroscopic infections, SMI) and also occult loaiosis (OL: *Loa loa* infection without detectable circulating microfilaria on standard microscopy). It provides therefore the issue of management of these kinds of infections with regard to the eradication

**i. Malaria:** Malaria is caused by *Plasmodium* parasites, of which there are about 200 species (Levine ND 1980). These protozoans belong to the *Apicomplexa* phylum, *Sporozoa*  class and *Haemosporidae* subclass (Levine ND 1970). They are obligatory intracellular parasites. Two successive hosts, humans and mosquitoes (Culicidea and Anophelinea), are necessary for their life cycle. Four main species infect humans, namely *Plasmodium falciparum*, *P. vivax*, *P. malariae* and *P. ovale*. A fifth species, *P. knowlesi*, is currently spreading in south-east Asia and Oceania. This species derived from chimpanzees has caused more than 250 human cases of malaria in Malaysia but is still considered to be zoonotic (Figtree et al. 2010). *P. falciparum* causes most life-threatening infections. Human is the intermediate host for malaria, wherein the asexual phase of the life cycle occurs. The sporozoïtes, inoculated by the infested female *Anopheles* mosquito, initiate this phase of the cycle from the liver, and continue within the red blood cells. From the mosquito bite, tens to few hundred invasive sporozoïtes are introduced into the skin. Following the intradermal drop, some sporozoïtes are destroyed locally by the immune cells, or enter into the lymphatic vessels, and some others can find blood circulation (Megumi L et al. 2007; Ashley M et al. 2008; Olivier S et al. 2008). The sporozoïtes that find peripheral blood circulation reach the liver within a few hours. It has been recently demonstrated that these sporozoïtes travel by a continuous sequence of stick-and-slip motility, using the thrombospondin-related anonymous protein (TRAP) family and an actin–myosin motor (Baum J et al. 2006; Megumi L et al. 2007; Münter S et al. 2009). The sporozoites migrate into hepatocytes and then grow within parasitophorous vacuoles and develop to the schizont stage which releases merozoites (Jones MK et al. 2006; Kebaier C et al. 2009). The entire pre-eryhrocytic phase lasts about 5–16 days depending

**1. Introduction** 

policy of such pathogens.

**1.1 Classification** 


## **Submicroscopic Human Parasitic Infections**

Fousseyni S. Touré Ndouo

*Medical Parasitology Unit, Centre International de Recherches Médicales de Franceville (CIRMF), Franceville Gabon* 

## **1. Introduction**

14 Will-be-set-by-IN-TECH

500 Polymerase Chain Reaction

Czopka, T., Von Holst, A., Schmidt, G., Ffrench-Constant, C. & Faissner, A. (2009) Tenascin

Dobbertin, A., Czvitkovich, S., Theocharidis, U., Garwood, J., Andrews, M.R., Properzi, F.,

Garcion, E., Faissner, A. & ffrench-Constant, C. (2001) Knockout mice reveal a contribution

Garcion, E., Halilagic, A., Faissner, A. & ffrench-Constant, C. (2004) Generation of an

Garwood, J., Theocharidis, U., Calco, V., Dobbertin, A. & Faissner, A. (2011) Existence of

Gates, M.A., Thomas, L.B., Howard, E.M., Laywell, E.D., Sajin, B., Faissner, A., Götz, B.,

Joester, A. & Faissner, A. (1999). Evidence for combinatorial variability of tenascin-C isoforms

Michele, M. & Faissner, A. (2009) Tenascin-C stimulates contactin-dependent neurite

Orend, G. & Chiquet-Ehrismann, R. (2006). Tenascin-C induced signaling in cancer. Cancer

Rigato, F., Garwood, J., Calco, V., Heck, N., Faivre-Sarrailh, C. & Faissner, A. (2002)

adhesion molecule F3/contactin. J Neurosci. 2002 Aug 1;22(15):6596-609. von Holst, A., Egbers, U., Prochiantz, A. & Faissner, A. (2007) Neural stem/progenitor cells

basic protein via a separate pathway. Glia. 2009 Dec;57(16):1790-801. Czopka, T., von Holst, A., ffrench-Constant, C. & Faissner, A. (2010) Regulatory

precursor differentiation. J Neurosci. 2010 Sep 15;30(37):12310-22.

fibronectin preparations. Nature. 1984 Sep 20-26;311(5983):267-9.

molecule tenascin C. Development. 2004 Jul;131(14):3423-32.

migration. Development. 2001 Jul;128(13):2485-96.

designation: Cell. Mol. Neurobiol. 32(2), 279-287

Aug;41(4):397-408. Epub 2009 Apr 24.

249-66.

274(24), 17144-51.

Lett, 244(2), 143-63.

282, 9172-9181.

C and tenascin R similarly prevent the formation of myelin membranes in a RhoA-dependent manner, but antagonistically regulate the expression of myelin

mechanisms that mediate tenascin C-dependent inhibition of oligodendrocyte

Lin, R., Fawcett, J.W. & Faissner, A. (2010) Analysis of combinatorial variability reveals selective accumulation of the fibronectin type III domains B and D of tenascin-C in injured brain. Exp Neurol. 2010 Sep;225(1):60-73. Epub 2010 May 5. Erickson, H.P. & Inglesias, J.L. (1984) A six-armed oligomer isolated from cell surface

of the extracellular matrix molecule tenascin-C to neural precursor proliferation and

environmental niche for neural stem cell development by the extracellular matrix

Tenascin-C Isoforms in Rat that Contain the Alternatively Spliced AD1 Domain are Developmentally Regulated During Hippocampal Development. Cell Mol Neurobiol. 2011 Oct 4. The paper has been published in (2012); add the correct

Silver, J. & Steindler, D.A. (1995). Cell and molecular analysis of the developing and adult mouse subventricular zone of the cerebral hemispheres. J Comp Neurol, 361(2),

and developmental regulation in the mouse central nervous system. J Biol Chem,

outgrowth via activation of phospholipase C. Mol Cell Neurosci. 2009

Tenascin-C promotes neurite outgrowth of embryonic hippocampal neurons through the alternatively spliced fibronectin type III BD domains via activation of the cell

express 20 tenascin C isoforms that are differentially regulated by Pax6. J Biol Chem,

Polymerase chain reaction (PCR) amplification provides a powerful tool for parasite detection. This chapter examines the use of PCR to diagnose malaria in patients with low parasite densities (submicroscopic infections, SMI) and also occult loaiosis (OL: *Loa loa* infection without detectable circulating microfilaria on standard microscopy). It provides therefore the issue of management of these kinds of infections with regard to the eradication policy of such pathogens.

#### **1.1 Classification**

**i. Malaria:** Malaria is caused by *Plasmodium* parasites, of which there are about 200 species (Levine ND 1980). These protozoans belong to the *Apicomplexa* phylum, *Sporozoa*  class and *Haemosporidae* subclass (Levine ND 1970). They are obligatory intracellular parasites. Two successive hosts, humans and mosquitoes (Culicidea and Anophelinea), are necessary for their life cycle. Four main species infect humans, namely *Plasmodium falciparum*, *P. vivax*, *P. malariae* and *P. ovale*. A fifth species, *P. knowlesi*, is currently spreading in south-east Asia and Oceania. This species derived from chimpanzees has caused more than 250 human cases of malaria in Malaysia but is still considered to be zoonotic (Figtree et al. 2010). *P. falciparum* causes most life-threatening infections.

Human is the intermediate host for malaria, wherein the asexual phase of the life cycle occurs. The sporozoïtes, inoculated by the infested female *Anopheles* mosquito, initiate this phase of the cycle from the liver, and continue within the red blood cells. From the mosquito bite, tens to few hundred invasive sporozoïtes are introduced into the skin. Following the intradermal drop, some sporozoïtes are destroyed locally by the immune cells, or enter into the lymphatic vessels, and some others can find blood circulation (Megumi L et al. 2007; Ashley M et al. 2008; Olivier S et al. 2008). The sporozoïtes that find peripheral blood circulation reach the liver within a few hours. It has been recently demonstrated that these sporozoïtes travel by a continuous sequence of stick-and-slip motility, using the thrombospondin-related anonymous protein (TRAP) family and an actin–myosin motor (Baum J et al. 2006; Megumi L et al. 2007; Münter S et al. 2009). The sporozoites migrate into hepatocytes and then grow within parasitophorous vacuoles and develop to the schizont stage which releases merozoites (Jones MK et al. 2006; Kebaier C et al. 2009). The entire pre-eryhrocytic phase lasts about 5–16 days depending

Submicroscopic Human Parasitic Infections 503

*Plasmodium* apoptosis-linked pathogenicity factors (PALPF).

passage of adult worms.

**ii. Loaiosis:** Loaiosis is characterized by calabar oedema (swelling) and conjunctivitis due to ocular passage of adult worms. Calabar oedema is transient and located on the face, limbs and back of the hands and fingers. *L. loa* is also called the "African eye worm". Meningoencephalic complications are an adverse effect of diethylcarbamazine treatment for hypermicrofilaremia. Other complications such as nephropathies, endocarditis, retinopathies, neuropathies and pneumonitis have been reported (Schofield et al. 1955; Hulin et al. 1994). Symptoms are more frequent in expatriates. Immunologically, loaiosis is characterized by hypergammaglobulinemia, hypereosinophilia and high IgE levels responsible for allergic symptoms. In endemic areas, loaiosis is the third reason for medical consultations in rural settings, although many microfilaremic subjects are asymptomatic. Occult loaiosis (amicrofilaremic infection) is defined as infection by the adult worm without peripheral microfilaremia on standard microscopy. Amicrofilaremic status is common among autochthonous residents and may be due to sequestration of microfilaria or to their massive destruction by the immune system, or to the presence of sterile adult worms. This form of infection is the most common in endemic areas. Other amicrofilaremic subjects are thought to be resistant. There is currently no way of discriminating between these two amicrofilaremic subgroups in the absence of (transient) ocular

criteria, Imbert et al 2002). The reasons why some non immune individuals infected by *P. falciparum* develop severe malaria and die, while others have only uncomplicated malaria or remain asymptomatic, remain unclear (Marsh et al 1988). Severe anemia and cerebral malaria are responsible for most of the morbidity and mortality related to this disease in children. Despite abundant research, the pathophysiological mechanisms underlying severe forms are poorly understood. Several studies have implicated sequestration of *P. falciparum*-parasitized red blood cells (PRBC) in the lungs and brain (Taylor et al. 2001). This sequestration is characterized by PRBC adhesion (or cytoadherence), agglutination and rosetting. Cytoadherence of PRBC to host endothelial cells (EC) in brain and lung capillaries can obstruct the microvasculature, a phenomenon accompanied by changes in the T cell repertoire and by cytokine production (Mazier et al. 2000). This adherence is modulated by platelets (Brown et al. 2000) and is mediated by EC receptors such as CD36, intracellular adhesion molecule 1 (ICAM1), vascular cellular adhesion molecule 1 (VCAM1), CD31, integrins and hyaluronic acid (Hunt et al. 2003). PRBC adhesion can induce over-expression of inflammatory cytokines (Mazier et al. 2000) and EC apoptosis (Pino et al. 2003). Approximately 20% of *P. falciparum* isolates from Franceville, Gabon (Central Africa), were show to induce human lung endothelial cell (HLEC) apoptosis by cytoadherence (Touré et al. 2008). In addition, apoptogenic isolates were more frequent in children with neurological signs (prostration or coma), supporting the hypothesis that PRBCmediated EC apoptosis could amplify blood-brain barrier disruption and dysfunction (Combes et al. 2005; Bisser et al. 2006). Whole transcriptome analysis revealed that 59 genes were more intensely transcribed in apoptogenic strains than in non apoptogenic strains (Siau et al. 2007). Knock-down of 8 of these genes by double-strand RNA interference significantly reduced the apoptogenic response in 5 genes (PF07\_0032, PF10255, PFI0130c, PFD0875c, and MAL13P1.206). These five genes are known as

on the parasite species (5-6 days for *P. falciparum*, 8 days for *P. vivax,* 9 days for *P. ovale,*  13 days for *P. malariae* and 8-9 days for *P. knowlesi.* The pre-erythrocytic phase remains a "silent" phase, with little pathology and no symptoms, as only a few hepatocytes are affected (Ashley M et al. 2008). This phase is a single cycle, contrasting to the next, erythrocytic stage, which occurs repeatedly.

**ii. Loaiosis:** Filariasis are typically chronic tropical diseases caused by nematodes of the *Filariidae* family, transmitted by flies or mosquitoes. Eight species are currently known to infect humans, namely *Wuchereria bancrofti*, *Brugia malayi, B. timoni, Onchocerca volvulus*, *Loa loa*, *Mansonella perstans*, *M. ozzardi*, and *M. streptocerca*. Three groups of filariasis have been distinguished on the basis of their human target tissues: lymphatic filariasis (wuchereriasis and brugiasis); cutaneous dermal filariasis (loaiosis, onchocerciasis, and *streptocerca* mansonelliasis) and serous filariasis (*perstans* and *ozzardi*  mansonelliasis) (Gentilini 1982). The vectors are blood-sucking flies and female mosquitoes. The microfilarial eggs or embryos are ingested by the vector when it bites an infected human. These microfilariae become infective stage L3 larvae after two successive mutes within the vector, and are transmitted to a new human host through a new blood meal or bite.

More than 3.3 billion people are exposed to filariosis, and an estimated 300 million people are infected. Loaiosis occurs in Africa, brugiaioses in South Asia, wuchereriasis in Africa and Asia, onchocerciasis in Africa, Central and South America and Asia (Yemen), *perstans* mansonelliasis in Africa and Central and South America, *ozzardi* mansonelliasis in Central and South America, and *streptocerca* mansonellaiosis in Africa.

*L. loa* infection (loaiosis) was initially described in 1770 by Mongin, in a female slave originating from West Africa and living on Saint Domingue island. Guyon et al. found the same worm in Gabon (Central Africa) in 1864. *L. loa* was first described in detail by Brumpt *et al*. in 1904, and then by Connors *et al.* in 1976. Although discovered in the Antilles, *L. loa* is restricted to Africa (Gentilini 1982). The adult worms live under the skin for about 15 years (Gentilini et al. 1982). The tabanides responsible for *L. loa* transmission are primarily *Chrysops dimidiata* and *silacea*, two forest species often present in the same hearth. Only the females are hematophagous, and they have diurnal activity.

## **1.2 Pathogenesis**

**i. Malaria:** *P. falciparum* is responsible for most complicated forms of malaria and causes about 800 000 deaths a year, mostly among children in sub-Saharan African countries (WHO 2009). Malaria symptoms generally occur in three phases. After an incubation period of 7 to 10 days, symptoms begin with fever, aches and digestive disorders (febrile stomach upset). Then, when schizont rupture becomes synchronous, patients enter the feverish reviviscent schizogonic phase (periodic fever) of uncomplicated malaria. This phase is characterized by fevers typically appearing every 24 hours (third fever in infection by *P. vivax* or *ovalae*, every 48 hours, quartan fever in infection by *P. malariae* or *P. falciparum*), accompanied by a triad of symptoms: shivers, fever and sweating. Destruction of parasitized red blood cells leads to the release of malarial toxins and to TNF alpha production. The third phase, mainly seen with *P. falciparum*, corresponds to severe malaria (pernicious access), which sometimes occurs rapidly after infection. Clinical and biological signs are used to classify malaria (WHO 2000 gravity

**ii. Loaiosis:** Filariasis are typically chronic tropical diseases caused by nematodes of the *Filariidae* family, transmitted by flies or mosquitoes. Eight species are currently known to infect humans, namely *Wuchereria bancrofti*, *Brugia malayi, B. timoni, Onchocerca volvulus*, *Loa loa*, *Mansonella perstans*, *M. ozzardi*, and *M. streptocerca*. Three groups of filariasis have been distinguished on the basis of their human target tissues: lymphatic filariasis (wuchereriasis and brugiasis); cutaneous dermal filariasis (loaiosis, onchocerciasis, and *streptocerca* mansonelliasis) and serous filariasis (*perstans* and *ozzardi*  mansonelliasis) (Gentilini 1982). The vectors are blood-sucking flies and female mosquitoes. The microfilarial eggs or embryos are ingested by the vector when it bites an infected human. These microfilariae become infective stage L3 larvae after two successive mutes within the vector, and are transmitted to a new human host through a

More than 3.3 billion people are exposed to filariosis, and an estimated 300 million people are infected. Loaiosis occurs in Africa, brugiaioses in South Asia, wuchereriasis in Africa and Asia, onchocerciasis in Africa, Central and South America and Asia (Yemen), *perstans* mansonelliasis in Africa and Central and South America, *ozzardi* mansonelliasis in Central

*L. loa* infection (loaiosis) was initially described in 1770 by Mongin, in a female slave originating from West Africa and living on Saint Domingue island. Guyon et al. found the same worm in Gabon (Central Africa) in 1864. *L. loa* was first described in detail by Brumpt *et al*. in 1904, and then by Connors *et al.* in 1976. Although discovered in the Antilles, *L. loa* is restricted to Africa (Gentilini 1982). The adult worms live under the skin for about 15 years (Gentilini et al. 1982). The tabanides responsible for *L. loa* transmission are primarily *Chrysops dimidiata* and *silacea*, two forest species often present in the same hearth. Only the

**i. Malaria:** *P. falciparum* is responsible for most complicated forms of malaria and causes about 800 000 deaths a year, mostly among children in sub-Saharan African countries (WHO 2009). Malaria symptoms generally occur in three phases. After an incubation period of 7 to 10 days, symptoms begin with fever, aches and digestive disorders (febrile stomach upset). Then, when schizont rupture becomes synchronous, patients enter the feverish reviviscent schizogonic phase (periodic fever) of uncomplicated malaria. This phase is characterized by fevers typically appearing every 24 hours (third fever in infection by *P. vivax* or *ovalae*, every 48 hours, quartan fever in infection by *P. malariae* or *P. falciparum*), accompanied by a triad of symptoms: shivers, fever and sweating. Destruction of parasitized red blood cells leads to the release of malarial toxins and to TNF alpha production. The third phase, mainly seen with *P. falciparum*, corresponds to severe malaria (pernicious access), which sometimes occurs rapidly after infection. Clinical and biological signs are used to classify malaria (WHO 2000 gravity

erythrocytic stage, which occurs repeatedly.

and South America, and *streptocerca* mansonellaiosis in Africa.

females are hematophagous, and they have diurnal activity.

new blood meal or bite.

**1.2 Pathogenesis** 

on the parasite species (5-6 days for *P. falciparum*, 8 days for *P. vivax,* 9 days for *P. ovale,*  13 days for *P. malariae* and 8-9 days for *P. knowlesi.* The pre-erythrocytic phase remains a "silent" phase, with little pathology and no symptoms, as only a few hepatocytes are affected (Ashley M et al. 2008). This phase is a single cycle, contrasting to the next, criteria, Imbert et al 2002). The reasons why some non immune individuals infected by *P. falciparum* develop severe malaria and die, while others have only uncomplicated malaria or remain asymptomatic, remain unclear (Marsh et al 1988). Severe anemia and cerebral malaria are responsible for most of the morbidity and mortality related to this disease in children. Despite abundant research, the pathophysiological mechanisms underlying severe forms are poorly understood. Several studies have implicated sequestration of *P. falciparum*-parasitized red blood cells (PRBC) in the lungs and brain (Taylor et al. 2001). This sequestration is characterized by PRBC adhesion (or cytoadherence), agglutination and rosetting. Cytoadherence of PRBC to host endothelial cells (EC) in brain and lung capillaries can obstruct the microvasculature, a phenomenon accompanied by changes in the T cell repertoire and by cytokine production (Mazier et al. 2000). This adherence is modulated by platelets (Brown et al. 2000) and is mediated by EC receptors such as CD36, intracellular adhesion molecule 1 (ICAM1), vascular cellular adhesion molecule 1 (VCAM1), CD31, integrins and hyaluronic acid (Hunt et al. 2003). PRBC adhesion can induce over-expression of inflammatory cytokines (Mazier et al. 2000) and EC apoptosis (Pino et al. 2003). Approximately 20% of *P. falciparum* isolates from Franceville, Gabon (Central Africa), were show to induce human lung endothelial cell (HLEC) apoptosis by cytoadherence (Touré et al. 2008). In addition, apoptogenic isolates were more frequent in children with neurological signs (prostration or coma), supporting the hypothesis that PRBCmediated EC apoptosis could amplify blood-brain barrier disruption and dysfunction (Combes et al. 2005; Bisser et al. 2006). Whole transcriptome analysis revealed that 59 genes were more intensely transcribed in apoptogenic strains than in non apoptogenic strains (Siau et al. 2007). Knock-down of 8 of these genes by double-strand RNA interference significantly reduced the apoptogenic response in 5 genes (PF07\_0032, PF10255, PFI0130c, PFD0875c, and MAL13P1.206). These five genes are known as *Plasmodium* apoptosis-linked pathogenicity factors (PALPF).

**ii. Loaiosis:** Loaiosis is characterized by calabar oedema (swelling) and conjunctivitis due to ocular passage of adult worms. Calabar oedema is transient and located on the face, limbs and back of the hands and fingers. *L. loa* is also called the "African eye worm". Meningoencephalic complications are an adverse effect of diethylcarbamazine treatment for hypermicrofilaremia. Other complications such as nephropathies, endocarditis, retinopathies, neuropathies and pneumonitis have been reported (Schofield et al. 1955; Hulin et al. 1994). Symptoms are more frequent in expatriates. Immunologically, loaiosis is characterized by hypergammaglobulinemia, hypereosinophilia and high IgE levels responsible for allergic symptoms. In endemic areas, loaiosis is the third reason for medical consultations in rural settings, although many microfilaremic subjects are asymptomatic. Occult loaiosis (amicrofilaremic infection) is defined as infection by the adult worm without peripheral microfilaremia on standard microscopy. Amicrofilaremic status is common among autochthonous residents and may be due to sequestration of microfilaria or to their massive destruction by the immune system, or to the presence of sterile adult worms. This form of infection is the most common in endemic areas. Other amicrofilaremic subjects are thought to be resistant. There is currently no way of discriminating between these two amicrofilaremic subgroups in the absence of (transient) ocular passage of adult worms.

Submicroscopic Human Parasitic Infections 505

hundred samples (Steenkeste N et al. 2009). The CYTB is a nested PCR based on Plasmodium cytochrome b gene followed by species detection using SNP (single nucleotide polymorphism) analysis. The usefulness of these methods in detecting malaria has been

Samples must be collected in sterile tubes. For example, peripheral blood is collected in tubes containing an anticoagulant such as EDTA. However, some anticoagulants, such as heparin, inhibit the action of Taq DNA polymerase and should thus be avoided. Blood samples can also be collected in the form of drops on calibrated pre-punched paper disks (Serobuvard, LDA 22H, Zoopole, Ploufragan, France) (Ouwe-Missi-

Thick and thin peripheral blood films were stained with Giemsa and examined by microscope. For microscope positive samples, the parasite load is expressed as the number of asexual forms of *P. falciparum*/µL of blood, assuming an average leukocyte

There are many useful techniques for DNA template processing. Plasmodial DNA extraction involves erythrocyte lysis and proteinase K digestion to prevent PCR

**i. DNeasyR Blood & Tissue Kit:** Whole blood (200 µl) is used for DNA extraction with the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). Briefly, DNA extraction is carried out as follows. To a 1.5-ml tube containing 200 µl of while blood are added 20 µl of proteinase K solution and 200 µl of AL buffer (a detergent included in the kit). The mixture is pulse vortexed for 15 seconds and incubated for 15 minutes at 56°C. Two hundred microliters of cold ethanol is then added and the mixture is vortexed for 15 seconds. The mixture is transferred to a mini-column assembled on a 2-ml tube and centrifuged for 1 min at 8000 rpm. After centrifugation the 2-ml tube is discarded OK. The mini-column is recovered and placed on a new 2-ml tube. The mini-column is then washed with 500 µL of AW1 buffer (available in the kit) by centrifugation at 8000 rpm for 1 min. This washing step is repeated with another 500 µL of AW2 buffer, followed by centrifugation for

The mini-column is placed on a 1.5-ml tube and 60 µl of AE elution buffer is added. This unit is left at room temperature for 10 min and then centrifuged for 1 min at 8000 rpm. The DNA is then recovered in the 1.5-ml tube and immediately used as a

**ii. Dried blood-spot method** (DBS)**:** DNA templates are extracted as described by Plowe CV et al in 1995. The dried blood spot is placed in 1 ml of phosphate buffered saline (PBS) containing 0.5% saponin and is incubated overnight at 4°C. The resulting brown solution is replaced with 1 ml of PBS and incubated for an additional 15-30 minutes at 4°C. Then, 200 µl of 5% Chelex 100 (Bio-Rad Laboratories, CA) is placed in clean tubes and heated to 100°C in a water bath. The disks are removed from the PBS and placed in the preheated 5% Chelex 100,

3 min at 14 000 rpm. The 2-ml tube is again discarded.

template or stored at 20°C.

demonstrated especially in low endemic areas.

**2.1 Materials and methods** 

Oukem-Boyer et al. 2005).

count of 8000/µL. **c. DNA template preparation** 

a. **Blood sampling**

b. **Microscopy** 

inhibition.

## **1.3 Diagnostic challenges**


## **2. PCR-based diagnosis of malaria**

In 1993 a PCR method targeting the small subunit of the ribosomal RNA (SSUrRNA) gene was developed for use as an alternative to microscopy for detecting the four main *Plasmodium* species (Snounou et al. 1993, 1994, 1995). Nested PCR was used for its high sensitivity and specificity (Snounou et al. 1993). However, the nested reaction requires five separate PCR reactions and is therefore time-consuming, expensive and not always feasible in developing world laboratories. Several variants of this nested PCR method, such as seminested multiplex and one-tube multiplex have been developed (Mixon-Hayden T et al. 2010). In 1998 Jarra and Snounou showed that *Plasmodium* DNA is cleared very quickly from the bloodstream and that positive PCR amplification is usually associated with the presence of viable parasites. PCR positivity therefore indicates active *Plasmodium* infection. Since 1997, several PCR methods targeting other *P. falciparum* genes have been developed (Cheng et al. 1997; Filisetti et al. 2002). Their sensitivity has been estimated at 71%, 83% and 100% for the *MSP-2, SSUrRNA* and *STEVOR* genes, respectively (Oyedeji et al. 2007).

Real-time PCR has been reported to be able to improve parasite detection. Compared to *SSUrRNA* nested PCR, the real-time assay had a sensitivity of 99.5% and specicity of 100% for the diagnosis of malaria (Farcas GA et al. 2004). The real-time PCR method, specic for all *Plasmodium* species, avoids post-amplication sample handling and electrophoresis, and the result can be ready within 45 min (Farcas GA et al. 2004). This method would be useful for monitoring antimalarial drug efficacy, especially in areas of drug resistance (Lee MA et al. 2002).

More recently, it has been shown that dot18S (18SrRNA gene) and CYTB, two new molecular methods, are highly sensitive and allow high-throughput scaling up for many hundred samples (Steenkeste N et al. 2009). The CYTB is a nested PCR based on Plasmodium cytochrome b gene followed by species detection using SNP (single nucleotide polymorphism) analysis. The usefulness of these methods in detecting malaria has been demonstrated especially in low endemic areas.

#### **2.1 Materials and methods**

#### a. **Blood sampling**

504 Polymerase Chain Reaction

**i. Malaria:** Light microscopy of blood smears remains the standard method for *Plasmodium* detection, both for clinical diagnosis and epidemiological surveys (Okell LC et al. 2009). However, sensitivity depends on parasite density in blood. In patients with low parasitemia, mixed infections, antimalarial treatment or chronic infection, microscopic diagnosis requires painstaking examination by an experienced technician. Low-density infections that cannot be detected by conventional microscopy are termed submicroscopic infections (SMI). *Plasmodium* species identification is mainly based on microscopic morphological characteristics but this is not entirely reliable (*Plasmodium vivax* resembles *P. ovale*). In addition, parasite morphology can be altered by drug

**ii. Loaiosis:** Human loaiosis differs from other filariasis by the fact that most patients have "occult" infection, with no circulating microfilaria. This peripheral amicrofilaremia can be due to microfilaria destruction by the immune system, and/or to their sequestration. These subjects cannot be diagnosed by microscopy and consequently go untreated, constituting a parasite reservoir. Before 1997, *L. loa* diagnosis was still based on microscopic examination and the prevalence was therefore underestimated. In contrast, because of cross-reactions, serological tests, and especially those based on total IgG detection, tend to overestimate prevalence. The existence of many cases of occult but symptomatic infection among residents in endemic areas implies the need for specific

In 1993 a PCR method targeting the small subunit of the ribosomal RNA (SSUrRNA) gene was developed for use as an alternative to microscopy for detecting the four main *Plasmodium* species (Snounou et al. 1993, 1994, 1995). Nested PCR was used for its high sensitivity and specificity (Snounou et al. 1993). However, the nested reaction requires five separate PCR reactions and is therefore time-consuming, expensive and not always feasible in developing world laboratories. Several variants of this nested PCR method, such as seminested multiplex and one-tube multiplex have been developed (Mixon-Hayden T et al. 2010). In 1998 Jarra and Snounou showed that *Plasmodium* DNA is cleared very quickly from the bloodstream and that positive PCR amplification is usually associated with the presence of viable parasites. PCR positivity therefore indicates active *Plasmodium* infection. Since 1997, several PCR methods targeting other *P. falciparum* genes have been developed (Cheng et al. 1997; Filisetti et al. 2002). Their sensitivity has been estimated at 71%, 83% and 100% for

Real-time PCR has been reported to be able to improve parasite detection. Compared to *SSUrRNA* nested PCR, the real-time assay had a sensitivity of 99.5% and specicity of 100% for the diagnosis of malaria (Farcas GA et al. 2004). The real-time PCR method, specic for all *Plasmodium* species, avoids post-amplication sample handling and electrophoresis, and the result can be ready within 45 min (Farcas GA et al. 2004). This method would be useful for monitoring antimalarial drug efficacy, especially in areas of drug resistance (Lee MA et

More recently, it has been shown that dot18S (18SrRNA gene) and CYTB, two new molecular methods, are highly sensitive and allow high-throughput scaling up for many

the *MSP-2, SSUrRNA* and *STEVOR* genes, respectively (Oyedeji et al. 2007).

**1.3 Diagnostic challenges** 

and sensitive detection.

al. 2002).

**2. PCR-based diagnosis of malaria** 

treatment and/or sample storage conditions.

Samples must be collected in sterile tubes. For example, peripheral blood is collected in tubes containing an anticoagulant such as EDTA. However, some anticoagulants, such as heparin, inhibit the action of Taq DNA polymerase and should thus be avoided. Blood samples can also be collected in the form of drops on calibrated pre-punched paper disks (Serobuvard, LDA 22H, Zoopole, Ploufragan, France) (Ouwe-Missi-Oukem-Boyer et al. 2005).

#### b. **Microscopy**

Thick and thin peripheral blood films were stained with Giemsa and examined by microscope. For microscope positive samples, the parasite load is expressed as the number of asexual forms of *P. falciparum*/µL of blood, assuming an average leukocyte count of 8000/µL.

#### **c. DNA template preparation**

There are many useful techniques for DNA template processing. Plasmodial DNA extraction involves erythrocyte lysis and proteinase K digestion to prevent PCR inhibition.

**i. DNeasyR Blood & Tissue Kit:** Whole blood (200 µl) is used for DNA extraction with the DNeasy Blood & Tissue kit (QIAGEN, Hilden, Germany). Briefly, DNA extraction is carried out as follows. To a 1.5-ml tube containing 200 µl of while blood are added 20 µl of proteinase K solution and 200 µl of AL buffer (a detergent included in the kit). The mixture is pulse vortexed for 15 seconds and incubated for 15 minutes at 56°C. Two hundred microliters of cold ethanol is then added and the mixture is vortexed for 15 seconds. The mixture is transferred to a mini-column assembled on a 2-ml tube and centrifuged for 1 min at 8000 rpm. After centrifugation the 2-ml tube is discarded OK. The mini-column is recovered and placed on a new 2-ml tube. The mini-column is then washed with 500 µL of AW1 buffer (available in the kit) by centrifugation at 8000 rpm for 1 min. This washing step is repeated with another 500 µL of AW2 buffer, followed by centrifugation for 3 min at 14 000 rpm. The 2-ml tube is again discarded.

The mini-column is placed on a 1.5-ml tube and 60 µl of AE elution buffer is added. This unit is left at room temperature for 10 min and then centrifuged for 1 min at 8000 rpm. The DNA is then recovered in the 1.5-ml tube and immediately used as a template or stored at 20°C.

**ii. Dried blood-spot method** (DBS)**:** DNA templates are extracted as described by Plowe CV et al in 1995. The dried blood spot is placed in 1 ml of phosphate buffered saline (PBS) containing 0.5% saponin and is incubated overnight at 4°C. The resulting brown solution is replaced with 1 ml of PBS and incubated for an additional 15-30 minutes at 4°C. Then, 200 µl of 5% Chelex 100 (Bio-Rad Laboratories, CA) is placed in clean tubes and heated to 100°C in a water bath. The disks are removed from the PBS and placed in the preheated 5% Chelex 100,

Submicroscopic Human Parasitic Infections 507

DNA polymerase, 0.4 pM each primer (P5, P18, P19 and P20) (P5 5'-GGG AAT TCT TTA TTT GAT GAA GAT G-3', P18 5'-TTT CA(C/T) CAC CAA ACA TTT CTT-3', P19 5'-AAT CCA CAT TAT CAC AAT GA-3', P20 5'-CCG ATT TTA ACA TAA TAT GA-3') and 5 μl of DNA template. The PCR program is as follows: denaturation at 93°C for 3 min followed by 25 cycles of 30 s at 93°C, 50 s at 50°C and 30 s at 72°C, with a final extension step of 3 min at 72°C. Two microliters of the first-round PCR product is used for the second round of amplification, with a reaction mixture of 50 μl containing 5.0 μl of 10X reaction buffer, 200 μM each dNTP, 1.25 units of Taq DNA polymerase and 0.4 pM each primer (P17 and P24) (P17 5'-ACA TTA TCA TAA TGA (C/T) CC AGA ACT-3', P24 5'-GTT TGC AAT AAT TCT TTT TCT AGC-3'). The PCR conditions for the nested reaction are as follows: denaturation at 93°C for 3 s, followed by 25 cycles of 30 s at 93°C, 50 s at 55°C and 30 s at 72°C, with a final

**Analysis of PCR products:** After amplification, 10 µl of each PCR product is mixed with 1 µl of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 40% w/v sucrose in water) and analyzed by electrophoresis on 1.5% agarose gel. The gel is stained with ethidium bromide or FluoProbes Gel Red (Interchim Montlucon, France) and the DNA is

**M 1 2 3 4 5 6 7 8 9 10 11** 

Fig. 1. Detection and speciation of *Plasmodium* by nested PCR using genus-specific primers and 1.5% agarose gel electrophoresis. Lanes 1 and 8: PCR-negative controls; lane 2: an individual with submicroscopic infection by *P. malariae* (size: 144 base pairs); lanes 3, 5, 7 and 10: PCR-negative individuals; lanes 4, 6, 9 and 11: individuals with submicroscopic coinfection with *P. falciparum* (size: 205 bp) and *P. malariae*. Lane M represents the DNA

Theoretically, three specific bands between 189-700 base pairs are generated using nested primers. We obtained a specific band of 250 bp for all *P. falciparum* isolates tested in

extension step of 3 min at 72°C.

*Plasmodium* **SSUrRNA gene:** 

molecular weight marker (100 bp).

*P. falciparum* **STEVOR gene** 

Franceville, southeastern Gabon.

visualized and photographed under ultraviolet light.

**iii. Detection procedures** 

vortexed at high speed for 30 seconds and placed in a water bath at 100°C for 10 minutes with gentle agitation. The samples are then centrifuged at 10 000 *g* for 2 minutes, and the supernatant is removed and centrifuged as before. The supernatant is then collected in a clean tube and immediately used for PCR or stored at 20°C until use.

DNA can be also extracted from dried blood spots with several other methods, such as the QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany).

## **2.2** *P. falciparum* **DNA amplification and detection**

### **i. SSUrRNA gene amplification**

Two microliters of DNA extract is amplified in a final volume of 25 μl containing 2.5 μl of 10X reaction buffer, 100 μM each dNTP (dATP, dGTP, dTTP, and dCTP), 0.5 pM each primer (rPLU5/rPLU6 (rPLU5 5'-CCT GTT GTT GCC TTA AAC TTC-3' and rPLU6 5'-TTA AAA TTG TTG CAG TTA AAA CG-3') for the primary reaction, and rFAL1/rFAL2 (rFAL 1 5'-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT-3' and rFAL 2 5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3') for the nested reaction) and 0.75 units of Taq DNA polymerase (QIAGEN, Hilden, Germany). The primer sequences (Table 1) are based on SSUrRNA sequences described elsewhere (Snounou et al. 1993). The PCR program is as follows: denaturation at 95°C for 5 min followed by 25 cycles (30 cycles in nested PCR) at 94°C for 1 min, 60°C for 2 min and 72°C for 2 min, with a final extension step of 5 min at 72°C.

### **ii. STEVOR gene amplification**

The first round of amplification is performed with a reaction mix of 50 μl containing 5.0 μl of 10X reaction buffer, 200 μM each dNTP (dATP, dGTP, dTTP, and dCTP), 1.25 units of Taq

Schema 1. Schematic representation of the STEVOR PCR methodology (CHENG et al 1997).

DNA can be also extracted from dried blood spots with several other methods, such as the

Two microliters of DNA extract is amplified in a final volume of 25 μl containing 2.5 μl of 10X reaction buffer, 100 μM each dNTP (dATP, dGTP, dTTP, and dCTP), 0.5 pM each primer (rPLU5/rPLU6 (rPLU5 5'-CCT GTT GTT GCC TTA AAC TTC-3' and rPLU6 5'-TTA AAA TTG TTG CAG TTA AAA CG-3') for the primary reaction, and rFAL1/rFAL2 (rFAL 1 5'-TTA AAC TGG TTT GGG AAA ACC AAA TAT ATT-3' and rFAL 2 5'-ACA CAA TGA ACT CAA TCA TGA CTA CCC GTC-3') for the nested reaction) and 0.75 units of Taq DNA polymerase (QIAGEN, Hilden, Germany). The primer sequences (Table 1) are based on SSUrRNA sequences described elsewhere (Snounou et al. 1993). The PCR program is as follows: denaturation at 95°C for 5 min followed by 25 cycles (30 cycles in nested PCR) at 94°C for 1 min, 60°C for 2 min and 72°C for 2 min, with a final extension step of 5 min at 72°C.

The first round of amplification is performed with a reaction mix of 50 μl containing 5.0 μl of 10X reaction buffer, 200 μM each dNTP (dATP, dGTP, dTTP, and dCTP), 1.25 units of Taq

Schema 1. Schematic representation of the STEVOR PCR methodology (CHENG et al 1997).

stored at 20°C until use.

**i. SSUrRNA gene amplification** 

**ii. STEVOR gene amplification** 

QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany).

**2.2** *P. falciparum* **DNA amplification and detection** 

vortexed at high speed for 30 seconds and placed in a water bath at 100°C for 10 minutes with gentle agitation. The samples are then centrifuged at 10 000 *g* for 2 minutes, and the supernatant is removed and centrifuged as before. The supernatant is then collected in a clean tube and immediately used for PCR or DNA polymerase, 0.4 pM each primer (P5, P18, P19 and P20) (P5 5'-GGG AAT TCT TTA TTT GAT GAA GAT G-3', P18 5'-TTT CA(C/T) CAC CAA ACA TTT CTT-3', P19 5'-AAT CCA CAT TAT CAC AAT GA-3', P20 5'-CCG ATT TTA ACA TAA TAT GA-3') and 5 μl of DNA template. The PCR program is as follows: denaturation at 93°C for 3 min followed by 25 cycles of 30 s at 93°C, 50 s at 50°C and 30 s at 72°C, with a final extension step of 3 min at 72°C. Two microliters of the first-round PCR product is used for the second round of amplification, with a reaction mixture of 50 μl containing 5.0 μl of 10X reaction buffer, 200 μM each dNTP, 1.25 units of Taq DNA polymerase and 0.4 pM each primer (P17 and P24) (P17 5'-ACA TTA TCA TAA TGA (C/T) CC AGA ACT-3', P24 5'-GTT TGC AAT AAT TCT TTT TCT AGC-3'). The PCR conditions for the nested reaction are as follows: denaturation at 93°C for 3 s, followed by 25 cycles of 30 s at 93°C, 50 s at 55°C and 30 s at 72°C, with a final extension step of 3 min at 72°C.

#### **iii. Detection procedures**

**Analysis of PCR products:** After amplification, 10 µl of each PCR product is mixed with 1 µl of loading dye (0.25% bromophenol blue, 0.25% xylene cyanol and 40% w/v sucrose in water) and analyzed by electrophoresis on 1.5% agarose gel. The gel is stained with ethidium bromide or FluoProbes Gel Red (Interchim Montlucon, France) and the DNA is visualized and photographed under ultraviolet light.

**M 1 2 3 4 5 6 7 8 9 10 11** 

*Plasmodium* **SSUrRNA gene:** 

Fig. 1. Detection and speciation of *Plasmodium* by nested PCR using genus-specific primers and 1.5% agarose gel electrophoresis. Lanes 1 and 8: PCR-negative controls; lane 2: an individual with submicroscopic infection by *P. malariae* (size: 144 base pairs); lanes 3, 5, 7 and 10: PCR-negative individuals; lanes 4, 6, 9 and 11: individuals with submicroscopic coinfection with *P. falciparum* (size: 205 bp) and *P. malariae*. Lane M represents the DNA molecular weight marker (100 bp).

#### *P. falciparum* **STEVOR gene**

Theoretically, three specific bands between 189-700 base pairs are generated using nested primers. We obtained a specific band of 250 bp for all *P. falciparum* isolates tested in Franceville, southeastern Gabon.

Submicroscopic Human Parasitic Infections 509

sheath (*L. loa)*.

required.

c. Whole blood lysate processing

d. *L. loa* 15r3 gene amplification and detection

M: DNA molecular weight marker VI (Boehringer).

method is also used to detect *L. loa* microfilariae.

during the day, given the diurnal periodicity of human loaiosis.

*loa* and *M. perstans* microfilariae is based on size, motility, and by the presence of a

Thick smears can be also prepared with venous blood and stained with Giemsa or hematoxylin-eosin to detect microfilariae. The QBC (Quantitative Coating Buffer)

For microscopic detection of *L. loa* microfilariae, blood samples must be collected

Whole blood (100 µl) is mixed with 500 µl of TE buffer (10 mM Tris pH 8; 0.1 mM EDTA pH 8) and spun at 10 000 *g* twice for 2 min, discarding the supernatant at each step. The pellet is resuspended in 500 µl of sucrose buffer (10 mM Tris pH 7.6, 5 mM MgCl2 1 M sucrose and 1% Triton X 100) and spun at 10 000 *g* twice for 2 min. After the final wash, the supernatant is discarded and the pellet is resuspended with 200 µl of prewarmed (56°C) proteinase K buffer (containing 20 mM Tris pH 8, 50 mM KCl, 2.5 mM MgCl2, 100 µg/ml proteinase K and 0.5% Tween20), incubated at 56°C for two hours, then held at 90°C for 10 min. The DNA can be stored at 4°C for several days or at -20°C until

Primers corresponding to the 5' and 3' ends of the repeat 3 sequence of the gene coding for *L. loa* 15 kDa allergenic polyprotein are used. Primary amplification is done with a reaction mixture of 50 µl containing 2 µl of blood lysate, 1X PCR buffer (supplied by the manufacturer: 200 mM Tris-HCl pH 8.7, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton x100, 1 mg/ml bovine serum albumin), 200 µM each dATP, dCTP, dGTP and dTTP, 1 µM each primer (15r3-1: 5'-AAT-CAG-GCA-AAT-AAT-GGC-ACA-AAA-3', 15r3-2: 5'-GCG-TTT-TCT-TCT-CAC-CAG-CTG-TCT-3') and 1 unit of DNA polymerase. Amplification is performed with a Perkin Elmer thermal cycler for 40 cycles: 94°C for 1 min (denaturation), 65°C for 1 min (annealing) and 72°C for 2 min

Fig. 3. Representative 1.5% agarose gel electrophoresis patterns of nested 15r3 PCR

products. Lanes 1, 2 and 7: *L. loa* amicrofilaremic individuals (AMF) positive by PCR. Lanes 4 and 5: individuals negative by PCR. Lane 8: an individual with 100 *L. loa* microfilariae per ml and positive by PCR. Lanes 3 and 6: PCR-negative controls (no template); 5 µl of each nested PCR product was applied to each lane and revealed using UV transillumination after ethidium bromide staining. A fragment of 366 bp was observed with positive samples. Lane

Fig. 2. Detection of the *Plasmodium* STEVOR gene by nested PCR using specific primers and 1.5% agarose gel electrophoresis. Lanes 1 and 10: PCR-negative controls; lanes 2, 3, 4, 6, 8, 9 and 11: PCR-negative samples; lanes 5 and 7: PCR-positive samples; lane 12: PCR-positive control; lane M: DNA molecular weight marker (123 bp).

## **3. PCR based diagnosis of** *Loa loa*

In 1997, a PCR method (15r3-PCR) was developed to detect the repeat 3 region of the gene encoding the *L. loa* polyprotein in blood samples (Touré et al. 1997a, 1997b). Amicrofilaremic status is generally due to massive destruction of microfilaria, releasing parasite DNA into the bloodstream. These molecules may exist free in plasma, or be associated with cellsurface proteins, or even be contained in phagocytic cells. In addition, the adult worms can release DNA when they produce nonviable eggs or when they die after immune attack. The quantity of DNA released, whether from eggs, microfilaria or adult worms, is related to the parasite load of adult worms. The 15r3-PCR assay had a sensitivity of 95% with respect to detection of ocular passage of *L. loa* adult worms, and 100% compared to detection of microfilaremia.

#### **3.1 Materials and methods**

a. Blood sampling

As previously mentioned blood samples must be collected by venipuncture into Vacutainer tubes containing an adequate anticoagulant such as EDTA.

b. Leukoconcentration

Clinically, *L. loa* infection is diagnosed when migration of adult worms under the conjunctiva and/or skin is observed, or when a patient presents with classical symptoms. Diagnosis is classically based on standard microscopy. Microfilariae are the blood stage of *L. loa*. One milliliter of each blood sample is added to a 15-ml tube containing 9 ml of phosphate buffered saline (PBS), in duplicate. The mixture is treated with 600 µl of 2% saponin at room temperature for 15 min to lyse red blood cells, followed by centrifugation at 1000 *g* for 15 minutes at 4°C. The supernatant is discarded and the pellet is examined microscopically for microfilariae. The distinction between *L.* 

**M 1 2 3 4 5 6 7 8 9 10 11 12** 

Fig. 2. Detection of the *Plasmodium* STEVOR gene by nested PCR using specific primers and 1.5% agarose gel electrophoresis. Lanes 1 and 10: PCR-negative controls; lanes 2, 3, 4, 6, 8, 9 and 11: PCR-negative samples; lanes 5 and 7: PCR-positive samples; lane 12: PCR-positive

In 1997, a PCR method (15r3-PCR) was developed to detect the repeat 3 region of the gene encoding the *L. loa* polyprotein in blood samples (Touré et al. 1997a, 1997b). Amicrofilaremic status is generally due to massive destruction of microfilaria, releasing parasite DNA into the bloodstream. These molecules may exist free in plasma, or be associated with cellsurface proteins, or even be contained in phagocytic cells. In addition, the adult worms can release DNA when they produce nonviable eggs or when they die after immune attack. The quantity of DNA released, whether from eggs, microfilaria or adult worms, is related to the parasite load of adult worms. The 15r3-PCR assay had a sensitivity of 95% with respect to detection of ocular passage of *L. loa* adult worms, and 100% compared to detection of

As previously mentioned blood samples must be collected by venipuncture into

Clinically, *L. loa* infection is diagnosed when migration of adult worms under the conjunctiva and/or skin is observed, or when a patient presents with classical symptoms. Diagnosis is classically based on standard microscopy. Microfilariae are the blood stage of *L. loa*. One milliliter of each blood sample is added to a 15-ml tube containing 9 ml of phosphate buffered saline (PBS), in duplicate. The mixture is treated with 600 µl of 2% saponin at room temperature for 15 min to lyse red blood cells, followed by centrifugation at 1000 *g* for 15 minutes at 4°C. The supernatant is discarded and the pellet is examined microscopically for microfilariae. The distinction between *L.* 

Vacutainer tubes containing an adequate anticoagulant such as EDTA.

control; lane M: DNA molecular weight marker (123 bp).

**3. PCR based diagnosis of** *Loa loa*

microfilaremia.

a. Blood sampling

b. Leukoconcentration

**3.1 Materials and methods** 

*loa* and *M. perstans* microfilariae is based on size, motility, and by the presence of a sheath (*L. loa)*.

Thick smears can be also prepared with venous blood and stained with Giemsa or hematoxylin-eosin to detect microfilariae. The QBC (Quantitative Coating Buffer) method is also used to detect *L. loa* microfilariae.

For microscopic detection of *L. loa* microfilariae, blood samples must be collected during the day, given the diurnal periodicity of human loaiosis.

c. Whole blood lysate processing

Whole blood (100 µl) is mixed with 500 µl of TE buffer (10 mM Tris pH 8; 0.1 mM EDTA pH 8) and spun at 10 000 *g* twice for 2 min, discarding the supernatant at each step. The pellet is resuspended in 500 µl of sucrose buffer (10 mM Tris pH 7.6, 5 mM MgCl2 1 M sucrose and 1% Triton X 100) and spun at 10 000 *g* twice for 2 min. After the final wash, the supernatant is discarded and the pellet is resuspended with 200 µl of prewarmed (56°C) proteinase K buffer (containing 20 mM Tris pH 8, 50 mM KCl, 2.5 mM MgCl2, 100 µg/ml proteinase K and 0.5% Tween20), incubated at 56°C for two hours, then held at 90°C for 10 min. The DNA can be stored at 4°C for several days or at -20°C until required.

d. *L. loa* 15r3 gene amplification and detection

Primers corresponding to the 5' and 3' ends of the repeat 3 sequence of the gene coding for *L. loa* 15 kDa allergenic polyprotein are used. Primary amplification is done with a reaction mixture of 50 µl containing 2 µl of blood lysate, 1X PCR buffer (supplied by the manufacturer: 200 mM Tris-HCl pH 8.7, 100 mM KCl, 100 mM (NH4)2SO4, 20 mM MgSO4, 1% Triton x100, 1 mg/ml bovine serum albumin), 200 µM each dATP, dCTP, dGTP and dTTP, 1 µM each primer (15r3-1: 5'-AAT-CAG-GCA-AAT-AAT-GGC-ACA-AAA-3', 15r3-2: 5'-GCG-TTT-TCT-TCT-CAC-CAG-CTG-TCT-3') and 1 unit of DNA polymerase. Amplification is performed with a Perkin Elmer thermal cycler for 40 cycles: 94°C for 1 min (denaturation), 65°C for 1 min (annealing) and 72°C for 2 min

Fig. 3. Representative 1.5% agarose gel electrophoresis patterns of nested 15r3 PCR products. Lanes 1, 2 and 7: *L. loa* amicrofilaremic individuals (AMF) positive by PCR. Lanes 4 and 5: individuals negative by PCR. Lane 8: an individual with 100 *L. loa* microfilariae per ml and positive by PCR. Lanes 3 and 6: PCR-negative controls (no template); 5 µl of each nested PCR product was applied to each lane and revealed using UV transillumination after ethidium bromide staining. A fragment of 366 bp was observed with positive samples. Lane M: DNA molecular weight marker VI (Boehringer).

Submicroscopic Human Parasitic Infections 511

gametocyte reservoir may sustain malaria transmission despite efforts to fight malaria in endemic areas (Karl S et al. 2011). The prevalence of SMI, including submicroscopic gametocytes, must be assessed and taken into account in malaria control programs (Okell

Only patients with positive blood smears and/or rapid diagnostic tests (RDT) are routinely treated, while the treatment of patients negative by both methods depends on clinical signs and the physician's appreciation. These patients, including those with SMI, may represent more than 10% of infected individuals. In Gabon, SMI currently tends to be more frequent than microscopic infection, possibly due to better preventive policies and/or case management (Bouyou-Akotet et al. 2010). Treatment of all infected subjects, including those with SMI and submicroscopic gametocytes, would reduce the community parasite burden. Indeed, it has been shown that intermittent preventive treatment can reduce the prevalence

Human loaiosis differs from other filariasis by the fact that most infected individuals do not have blood microfilariae detectable by standard microscopy. Since the first description of this filariasis, many epidemiologists have found a low prevalence of microfilaria despite local vector abundance. The notion that most patients clear their microfilaremia but continue to have (occult) infection is primarily based on the observation of adult worms during eye passage. The assumption that endemic resistant subjects also may exist (subjects able to completely eliminate *L. loa* infection) is still based on the same observations. Only a sensitive diagnostic test can confirm these assumptions. Our results have shown that 15r3- PCR is suitable for discriminating among endemic groups (microfilaremics, occult infected individuals (occults) and resistant subjects), as the results should be positive in the first two groups and negative in the last. Indeed, two-thirds of infected individuals in southeastern Gabon have occult loaiosis (OL) Touré et al. (1998, 1999a). This needs to be shown in a longitudinal study, however, as *L. loa* infection is characterized by its relative stability in humans and mandrills, the adult worm having a lifespan of about 15 years (Gentillini 1982,

This implies that the prevalence of loaiosis would be underestimated by microscopy. If *L. loa* DNA detection is a marker of active infection, all subjects positive by PCR should be treated. This would not have a major impact on health at the individual level but could reduce the parasite burden in the community, in turn reducing the intensity of transmission and

Studies of resistant individuals may provide interesting immunological information. Marked differences in humoral and cellular immune responses have already been noted between microfilaremic and amicrofilaremic patients (Pinder 1988, Akué 1997, Baize et al. 1997), as well as in the mandrill model (Leroy 1997). However, lacking a reliable method for diagnosing occult infection, it is not known if this difference is due to immunity directed against adult worms or against microfilaria. The identification of endemic groups ("microfilaremics", "occults" and "resistants") by 15r3-PCR method should allow immunological studies to be carried out with sera and cells from each endemic group, using antigens of each developmental stage of *L. loa*, and particularly infective larvae and adult worms. Such studies could help to identify possible cellular or humoral markers involved in

and genetic diversity of *P. falciparum* malaria (Liljander A et al. 2010).

LC et al. 2009, Karl S et al. 2011).

**ii.** *Loa loa*

Pinder 1994).

resulting in public health benefits.

(extension), preceded by a "hot start" cycle at 96°C for 10 min, 80°C for 5 min and 94°C for 30 s. One microliter of product from the first-round amplification is used for a second round in the above conditions for 30 cycles. The following primers are used: 15r3-3: 5'GGC ACA AAA CAC TGC AGC AGT CCT3', and 15r3-4: 5'CAG CTG TCT CAA ATC GAA GAT TCT 3.'

## **4. Submicroscopic infection and disease management and control**

#### **i. Malaria:**

The global strategy for malaria control is based on prevention, early diagnosis and prompt treatment. The detection limit of routine microscopy has been estimated to be about 100 parasites/milliliter, whereas PCR can detect as little as 0.01 parasite /micro liter (Mockenhaupt FP et al. 2002). Submicroscopic infection (SMI) including submicroscopic gametocytes is common in both symptomatic and asymptomatic individuals with malaria. A systematic review and analysis of field data carried out by Okell LC et al. in 2009 showed that the prevalence of *P. falciparum* was twice as high with PCR as with microscopy. In a village in Dienga, southeastern Gabon, PCR was performed on blood samples from asymptomatic individuals negative by microscopy: the prevalence of SMI (PCR positivity) was 13.7% by PCR and 7.2% by microscopy (Touré et al. 2006). A study carried out by Bouyou-Akotet et al. in 2010 in Libreville (capital of Gabon) showed an 18.2% prevalence of SMI in pregnant women. Recently, SMI was detected in 18% of symptomatic individuals in Franceville, southeastern Gabon, whereas the microscopic prevalence was 23% (author's personal data). It has been estimated that as many as 88% of infections remain undetectable by microscopy in low-transmission areas, where the PCR prevalence is generally under 10% (Okell LC et al. 2009). Thus, a high rate of SMI could undermine disease control programs. In endemic areas, it has been shown that *P. falciparum* SMI contributes to acute disease (Rogier C et al. 1996), and to malaria-associated anemia and inflammation (Mockenhaupt FP et al. 2002). It has also been shown that cerebral malaria is frequently associated with SMI in semi-immune individuals (Giha HA et al. 2005). Finally, Bouyou-Akotet et al. 2010 have demonstrated that SMI during pregnancy is associated with low birth weight, especially in primagravidae. As parasite resistance to antimalarial drugs is currently widespread and increasing, it is very important to identify resistant parasites in patients with SMI. Two major genes have been implicated in *P. falciparum* resistance to quinoline, namely *Pfcrt* (*P. falciparum* chloroquine resistance transporter) and *Pfmdr1* (*P. falciparum* multidrug resistance gene 1). Single-nucleotide polymorphisms (SNPs) in these genes are associated with resistance both *in vitro* and *in vivo* (Wongsrichanalai et al. 2002). Therefore, *P. falciparum* drug resistance is linked to particular parasite genotypes (Duraisingh et al. 1997). *P. falciparum* infection is generally polyclonal, and may thus involve both drug-sensitive and resistant genotypes. SMI detection can be used to evaluate the therapeutic effectiveness of anti-malarial drugs during mass treatments and preclinical trials.

SMI individuals are capable of infecting mosquitoes and contributing to human transmission (Coleman RE et al. 2004), mainly in areas of seasonal transmission (Nwakanma D et al. 2008). Microscopy fails to detect the parasite in 49.2% of all malaria cases and in 91.3% of gametocytemic individuals (Okell LC et al. 2009). Individuals whose blood smears are negative for gametocytes (submicroscopic gametocyte) are equally able to transmit the infection to mosquitoes as slide-positive individuals (Coleman RE et al. 2004). Thus, the SMI gametocyte reservoir may sustain malaria transmission despite efforts to fight malaria in endemic areas (Karl S et al. 2011). The prevalence of SMI, including submicroscopic gametocytes, must be assessed and taken into account in malaria control programs (Okell LC et al. 2009, Karl S et al. 2011).

Only patients with positive blood smears and/or rapid diagnostic tests (RDT) are routinely treated, while the treatment of patients negative by both methods depends on clinical signs and the physician's appreciation. These patients, including those with SMI, may represent more than 10% of infected individuals. In Gabon, SMI currently tends to be more frequent than microscopic infection, possibly due to better preventive policies and/or case management (Bouyou-Akotet et al. 2010). Treatment of all infected subjects, including those with SMI and submicroscopic gametocytes, would reduce the community parasite burden. Indeed, it has been shown that intermittent preventive treatment can reduce the prevalence and genetic diversity of *P. falciparum* malaria (Liljander A et al. 2010).

#### **ii.** *Loa loa*

510 Polymerase Chain Reaction

The global strategy for malaria control is based on prevention, early diagnosis and prompt treatment. The detection limit of routine microscopy has been estimated to be about 100 parasites/milliliter, whereas PCR can detect as little as 0.01 parasite /micro liter (Mockenhaupt FP et al. 2002). Submicroscopic infection (SMI) including submicroscopic gametocytes is common in both symptomatic and asymptomatic individuals with malaria. A systematic review and analysis of field data carried out by Okell LC et al. in 2009 showed that the prevalence of *P. falciparum* was twice as high with PCR as with microscopy. In a village in Dienga, southeastern Gabon, PCR was performed on blood samples from asymptomatic individuals negative by microscopy: the prevalence of SMI (PCR positivity) was 13.7% by PCR and 7.2% by microscopy (Touré et al. 2006). A study carried out by Bouyou-Akotet et al. in 2010 in Libreville (capital of Gabon) showed an 18.2% prevalence of SMI in pregnant women. Recently, SMI was detected in 18% of symptomatic individuals in Franceville, southeastern Gabon, whereas the microscopic prevalence was 23% (author's personal data). It has been estimated that as many as 88% of infections remain undetectable by microscopy in low-transmission areas, where the PCR prevalence is generally under 10% (Okell LC et al. 2009). Thus, a high rate of SMI could undermine disease control programs. In endemic areas, it has been shown that *P. falciparum* SMI contributes to acute disease (Rogier C et al. 1996), and to malaria-associated anemia and inflammation (Mockenhaupt FP et al. 2002). It has also been shown that cerebral malaria is frequently associated with SMI in semi-immune individuals (Giha HA et al. 2005). Finally, Bouyou-Akotet et al. 2010 have demonstrated that SMI during pregnancy is associated with low birth weight, especially in primagravidae. As parasite resistance to antimalarial drugs is currently widespread and increasing, it is very important to identify resistant parasites in patients with SMI. Two major genes have been implicated in *P. falciparum* resistance to quinoline, namely *Pfcrt* (*P. falciparum* chloroquine resistance transporter) and *Pfmdr1* (*P. falciparum* multidrug resistance gene 1). Single-nucleotide polymorphisms (SNPs) in these genes are associated with resistance both *in vitro* and *in vivo* (Wongsrichanalai et al. 2002). Therefore, *P. falciparum* drug resistance is linked to particular parasite genotypes (Duraisingh et al. 1997). *P. falciparum* infection is generally polyclonal, and may thus involve both drug-sensitive and resistant genotypes. SMI detection can be used to evaluate the therapeutic effectiveness of

**4. Submicroscopic infection and disease management and control** 

anti-malarial drugs during mass treatments and preclinical trials.

SMI individuals are capable of infecting mosquitoes and contributing to human transmission (Coleman RE et al. 2004), mainly in areas of seasonal transmission (Nwakanma D et al. 2008). Microscopy fails to detect the parasite in 49.2% of all malaria cases and in 91.3% of gametocytemic individuals (Okell LC et al. 2009). Individuals whose blood smears are negative for gametocytes (submicroscopic gametocyte) are equally able to transmit the infection to mosquitoes as slide-positive individuals (Coleman RE et al. 2004). Thus, the SMI

CAA ATC GAA GAT TCT 3.'

**i. Malaria:** 

(extension), preceded by a "hot start" cycle at 96°C for 10 min, 80°C for 5 min and 94°C for 30 s. One microliter of product from the first-round amplification is used for a second round in the above conditions for 30 cycles. The following primers are used: 15r3-3: 5'GGC ACA AAA CAC TGC AGC AGT CCT3', and 15r3-4: 5'CAG CTG TCT

> Human loaiosis differs from other filariasis by the fact that most infected individuals do not have blood microfilariae detectable by standard microscopy. Since the first description of this filariasis, many epidemiologists have found a low prevalence of microfilaria despite local vector abundance. The notion that most patients clear their microfilaremia but continue to have (occult) infection is primarily based on the observation of adult worms during eye passage. The assumption that endemic resistant subjects also may exist (subjects able to completely eliminate *L. loa* infection) is still based on the same observations. Only a sensitive diagnostic test can confirm these assumptions. Our results have shown that 15r3- PCR is suitable for discriminating among endemic groups (microfilaremics, occult infected individuals (occults) and resistant subjects), as the results should be positive in the first two groups and negative in the last. Indeed, two-thirds of infected individuals in southeastern Gabon have occult loaiosis (OL) Touré et al. (1998, 1999a). This needs to be shown in a longitudinal study, however, as *L. loa* infection is characterized by its relative stability in humans and mandrills, the adult worm having a lifespan of about 15 years (Gentillini 1982, Pinder 1994).

> This implies that the prevalence of loaiosis would be underestimated by microscopy. If *L. loa* DNA detection is a marker of active infection, all subjects positive by PCR should be treated. This would not have a major impact on health at the individual level but could reduce the parasite burden in the community, in turn reducing the intensity of transmission and resulting in public health benefits.

> Studies of resistant individuals may provide interesting immunological information. Marked differences in humoral and cellular immune responses have already been noted between microfilaremic and amicrofilaremic patients (Pinder 1988, Akué 1997, Baize et al. 1997), as well as in the mandrill model (Leroy 1997). However, lacking a reliable method for diagnosing occult infection, it is not known if this difference is due to immunity directed against adult worms or against microfilaria. The identification of endemic groups ("microfilaremics", "occults" and "resistants") by 15r3-PCR method should allow immunological studies to be carried out with sera and cells from each endemic group, using antigens of each developmental stage of *L. loa*, and particularly infective larvae and adult worms. Such studies could help to identify possible cellular or humoral markers involved in

Submicroscopic Human Parasitic Infections 513

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resistance to infection, as well as the underlying mechanisms, including host genetic factors. These studies would open the way to investigations of the underlying molecular mechanisms.

In addition, the detection of OL by PCR will allow precise evaluation of filaricide effectiveness during mass treatment, and also that of new drugs in animal models. Pinder et al. showed in 1994 that experimental mandrill infection (*Mandrillus sphinx*) by human *L. loa* isolates led to the same parasitologic characteristics as the natural human infection. Thus, mandrills with occult infection (absence of microfilarae but presence of adult worms, as shown by 15r3-PCR positivity; Touré et al. 1998) can be used to evaluate macrofilaricidal drugs. It has been demonstrated that the 15-kDa polyprotein is conserved within human and simian *L. loa* (Touré et al. 1999b). L.15r3-PCR also detects simian occult *L. loa* and could be used to identify infected animals before their inclusion in preclinical trials.

Finally, serological tests using purified recombinant antigens or peptides offer much better specificity than those using crude antigens. When these antigens become available for loaiosis, immunoenzymatic methods like IgG4 ELISA will reach acceptable specificity. Comparison of ELISA and PCR results sould show whether or not specific IgG4 antibodies are markers of active *L. loa* infection.

## **5. Conclusion**

The global strategy of eliminating the parasitic diseases especially malaria and filariasis is mainly based on prevention, early diagnosis and prompt treatment. However, most decisions still rely on microscopy diagnosis which is not always adapted in detecting all infections. Indeed, the success of any intervention depends of the effectiveness of tools and methods especially those allowing proper detection of parasites. PCR offers an exciting opportunity to diagnose submicroscopic malaria infections and occult loaiosis which may constitute a hidden reservoir of disease transmission. The detection of such infections would allow the accurate management of all cases necessary to progress from disease control to elimination.

## **6. References**


resistance to infection, as well as the underlying mechanisms, including host genetic factors. These studies would open the way to investigations of the underlying molecular

In addition, the detection of OL by PCR will allow precise evaluation of filaricide effectiveness during mass treatment, and also that of new drugs in animal models. Pinder et al. showed in 1994 that experimental mandrill infection (*Mandrillus sphinx*) by human *L. loa* isolates led to the same parasitologic characteristics as the natural human infection. Thus, mandrills with occult infection (absence of microfilarae but presence of adult worms, as shown by 15r3-PCR positivity; Touré et al. 1998) can be used to evaluate macrofilaricidal drugs. It has been demonstrated that the 15-kDa polyprotein is conserved within human and simian *L. loa* (Touré et al. 1999b). L.15r3-PCR also detects simian occult *L. loa* and could

Finally, serological tests using purified recombinant antigens or peptides offer much better specificity than those using crude antigens. When these antigens become available for loaiosis, immunoenzymatic methods like IgG4 ELISA will reach acceptable specificity. Comparison of ELISA and PCR results sould show whether or not specific IgG4 antibodies

The global strategy of eliminating the parasitic diseases especially malaria and filariasis is mainly based on prevention, early diagnosis and prompt treatment. However, most decisions still rely on microscopy diagnosis which is not always adapted in detecting all infections. Indeed, the success of any intervention depends of the effectiveness of tools and methods especially those allowing proper detection of parasites. PCR offers an exciting opportunity to diagnose submicroscopic malaria infections and occult loaiosis which may constitute a hidden reservoir of disease transmission. The detection of such infections would allow the accurate management of all cases necessary to progress from disease control to

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**25** 

*India* 

**Identification of Genetic Markers** 

*PG & Research Department of Zoology and Biotechnology,* 

 **in Graves' Hyperthyroidism** 

P. Veeramuthumari and W. Isabel

*Lady Doak College, Madurai, Tamil Nadu* 

**Using Polymerase Chain Reaction (PCR)** 

Thyroid is a butterfly shaped gland composed of two encapsulated lobes, located on either side of the trachea just below the cricoid cartilage. This is connected by thin isthmus and is composed of spherical thyroid follicles, which contain the hormone in colloidal form. T3 and T4 are active hormones secreted under the control of TSH from adenohypophysis of pituitary gland. T3 is three to four fold more potent than T4. It is involved in normal growth and development in children temperature regulation, metabolism, energy production and intelligence in both children adults. It ensures normal growth and development of nervous

Fig. 1. Diagrammatic representation of variation of thyroid hormones in hypo and hyper

**1. Introduction** 

system [1].

thyroidism


## **Identification of Genetic Markers Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism**

P. Veeramuthumari and W. Isabel

*PG & Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, Tamil Nadu India* 

## **1. Introduction**

516 Polymerase Chain Reaction

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microfilariae the recognition of which correlates with the amicrofilaremic state in

in human *Loa loa* infection: defective specific proliferation and cytokine production by CD4+ T cells from microfilaremic subjects compared with amicrofilaremics. *Clin.* 

nonhuman primate with Loa loa induces transient strong immune activation followed by peripheral unresponsiveness of helper T cells. *Infection Immunity*. 65 Thyroid is a butterfly shaped gland composed of two encapsulated lobes, located on either side of the trachea just below the cricoid cartilage. This is connected by thin isthmus and is composed of spherical thyroid follicles, which contain the hormone in colloidal form. T3 and T4 are active hormones secreted under the control of TSH from adenohypophysis of pituitary gland. T3 is three to four fold more potent than T4. It is involved in normal growth and development in children temperature regulation, metabolism, energy production and intelligence in both children adults. It ensures normal growth and development of nervous system [1].

Fig. 1. Diagrammatic representation of variation of thyroid hormones in hypo and hyper thyroidism

Identification of Genetic Markers

Fig. 3.

**2. Cytogenetic location of CTLA-4 gene** 

Molecular Location on chromosome 2: base pairs 204,732,510 to 204,738,682

Fig. 4. The CTLA4 gene is located on the long (q) arm of chromosome 2 at position 33.

More precisely, the *CTLA4* gene is located from base pair 204,732,510 to base pair

Cytogenetic Location: 2q33

204,738,682 on chromosome 2.

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 519

The normal range of T4 is suggested to be 77-155nmol/L, T3 to 1.2 -2.8nmol/L ) and TSH to be 0.3-4 mU/L [2]. If the hormone levels are above or below the normal range, it leads to hyperthyroidism or hypothyroidism. The most common hypothyroid condition is Hashimoto's thyroiditis in adults and congenital hypothyroidism in children. Hyperthyroid conditions include Graves' disease, postpartum thyroiditis and thyrotoxicosis factitia.

Hyperthyroidism also leads to a number of complications like heart problems, brittle bones (Osteoporosis), eye problems (Graves' opthalmopathy) (Figure:2).

Fig. 2. Symptoms of Graves' disease

Hypothyroidism describes an under active thyroid gland that is producing low level of thyroid hormone. Hypothyroid patients experience a variety of symptoms, including weight gain, intolerance to cold, goiter (enlarged thyroid), dry coarse, skin, fatigue, constipation, decreased heart rate, poor memory and depression.

The most common form of hyperthyroidism is Graves' disease (GD), an autoimmune disorder accounting for 60-80 % of all cases, in which the antibodies produced by immune system stimulates thyroid gland to produce excess of thyroxine. Normally, the immune system uses antibodies to protect against viruses, bacteria and other foreign substances that enter the body system. In GD, the antibodies mistakenly attack the thyroid gland and occasionally the tissues behind the eyes and the skin of lower legs over the shins. Though the exact cause of GD is not known, several factors including a genetic predisposition are likely to be involved **(Figure:3).** 

GD is an organ specific heterogeneous autoimmune disorder associated with T-lymphocyte abnormality affecting the thyroid eyes and skin. GD is also multifactorial disease that develops as a result of complex interaction between genetic susceptibility genes and environmental factors. Human leucocyte antigen (HLA) and cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) are susceptibility candidates. CTLA\_4 gene plays an important role in the development of GD, which is located on chromosome 2 q33.

Fig. 3.

The normal range of T4 is suggested to be 77-155nmol/L, T3 to 1.2 -2.8nmol/L ) and TSH to be 0.3-4 mU/L [2]. If the hormone levels are above or below the normal range, it leads to hyperthyroidism or hypothyroidism. The most common hypothyroid condition is Hashimoto's thyroiditis in adults and congenital hypothyroidism in children. Hyperthyroid conditions include Graves' disease, postpartum thyroiditis and

Hyperthyroidism also leads to a number of complications like heart problems, brittle bones

Hypothyroidism describes an under active thyroid gland that is producing low level of thyroid hormone. Hypothyroid patients experience a variety of symptoms, including weight gain, intolerance to cold, goiter (enlarged thyroid), dry coarse, skin, fatigue, constipation,

The most common form of hyperthyroidism is Graves' disease (GD), an autoimmune disorder accounting for 60-80 % of all cases, in which the antibodies produced by immune system stimulates thyroid gland to produce excess of thyroxine. Normally, the immune system uses antibodies to protect against viruses, bacteria and other foreign substances that enter the body system. In GD, the antibodies mistakenly attack the thyroid gland and occasionally the tissues behind the eyes and the skin of lower legs over the shins. Though the exact cause of GD is not known, several factors including a genetic predisposition are

GD is an organ specific heterogeneous autoimmune disorder associated with T-lymphocyte abnormality affecting the thyroid eyes and skin. GD is also multifactorial disease that develops as a result of complex interaction between genetic susceptibility genes and environmental factors. Human leucocyte antigen (HLA) and cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) are susceptibility candidates. CTLA\_4 gene plays an

important role in the development of GD, which is located on chromosome 2 q33.

(Osteoporosis), eye problems (Graves' opthalmopathy) (Figure:2).

thyrotoxicosis factitia.

Fig. 2. Symptoms of Graves' disease

likely to be involved **(Figure:3).** 

decreased heart rate, poor memory and depression.

## **2. Cytogenetic location of CTLA-4 gene**

Cytogenetic Location: 2q33

Molecular Location on chromosome 2: base pairs 204,732,510 to 204,738,682

Fig. 4. The CTLA4 gene is located on the long (q) arm of chromosome 2 at position 33. More precisely, the *CTLA4* gene is located from base pair 204,732,510 to base pair 204,738,682 on chromosome 2.

Identification of Genetic Markers

**Polymerase Chain Reaction** 

tool for any molecular biologist.

primer pairs from a template sequence.

**3.1 Guidelines for primer design** 

potential for secondary annealing.

temperature of the product.

regions of homology

**3.2 Software for primer design** 

more each can lead to no amplification.

primers can lead to poor or no yield of the product.

considered:

**3. Primer designing [13]** 

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 521

The polymerase chain reaction (PCR) is a laboratory (in vitro) technique for generating large quantities of a specified DNA. Obviously, PCR is a cell-free amplification technique for synthesizing multiple identical copies (billions) of any DNA of interest, which was developed in 1994 by Karry Mullis (Nobel Prize, 1993). PCR is now considered as a basic

As oligonucleotide primers are useful for polymerase chain reaction (PCR), oligo hybridization and DNA sequencing, proper primer designing is actually one of the most important factors/steps. Various bioinformatics programs are available for selection of

When choosing two PCR amplification primers, the following guidelines should be

**Primer length:** It is accepted that optimal length of PCR primers is 18-22 bp (Wu et al., 1991) **Melting temperature (Tm):** It can be calculated using the formula of Wallace et al., 1997, Tm (oC) = 2(A+T)+4(G+C). The optimal melting temperature for primers ranges between 52- 58oC. Primers with melting temperature above 65oC should also be avoided because of

**Primer annealing temperature:** The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5oC or

() ( ) T 0.3 x T primer 0.7 a m <sup>m</sup> =+ = T primer 14.9

Where, Tm (primer) = Melting Temperature of the primers, Tm (product) = Melting

**GC Content:** Primers should have GC content between 45 and 60 percent. GC content, melting temperature and annealing temperature are strictly dependent on one another

.Dimers and false priming because misleading results: Presence of the secondary structures such as hairpins, self dimer produced by intermolecular or intramolecular interactions in

**Avoid Cross homology:** To improve specificity of the primers it is necessary to avoid

**NETPRIMER** is software used to design and analyze the parameters of designed primer sequences using the following link http://premierbiosoft.com/netprimer/index.html

Activation of T cells requires 2 signals transduced by the antigen specific TCR and co stimulatory ligand such as CD28. CTLA-4, which is expressed on activated T cells, bind to B7 present on antigen presenting cells and functions as a negative regulator of T cell activation. CTLA-4 gene polymorphism confers susceptibility to several autoimmune diseases, such as Graves' disease (GD), Hashimoto's thyroiditis (HT), Addison's disease (AD), Insulin-dependent diabetes mellitus (IDDM), Rheumatoid arthritis (RA) and Multiple sclerosis.

The activity of T cells requires a co stimulatory signal mediated by CD28/B7 interaction. The CTLA-4 gene product delivers a negative signal to T cells and mediates apoptosis. This CTLA-4 gene product is a T cell surface molecule that binds to the B 7 molecule on the antigen presenting cells (APCs). The CTLA-4 gene expression on T cells may affect the course of ongoing immune process. TSH receptor antibody (TRAb) causes Graves' hyperthyroidism.

The GD will go into remission during antithyroid drug (ATD) treatment. Remission of GD is predicted by a smooth decrease in TRAb during (ATD) treatment. Treatment of GD may involve surgery or use of radioactive iodine or use of ATD like propylthiouracil, methimazole and carbimazole. The genetic susceptibility to GD is also conferred by genes in human leucocyte antigen (HLA) and several other genes that are not linked to HLA. The present paper describes the association of GD with the CTLA-4 gene.

The prevalence of hyperthyroidism has been reported to be 3.63% and hypothyroidism to be 2.97% especially the females being more affected by hyperthyroidism [3]. Hence the current study deals with A/G single nucleotide polymorphism (SNP) at position 49 (exon1, codon 17) of the CTLA-4 gene where in Thr/Ala substitution and can be a function related marker. It has been shown to be associated with GD in Caucasians, Japanese, Koreans, Tunisians, Hong Kong Chinese children [2,4,5,6,7,8,9,10] and South Indains [11,12].

The polymorphism cited (A/G polymorphism in exon 1, C/T polymorphism in the promoter, and micro satellite repeat in 3'-untranslated region of exon 4) in CTLA-4 gene have been reported to be associated with autoimmune endocrine disorder.

**A/G polymorphism** at position 49 in exon 1 of the CTLA-4 gene among South Indian population with Graves' hyperthyroidism has revealed the frequencies of the GG genotype and "G" allele to the significantly higher in GD patients. The study has also demonstrated that GD patients had higher frequencies of "G" allele (GG genotype) and lower frequencies of "A" allele (AA genotype) than control group.

Kinjo *et al.,* (2000) have also reported the relationship between the CTLA-4 gene type and severity of the thyroid dysfunction. At diagnosis, free T4 concentrations were shown to be more in patients with the GG genotype and low in patients with the AA genotype. GD patients were reported to have more "G" allele than the control, suggesting that the CTLA-4 GG genotype might induce down regulation of T-cell activation. If the function of CTLA-4 with "G" alleles at position 49 in exon 1 is impaired CTLA-4 function may have d\difficulty in achieving remission.

#### **Identification of SNP**

We can analyze and identify all types of gene SNPs by Polymerase Chain Reaction (PCR) thermal cycler.

520 Polymerase Chain Reaction

Activation of T cells requires 2 signals transduced by the antigen specific TCR and co stimulatory ligand such as CD28. CTLA-4, which is expressed on activated T cells, bind to B7 present on antigen presenting cells and functions as a negative regulator of T cell activation. CTLA-4 gene polymorphism confers susceptibility to several autoimmune diseases, such as Graves' disease (GD), Hashimoto's thyroiditis (HT), Addison's disease (AD), Insulin-dependent diabetes mellitus (IDDM), Rheumatoid arthritis (RA) and Multiple

The activity of T cells requires a co stimulatory signal mediated by CD28/B7 interaction. The CTLA-4 gene product delivers a negative signal to T cells and mediates apoptosis. This CTLA-4 gene product is a T cell surface molecule that binds to the B 7 molecule on the antigen presenting cells (APCs). The CTLA-4 gene expression on T cells may affect the course of ongoing immune process. TSH receptor antibody (TRAb) causes Graves'

The GD will go into remission during antithyroid drug (ATD) treatment. Remission of GD is predicted by a smooth decrease in TRAb during (ATD) treatment. Treatment of GD may involve surgery or use of radioactive iodine or use of ATD like propylthiouracil, methimazole and carbimazole. The genetic susceptibility to GD is also conferred by genes in human leucocyte antigen (HLA) and several other genes that are not linked to HLA. The

The prevalence of hyperthyroidism has been reported to be 3.63% and hypothyroidism to be 2.97% especially the females being more affected by hyperthyroidism [3]. Hence the current study deals with A/G single nucleotide polymorphism (SNP) at position 49 (exon1, codon 17) of the CTLA-4 gene where in Thr/Ala substitution and can be a function related marker. It has been shown to be associated with GD in Caucasians, Japanese, Koreans, Tunisians,

The polymorphism cited (A/G polymorphism in exon 1, C/T polymorphism in the promoter, and micro satellite repeat in 3'-untranslated region of exon 4) in CTLA-4 gene

**A/G polymorphism** at position 49 in exon 1 of the CTLA-4 gene among South Indian population with Graves' hyperthyroidism has revealed the frequencies of the GG genotype and "G" allele to the significantly higher in GD patients. The study has also demonstrated that GD patients had higher frequencies of "G" allele (GG genotype) and lower frequencies

Kinjo *et al.,* (2000) have also reported the relationship between the CTLA-4 gene type and severity of the thyroid dysfunction. At diagnosis, free T4 concentrations were shown to be more in patients with the GG genotype and low in patients with the AA genotype. GD patients were reported to have more "G" allele than the control, suggesting that the CTLA-4 GG genotype might induce down regulation of T-cell activation. If the function of CTLA-4 with "G" alleles at position 49 in exon 1 is impaired CTLA-4 function may have d\difficulty

We can analyze and identify all types of gene SNPs by Polymerase Chain Reaction (PCR)

present paper describes the association of GD with the CTLA-4 gene.

Hong Kong Chinese children [2,4,5,6,7,8,9,10] and South Indains [11,12].

have been reported to be associated with autoimmune endocrine disorder.

of "A" allele (AA genotype) than control group.

in achieving remission. **Identification of SNP** 

thermal cycler.

sclerosis.

hyperthyroidism.

The polymerase chain reaction (PCR) is a laboratory (in vitro) technique for generating large quantities of a specified DNA. Obviously, PCR is a cell-free amplification technique for synthesizing multiple identical copies (billions) of any DNA of interest, which was developed in 1994 by Karry Mullis (Nobel Prize, 1993). PCR is now considered as a basic tool for any molecular biologist.

## **3. Primer designing [13]**

As oligonucleotide primers are useful for polymerase chain reaction (PCR), oligo hybridization and DNA sequencing, proper primer designing is actually one of the most important factors/steps. Various bioinformatics programs are available for selection of primer pairs from a template sequence.

## **3.1 Guidelines for primer design**

When choosing two PCR amplification primers, the following guidelines should be considered:

**Primer length:** It is accepted that optimal length of PCR primers is 18-22 bp (Wu et al., 1991)

**Melting temperature (Tm):** It can be calculated using the formula of Wallace et al., 1997, Tm (oC) = 2(A+T)+4(G+C). The optimal melting temperature for primers ranges between 52- 58oC. Primers with melting temperature above 65oC should also be avoided because of potential for secondary annealing.

**Primer annealing temperature:** The two primers of a primer pair should have closely matched melting temperatures for maximizing PCR product yield. The difference of 5oC or more each can lead to no amplification.

() ( ) T 0.3 x T primer 0.7 a m <sup>m</sup> =+ = T primer 14.9

Where, Tm (primer) = Melting Temperature of the primers, Tm (product) = Melting temperature of the product.

**GC Content:** Primers should have GC content between 45 and 60 percent. GC content, melting temperature and annealing temperature are strictly dependent on one another

.Dimers and false priming because misleading results: Presence of the secondary structures such as hairpins, self dimer produced by intermolecular or intramolecular interactions in primers can lead to poor or no yield of the product.

**Avoid Cross homology:** To improve specificity of the primers it is necessary to avoid regions of homology

## **3.2 Software for primer design**

**NETPRIMER** is software used to design and analyze the parameters of designed primer sequences using the following link http://premierbiosoft.com/netprimer/index.html

Identification of Genetic Markers

Fig. 5.

shapes.

**3.4 Genetic marker [15,16,17,18]** 

**3.5 Some commonly used types of genetic markers**  RFLP (or Restriction fragment length polymorphism) SSLP (or Simple sequence length polymorphism) AFLP (or Amplified fragment length polymorphism) RAPD (or Random amplification of polymorphic DNA)

VNTR (or Variable number tandem repeat)

**Optimization of MgCl2 concentration:** 

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 523

Magnesium chloride is an essential component for PCR. It is a cofactor for Taq DNA polymerase. Mg++ promotes DNA/DNA interactions and forms complexes with dNTPs that are the actual substrates for Taq polymerase. When Mg++ is too low, primers fail to anneal to the target DNA. When Mg++ is too high, the base pairing becomes too strong and the amplicon fails to denature completely when you heat 94oC. MgCl2 concentration should be optimized for every PCR reaction. All the components of the reaction mixture can bind to magnesium ion, including primers, template, PCR products and dNTPs. Therefore, the concentration of MgCl2 has to be optimized for a new PCR. The most commonly used concentration of MgCl2 is 1.5mM and it can be optimized empirically between 1.5 and 4.0mM.

A **genetic marker** is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described and observed as a variation which may arise due to mutation or alteration in the genomic loci. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites. For many years, gene mapping was limited in most organisms by traditional genetic markers which include genes that encode easily observable characteristics such as blood types or seed

## **3.3 PCR standardization [13,14]**

PCR is a revolutionary technique used in almost all molecular biology experiments. In PCR, the repeated three-step process of denaturation, primer annealing and DNA polymerase extension results in exponential amplification of target DNA. Initially PCR was reported with E.Coli DNA polymerase Klenow fragment in 1985. In 1988, the first report on PCR using thermostable Taq DNA polymerase was published. Since then PCR has been extensively modified and used for various applications such as cloning, sequencing, sitedirected mutagenesis, diagnostics, genotyping, genome walking, amplification of RNA after reverse transcription for gene expression analysis amplification of a whole genome, etc.

The central components of a PCR reaction are oligonucleotide primers, thermostable DNA polymerase, target DNA, dNTPs and reaction buffer including MgCl2. When a new PCR has to be developed, suitable primer pairs should be designed based on the target sequence,. Subsequently, the concentration of PCR components and the cycling conditions should be optimized.

#### **Thermostable enzymes:**

Thermostable enzymes should be selected based on the applications. High fidelity Taq DNA polymerase and proofreading recombinant enzymes are required for the amplification of more than 3 kb target sequence. For a standard PCR, 2 to 5 units of Taq DNA polymerase are recommended for a typical 100µl PCR.

#### **Deoxynucleoside triphosphate (dNTPs):**

For a standard PCR, 100 to 200 µM concentrations of dNTPs is used. The balanced solutions of all four nucleotides should be used to minimize the error frequency. The concentrations may be increased for Multiplex PCR and Repetitive PCR, where more than one PCR amplicons are expected.

#### **Template DNA:**

The purity and concentration of the template DNA are critical for a successful PCR amplification. For initial experiments, 0.1 to 200ng of the template DNA, based on the type can be used. For example, if it is a plasmid 0.1 to 1ng is sufficient. If the template is human genomic DNA, upto 200ng can be used.

#### **Primer concentrations:**

The primer concentration can affect the PCR. If the primer concentration is too low, amplifications will be failed; and if the concentration is too high, non-specific amplification will occur. Therfore, the primer concentration should be optimized empirically between 0.1 to 1µM final concentrations. The most straightforward way of optimizing a PCR with a given primer pair is to change the concentration of MgCl2 or the annealing temperature or both.

#### **Optimization of primer annealing temperature:**

Optimization of the primer annealing temperature is the most critical step in PCR. The primer designing programs will suggest the Tm of the primers. In general, the annealing temperature should be set 2 to 5oC below the Tm of the primers. However, some oligonucleotides may not work optically at this temperature and hence the annealing temperature should be optimized using gradient PCR approach.

## **Optimization of MgCl2 concentration:**

Magnesium chloride is an essential component for PCR. It is a cofactor for Taq DNA polymerase. Mg++ promotes DNA/DNA interactions and forms complexes with dNTPs that are the actual substrates for Taq polymerase. When Mg++ is too low, primers fail to anneal to the target DNA. When Mg++ is too high, the base pairing becomes too strong and the amplicon fails to denature completely when you heat 94oC. MgCl2 concentration should be optimized for every PCR reaction. All the components of the reaction mixture can bind to magnesium ion, including primers, template, PCR products and dNTPs. Therefore, the concentration of MgCl2 has to be optimized for a new PCR. The most commonly used concentration of MgCl2 is 1.5mM and it can be optimized empirically between 1.5 and 4.0mM.

Fig. 5.

522 Polymerase Chain Reaction

PCR is a revolutionary technique used in almost all molecular biology experiments. In PCR, the repeated three-step process of denaturation, primer annealing and DNA polymerase extension results in exponential amplification of target DNA. Initially PCR was reported with E.Coli DNA polymerase Klenow fragment in 1985. In 1988, the first report on PCR using thermostable Taq DNA polymerase was published. Since then PCR has been extensively modified and used for various applications such as cloning, sequencing, sitedirected mutagenesis, diagnostics, genotyping, genome walking, amplification of RNA after reverse transcription for gene expression analysis amplification of a whole genome, etc.

The central components of a PCR reaction are oligonucleotide primers, thermostable DNA polymerase, target DNA, dNTPs and reaction buffer including MgCl2. When a new PCR has to be developed, suitable primer pairs should be designed based on the target sequence,. Subsequently, the concentration of PCR components and the cycling conditions should be

Thermostable enzymes should be selected based on the applications. High fidelity Taq DNA polymerase and proofreading recombinant enzymes are required for the amplification of more than 3 kb target sequence. For a standard PCR, 2 to 5 units of Taq DNA polymerase

For a standard PCR, 100 to 200 µM concentrations of dNTPs is used. The balanced solutions of all four nucleotides should be used to minimize the error frequency. The concentrations may be increased for Multiplex PCR and Repetitive PCR, where more than one PCR

The purity and concentration of the template DNA are critical for a successful PCR amplification. For initial experiments, 0.1 to 200ng of the template DNA, based on the type can be used. For example, if it is a plasmid 0.1 to 1ng is sufficient. If the template is human

The primer concentration can affect the PCR. If the primer concentration is too low, amplifications will be failed; and if the concentration is too high, non-specific amplification will occur. Therfore, the primer concentration should be optimized empirically between 0.1 to 1µM final concentrations. The most straightforward way of optimizing a PCR with a given primer pair is to change the concentration of MgCl2 or the annealing temperature or both.

Optimization of the primer annealing temperature is the most critical step in PCR. The primer designing programs will suggest the Tm of the primers. In general, the annealing temperature should be set 2 to 5oC below the Tm of the primers. However, some oligonucleotides may not work optically at this temperature and hence the annealing

**3.3 PCR standardization [13,14]** 

optimized.

**Thermostable enzymes:** 

amplicons are expected.

**Primer concentrations:** 

**Template DNA:** 

are recommended for a typical 100µl PCR. **Deoxynucleoside triphosphate (dNTPs):** 

genomic DNA, upto 200ng can be used.

**Optimization of primer annealing temperature:** 

temperature should be optimized using gradient PCR approach.

## **3.4 Genetic marker [15,16,17,18]**

A **genetic marker** is a gene or DNA sequence with a known location on a chromosome that can be used to identify cells, individuals or species. It can be described and observed as a variation which may arise due to mutation or alteration in the genomic loci. A genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change (single nucleotide polymorphism, SNP), or a long one, like minisatellites. For many years, gene mapping was limited in most organisms by traditional genetic markers which include genes that encode easily observable characteristics such as blood types or seed shapes.

## **3.5 Some commonly used types of genetic markers**

RFLP (or Restriction fragment length polymorphism) SSLP (or Simple sequence length polymorphism) AFLP (or Amplified fragment length polymorphism) RAPD (or Random amplification of polymorphic DNA) VNTR (or Variable number tandem repeat)

Identification of Genetic Markers

**4.2 Restriction digestion** 

proportionately.

**4.3 Results** 

electrophoresis **(Figure 8).** 

(13.75%) and A allele (37.5 %) (Table 1).

were obtained. This was confirmed by 2% agarose gel.

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 525

oligonucleotide primers (Forward, 5' – GCTCTACTTCCTGAAGACCT – 3' and Revers, 5' – AGTCTCACTCACCTTTGCAG – 5')[2]. PCR was performed by initial denaturation 30 sec for 5 min. annealing for 45 sec at 57oC, extension for 30 sec at 72oC, denaturation 30 sec at 94oC (for 20 cycles) and final extension for 7 min at 72oC. The PCR product was confirmed by agarose (1.8%) gel electrophoresis. The presence of G alleles was determined in each subject by PCR amplification of CTLA-4, followed by diffusion with *Bbv1,* which acts on the G variation, but not on the A variation. It a G allele was at position 49, 88/74 bp fragments

The amplified CTLA-4 gene should be digested with the restriction enzyme *Bbv1,*which is commercially available. A typical 30µl reaction mix was used . Modify the required volume

> PCR amplified product – 20. 0µl 10x buffer - 3.0µl Bbv1(10units/ul) - 1.0µl Deionized water - 6.0µl Total - 30.0µl

Incubated the reaction mixture at 37OC for 4 hrs and inactivated by heating at 70oC for 10

The presence of genomic DNA confirmed by subjecting the agarose gel electrophoresis (0.7%) **(Figure 6).** The genomic DNA was then subjected to PCR and 162 bp fragments were obtained **(Figure 7).** The amplified PCR product digested with enzyme *Bbv***1, t**he restriction enzyme acts on the G variation, but not on the A variation. If a G allele was at position 49, 88bp and 74bp fragments were obtained and the fragments were detected by 2% agarose gel

In the present study, the G/G genotype was observed in 32 (40 %) GD patients and in 26 (32.50 %) individuals of the control group, A/G genotype was found in 37 (46.25 %) patients and in 25 (31.25 %) persons of the control group, A/A genotype was observed in 11 (13.75 %) patients and in 29 (36.25 %) persons of the control group and G allele was found in 50 (62.5%) GD patients and in 38 (47.5 %) persons of the control group, and A allele was found in 30 (37.5 %) GD patients and 42 persons (52.5%) of the control group **(Table 1)**. There was significant difference (p <0.05) in genotype and allelic frequency between the control group and GD patients. The present study also demonstrates an association between the CTLA-4 gene polymorphism in Graves' disease and with the remission rate of Graves' hyperthyroidism. Among the GD cases studied, only 2% had remission. The frequencies of GG genotype (40 %) and G allele (62.5%) were higher when compared to A/A genotype

min. The product was confirmed using 2% agarose gel electrophoresis.

Microsatellite polymorphism, SSR (or Simple sequence repeat) SNP (or Single nucleotide polymorphism) STR (or Short tandem repeat) SFP (or Single feature polymorphism) DArT (or Diversity Arrays Technology) RAD markers (or Restriction site associated DNA markers) They can be further categorized as dominant or co-dominant.

**Dominant markers** allow for analyzing many loci at one time, e.g. RAPD. A primer amplifying a dominant marker could amplify at many loci in one sample of DNA with one PCR reaction. The dominant markers, as RAPDs and high-efficiency markers (like AFLPs and SMPLs), allow the analysis of many loci per experiment within requiring previous information about their sequence.

**Co-dominant markers** analyze one locus at a time. A primer amplifying a co-dominant marker would yield one targeted product. so they are more informative because the allelic variations of that locus can be distinguished. As a consequence, we can identify linkage groups between different genetic maps but, for their development it is necessary to know the sequence (which is still expensive and is considered one of their down sides). **Eg.** RFLPs, microsatellites, etc.,

## **3.6 Uses of genetic markers**


Hence, SNP (Single nucleotide polymorphism) in Graves' hyperthyroidism is used as marker to identify which mutation is responsible for causing GD and other hereditary diseases.

## **4. Analysis of CTLA-4 A/G polymorphism among South Indian population**

#### **4.1 Protocol used for A/G single nucleotide polymorphism (SNP) study in Graves' disease**

Genomic DNA was prepared from peripheral white cells using standardized protocol. We have analysed CTLA -4 genotypes and allele with PCR. PCR was performed with oligonucleotide primers (Forward, 5' – GCTCTACTTCCTGAAGACCT – 3' and Revers, 5' – AGTCTCACTCACCTTTGCAG – 5')[2]. PCR was performed by initial denaturation 30 sec for 5 min. annealing for 45 sec at 57oC, extension for 30 sec at 72oC, denaturation 30 sec at 94oC (for 20 cycles) and final extension for 7 min at 72oC. The PCR product was confirmed by agarose (1.8%) gel electrophoresis. The presence of G alleles was determined in each subject by PCR amplification of CTLA-4, followed by diffusion with *Bbv1,* which acts on the G variation, but not on the A variation. It a G allele was at position 49, 88/74 bp fragments were obtained. This was confirmed by 2% agarose gel.

## **4.2 Restriction digestion**

524 Polymerase Chain Reaction

**Dominant markers** allow for analyzing many loci at one time, e.g. RAPD. A primer amplifying a dominant marker could amplify at many loci in one sample of DNA with one PCR reaction. The dominant markers, as RAPDs and high-efficiency markers (like AFLPs and SMPLs), allow the analysis of many loci per experiment within requiring previous

**Co-dominant markers** analyze one locus at a time. A primer amplifying a co-dominant marker would yield one targeted product. so they are more informative because the allelic variations of that locus can be distinguished. As a consequence, we can identify linkage groups between different genetic maps but, for their development it is necessary to know the sequence (which is still expensive and is considered one of their down sides). **Eg.** RFLPs,

• Genetic markers can be used to study the relationship between an inherited disease and its genetic cause (for example, a particular mutation of a gene that results in a defective protein). It is known that pieces of DNA that lie near each other on a chromosome tend to be inherited together. This property enables the use of a marker, which can then be used to determine the precise inheritance pattern of the gene that has not yet been

• Genetic markers have to be easily identifiable, associated with a specific locus and

• Natural and artificial selection leads to a change in the genetic makeup of the cell. The presence of different alleles due to a distorted segregation at the genetic markers is

Hence, SNP (Single nucleotide polymorphism) in Graves' hyperthyroidism is used as marker to identify which mutation is responsible for causing GD and other hereditary

Genomic DNA was prepared from peripheral white cells using standardized protocol. We have analysed CTLA -4 genotypes and allele with PCR. PCR was performed with

**4. Analysis of CTLA-4 A/G polymorphism among South Indian population 4.1 Protocol used for A/G single nucleotide polymorphism (SNP) study in Graves'** 

highly polymorphic, because homozygotes do not provide any information. • Detection of the marker can be direct by RNA sequencing, or indirect using allozymes. • Genetic Markers have also been used to measure the genomic response to selection in

indicative of the difference between selected and non-selected livestock.

Microsatellite polymorphism, SSR (or Simple sequence repeat)

RAD markers (or Restriction site associated DNA markers) They can be further categorized as dominant or co-dominant.

SNP (or Single nucleotide polymorphism)

SFP (or Single feature polymorphism) DArT (or Diversity Arrays Technology)

information about their sequence.

microsatellites, etc.,

**3.6 Uses of genetic markers** 

exactly localized.

livestock.

diseases.

**disease** 

STR (or Short tandem repeat)

The amplified CTLA-4 gene should be digested with the restriction enzyme *Bbv1,*which is commercially available. A typical 30µl reaction mix was used . Modify the required volume proportionately.


Incubated the reaction mixture at 37OC for 4 hrs and inactivated by heating at 70oC for 10 min. The product was confirmed using 2% agarose gel electrophoresis.

## **4.3 Results**

The presence of genomic DNA confirmed by subjecting the agarose gel electrophoresis (0.7%) **(Figure 6).** The genomic DNA was then subjected to PCR and 162 bp fragments were obtained **(Figure 7).** The amplified PCR product digested with enzyme *Bbv***1, t**he restriction enzyme acts on the G variation, but not on the A variation. If a G allele was at position 49, 88bp and 74bp fragments were obtained and the fragments were detected by 2% agarose gel electrophoresis **(Figure 8).** 

In the present study, the G/G genotype was observed in 32 (40 %) GD patients and in 26 (32.50 %) individuals of the control group, A/G genotype was found in 37 (46.25 %) patients and in 25 (31.25 %) persons of the control group, A/A genotype was observed in 11 (13.75 %) patients and in 29 (36.25 %) persons of the control group and G allele was found in 50 (62.5%) GD patients and in 38 (47.5 %) persons of the control group, and A allele was found in 30 (37.5 %) GD patients and 42 persons (52.5%) of the control group **(Table 1)**. There was significant difference (p <0.05) in genotype and allelic frequency between the control group and GD patients. The present study also demonstrates an association between the CTLA-4 gene polymorphism in Graves' disease and with the remission rate of Graves' hyperthyroidism. Among the GD cases studied, only 2% had remission. The frequencies of GG genotype (40 %) and G allele (62.5%) were higher when compared to A/A genotype (13.75%) and A allele (37.5 %) (Table 1).

Identification of Genetic Markers

Genotype

detected by 2% Agarose gel electrophoresis**.**

contribute to the development of Graves' hyperthyroidism.

**4.4 Discussion** 

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 527

In the present study genomic DNA was isolated from patients and control groups and was subjected to Agarose gel electrophoresis (0.7%)**.** This enables easy visualization of DNA band patterns. After confirming the presence of genomic DNA, it was subjected to PCR and 162 bp fragments were obtained**.** The amplified PCR product was digested with enzyme *Bbv***1.** The restriction enzyme acts on the G variation, but not on the A variation. If a G allele was at position 49, 88bp and 74bp two fragments were obtained. The PCR products were

A/G polymorphism at position 49 in exon 1 of the CTLA-4 gene among Madurai population with Graves' hyperthyroidism revealed that the frequencies of the GG genotype and G allele were significantly higher in GD patients. This study has also revealed lower frequency (or absence) of A allele (AA genotype) than the control. CTLA-4 gene polymorphism has been reported to be associated with GD. CTLA-4 molecule is a member of the family of cell surface molecule CD28, which binds to B7. The CTLA-4/B7 complex competes with the CD28/B7 complex and delivers negative signals to the T-cells, which affects T-cell expansion, cytokine production, and immune responses as evidenced by Park *et.al.*[6] in Korean population, Yanagawa *et al.* [9] in Japanese population and Yanagawa *et al*. [8] in Caucasian population. However, we do not know how CTLA-4 gene polymorphisms may

Three polymorphism sites (A/G polymorphism in exon 1; C/T polymorphism in the promoter, and micro satellite repeat in the 3'-untranslated region of exon 4) in the CTLA-4 gene have been reported to be associated with autoimmune endocrine disorders. Kinjo *et. al*., [2] have reported the relationship between the CTLA-4 gene type and severity of the thyroid dysfunction. At diagnosis, free T4 concentrations were shown to be highest in patients with the GG genotype and lowest in patients with the AA genotype. GD patients have more G allele than control, suggesting that the CTLA-4 GG genotype might induce down regulation of T-cell activation. If the function of CTLA-4 with the G alleles at position 49 in exon 1 was impaired CTLA-4 function might have difficulty in achieving remission.

> **Controls % (**n = 110)

**Graves' Disease % (**n = 144)

of CTLA-4 gene in GD patients and controls among Japanese –population. [2]

G/G 50 (34.7) 26 (23.6) A/G 62 (43.1) 46 (41.8) A/A 32 (22.2) 38 (43.6)

Table 3. Frequency of the genotype and allele of A/G polymorphism at position 49 in exon 1

Bednarczuk *et. al.,* [4] analysed the association of CTLA-4 A49G polymorphism with Graves' disease in Caucasian and Japanese population. Their study also reveals that, CTLA –4 G allele and G/G genotype confer genetic susceptibility to GD in Caucasian and Japanese population. The study of Kouki *et. al*., [5] among patients with GD revealed there were more individuals with G/G (17.8 %GD vs 11.6% of controls) or A/G CTLA-4 exon 1 genotypes (64.4 % GD vs 53.5% control) and significantly fewer individuals with the A/A alleles (17.8 %GD vs 43.9

Fig. 6. Confirmation of human genomic DNA

Fig. 7. CTLA-4 gene amplification


Fig. 8. Restriction analysis of CTLA-4 gene


Table 2. Prevalence of CTLA-4 gene genotype and allele frequency among South Indian

### **4.4 Discussion**

526 Polymerase Chain Reaction

Fig. 6. Confirmation of human genomic DNA

Fig. 7. CTLA-4 gene amplification

Fig. 8. Restriction analysis of CTLA-4 gene

**Allele** 

**GENOTYPE GD patient (n=80)** *Control group* 

**G/G** 32 (40%) 26 (32.50%) **A/G** 37 (46.25%) 25 (31.25%) **A/A** 11 (13.75%) 29 (36.25%)

**G** 50 (62.5%) 38 (47.5%) **A** 30 (37.5%) 42 (52.5%) Table 2. Prevalence of CTLA-4 gene genotype and allele frequency among South Indian

**(n=80)**

In the present study genomic DNA was isolated from patients and control groups and was subjected to Agarose gel electrophoresis (0.7%)**.** This enables easy visualization of DNA band patterns. After confirming the presence of genomic DNA, it was subjected to PCR and 162 bp fragments were obtained**.** The amplified PCR product was digested with enzyme *Bbv***1.** The restriction enzyme acts on the G variation, but not on the A variation. If a G allele was at position 49, 88bp and 74bp two fragments were obtained. The PCR products were detected by 2% Agarose gel electrophoresis**.**

A/G polymorphism at position 49 in exon 1 of the CTLA-4 gene among Madurai population with Graves' hyperthyroidism revealed that the frequencies of the GG genotype and G allele were significantly higher in GD patients. This study has also revealed lower frequency (or absence) of A allele (AA genotype) than the control. CTLA-4 gene polymorphism has been reported to be associated with GD. CTLA-4 molecule is a member of the family of cell surface molecule CD28, which binds to B7. The CTLA-4/B7 complex competes with the CD28/B7 complex and delivers negative signals to the T-cells, which affects T-cell expansion, cytokine production, and immune responses as evidenced by Park *et.al.*[6] in Korean population, Yanagawa *et al.* [9] in Japanese population and Yanagawa *et al*. [8] in Caucasian population. However, we do not know how CTLA-4 gene polymorphisms may contribute to the development of Graves' hyperthyroidism.

Three polymorphism sites (A/G polymorphism in exon 1; C/T polymorphism in the promoter, and micro satellite repeat in the 3'-untranslated region of exon 4) in the CTLA-4 gene have been reported to be associated with autoimmune endocrine disorders. Kinjo *et. al*., [2] have reported the relationship between the CTLA-4 gene type and severity of the thyroid dysfunction. At diagnosis, free T4 concentrations were shown to be highest in patients with the GG genotype and lowest in patients with the AA genotype. GD patients have more G allele than control, suggesting that the CTLA-4 GG genotype might induce down regulation of T-cell activation. If the function of CTLA-4 with the G alleles at position 49 in exon 1 was impaired CTLA-4 function might have difficulty in achieving remission.



Bednarczuk *et. al.,* [4] analysed the association of CTLA-4 A49G polymorphism with Graves' disease in Caucasian and Japanese population. Their study also reveals that, CTLA –4 G allele and G/G genotype confer genetic susceptibility to GD in Caucasian and Japanese population.

The study of Kouki *et. al*., [5] among patients with GD revealed there were more individuals with G/G (17.8 %GD vs 11.6% of controls) or A/G CTLA-4 exon 1 genotypes (64.4 % GD vs 53.5% control) and significantly fewer individuals with the A/A alleles (17.8 %GD vs 43.9

Identification of Genetic Markers

Fig. 10. Restriction digestion PKD1 gene

genetic disorders.

**7. References** 

screen. 17-21.

148:13-18.

Immunol. 165:6606-6611.

**6. Acknowledgement** 

Using Polymerase Chain Reaction (PCR) in Graves' Hyperthyroidism 529

Hope this chapter will provide an insight on genetic screening of different disease and

We would like to acknowledge Gunasekaran P, Dr.Sujatha K, Dr.Mahalakshmi A, UGC-NRCBS, School of Biological Science, Madurai Kamaraj University, Madurai, for valuable guidance in standardization of PCR and Primer designing. And also we like to acknowledge Dr.R. Shenbagarathai & faculty in PG & Research Department of Zoology and

[2] Kinjo Y., Takasu N., Komiya I., Tomoyose T., Takara M., Kouki T., Shimajiri Y., Yabiku

[3] Velumani A., Kadival GV., Nirmala R. and Lele RD. (2005) Hyperthyroidism. Health

[4] Bednarczuk T., Hiromatsu Y., Fukutani T., Jazdzewski K., Miskiewicz P., Osikowska M.

[5] Kouki T., Sawai Y., Gardine C.A., Fisfalen M-E., Alegre M-L. and Degroot L.J. (2000)

[6] Park Y.J., Chung H.K., Park D.J., Kim W.B., Kim S.W., Koh J.J. and Cho B.Y. (2000)

[7] Vaidya B., Imrie H. and Perros P. (1999) The cytotoxic T-lymphocyte antigen –4 is a

major Graves disease locus. Hum. Mol. Genet. 8:1195-99.

molecule-4 gene. J. of clin. Endocrinol. and Metab. 87(6): 2593-2596.

K., and Yoshimura H. (2002) Remission of Graves' hyperthyroidism and A/G polymorphism at position 49 in exon 1 of Cytotoxic T- lymphocyte-associated

and Nauman J. (2003) Association of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) gene polymorphism and non-genetic factors with Graves' ophthalmophathy in European and Japanese populations. European J. of Endocrin.

CTLA-4 gene polymorphism at Position 49 in Exon 1 reduces the inhibitory function of CTLA-4 and Contributes to the Pathogens of Graves, disease. The J. of

Polymorphism in the promotor and exon 1 of the cytotoxic T lymphocyte antigen-4 gene associated with autoimmune thyroid disease in Koreans. Thyroid. 10:453 –459.

Biotechnology, Lady Doak College, Madurai, for their continuous support.

[1] Guyton. (1991) Text book of medical physiology, 1091 – 95.

%control) when compared with controls. According to their findings, the frequency of the G allele was higher in GD patients (50%) than in controls (38.4%) in their population.

There was significant difference between the control group and GD patients both in genotype and allelic frequency. Therefore, in accordance with previously published results, the present study also demonstrates an association between the CTLA-4 gene polymorphism in Graves' disease and with the remission rate of Graves' hyperthyroidism. Among the GD cases studied, only 2% had remission and the frequencies of GG genotype and G allele were higher when compared to A/A genotype and A allele. GD patient with G allele in exon 1 of the CTLA-4 gene were required to continue Anti thyroid drug (ATD) treatment [19] for longer periods to achieve remission. Further studies will be required to determine a clear association of the CTLA-4 gene polymorphism with the remission of GD.

We have studied another gene polymorphism called PKD1 (C/T) at position 4058 in exon 45 which is responsible for causing autosomal polycystic kidney disease (ADPKD) among South Indian.

## **5. Short summary of C/T polymorphism in PKD1 gene**

Polycystic kidney disease (PKD) is a group of monogenic disorders that result in renal cyst development in kidney leads to kidney failure. Autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) are two forms of PKD, which are largely limited to the kidney and liver, which extends from neonates to old age. ADPKD is a commonly inherited disorder in humans, with a frequency among the general population of 1 in 500. ADPKD caused by mutations in PKD1 gene (85%) located on human chromosome 16p13.3; the remaining 15% are caused by mutations in the PKD2 gene, located on human chromosome 4q21-23. A total of 60 ADPKD patients among South Indian (Madurai) population were analyzed. In genetic study, the genomic DNA was isolated, which would be subjects into PCR (Figure:9) and RFLP analysis (Figure:10). C/T polymorphism at position 4058 in exon 45 of the PKD1 gene among South Indian (Madurai) population with ADPKD revealed that the "TT" "CT' genotype and the frequency of "T" allele was found be significantly (at p=0.001) higher in the patients compared to control subjects. The study was demonstrated that ADPKD patients had higher frequencies of "T" allele and lower frequency of "C" allele than control subjects. The present study also has been supported by Constantinides *et al.,*[20]. Therefore, the study reveals that there was an association of C/T polymorphism in ADPKD and the prevalence of ADPKD among South Indian (Madurai) population.

Fig. 9. PKD1 gene amplification

Fig. 10. Restriction digestion PKD1 gene

Hope this chapter will provide an insight on genetic screening of different disease and genetic disorders.

## **6. Acknowledgement**

528 Polymerase Chain Reaction

%control) when compared with controls. According to their findings, the frequency of the G

There was significant difference between the control group and GD patients both in genotype and allelic frequency. Therefore, in accordance with previously published results, the present study also demonstrates an association between the CTLA-4 gene polymorphism in Graves' disease and with the remission rate of Graves' hyperthyroidism. Among the GD cases studied, only 2% had remission and the frequencies of GG genotype and G allele were higher when compared to A/A genotype and A allele. GD patient with G allele in exon 1 of the CTLA-4 gene were required to continue Anti thyroid drug (ATD) treatment [19] for longer periods to achieve remission. Further studies will be required to determine a clear association of the CTLA-4 gene polymorphism with the remission of GD. We have studied another gene polymorphism called PKD1 (C/T) at position 4058 in exon 45 which is responsible for causing autosomal polycystic kidney disease (ADPKD) among

Polycystic kidney disease (PKD) is a group of monogenic disorders that result in renal cyst development in kidney leads to kidney failure. Autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD) are two forms of PKD, which are largely limited to the kidney and liver, which extends from neonates to old age. ADPKD is a commonly inherited disorder in humans, with a frequency among the general population of 1 in 500. ADPKD caused by mutations in PKD1 gene (85%) located on human chromosome 16p13.3; the remaining 15% are caused by mutations in the PKD2 gene, located on human chromosome 4q21-23. A total of 60 ADPKD patients among South Indian (Madurai) population were analyzed. In genetic study, the genomic DNA was isolated, which would be subjects into PCR (Figure:9) and RFLP analysis (Figure:10). C/T polymorphism at position 4058 in exon 45 of the PKD1 gene among South Indian (Madurai) population with ADPKD revealed that the "TT" "CT' genotype and the frequency of "T" allele was found be significantly (at p=0.001) higher in the patients compared to control subjects. The study was demonstrated that ADPKD patients had higher frequencies of "T" allele and lower frequency of "C" allele than control subjects. The present study also has been supported by Constantinides *et al.,*[20]. Therefore, the study reveals that there was an association of C/T polymorphism in ADPKD and the prevalence of ADPKD among South

allele was higher in GD patients (50%) than in controls (38.4%) in their population.

**5. Short summary of C/T polymorphism in PKD1 gene** 

South Indian.

Indian (Madurai) population.

Fig. 9. PKD1 gene amplification

We would like to acknowledge Gunasekaran P, Dr.Sujatha K, Dr.Mahalakshmi A, UGC-NRCBS, School of Biological Science, Madurai Kamaraj University, Madurai, for valuable guidance in standardization of PCR and Primer designing. And also we like to acknowledge Dr.R. Shenbagarathai & faculty in PG & Research Department of Zoology and Biotechnology, Lady Doak College, Madurai, for their continuous support.

## **7. References**


**26** 

**Detection of Bacterial Pathogens** 

C. N. Wose Kinge1, M. Mbewe2 and N. P. Sithebe1

*North-West University, Mafikeng Campus, Mmabatho,* 

*Mafikeng Campus, Mmabatho* 

*South Africa* 

**in River Water Using Multiplex-PCR** 

*1Department of Biological Sciences, School of Environmental and Health Sciences,* 

*2Animal Health Programme, School of Agricultural Sciences, North-West University,* 

The aquatic environments receive a signicant number of human microbial pathogens from point and non-point sources of pollution. Point-source pollution enters the environment at different locations, through a direct route of discharge of treated or untreated domestic sewage, industrial effluent and acid mine drainage (State of the Environment Report [SER], 2002). Non-point (or diffuse) sources of pollution comprises up to 80 % of the pollution entering major river systems thus are of signicant concern with respect to the dissemination of pathogens and their indicators in water systems. They may be attributable to the run-off from urban and agricultural areas, leakage from sewers and septic systems, insecticides and herbicides from agricultural land, and sewer overows (Stewart et al., 2008). Although majority of pathogenic microbes can be eliminated by sewage treatment, many end up in the effluent which is then discharged into receiving bodies of water. These pathogenic microbes have been implicated in human diseases linked with the use of contaminated water and food. Adequate sanitation and clean water, being two critical factors in ensuring human health, protects against a wide range of water-related diseases. These include diarrhoea, cholera, trachoma, dysentery, typhoid, hepatitis, polio, malaria, and filariasis (United Nations Department of Public Information

Water is a vital natural resource because of its basic role to life, quality of life, the environment, food production, hygiene, industry, and power generation (Meays et al., 2004). With the rapid increase in world population and increased urbanisation, there is a massive strain on the existing water supply and sanitation facilities (UNDPI, 2005). In the developing world, poor access to safe water and inadequate sanitation continues to be a danger to human health (World Health Organisation [WHO], 2004). The water situation, in the African continent, has attracted a lot of concern from all sectors of government as it is estimated that more than 300 million out of the 800 million people who live on the continent are in water-scarce environments (United Nations Educational, Scientific and Cultural Organisation [UNESCO], 2004). In Northern Africa, the present water supply is unstable as population growth and economic development have surpassed the traditional water

**1. Introduction** 

[UNDPI], 2005).


## **Detection of Bacterial Pathogens in River Water Using Multiplex-PCR**

C. N. Wose Kinge1, M. Mbewe2 and N. P. Sithebe1 *1Department of Biological Sciences, School of Environmental and Health Sciences, North-West University, Mafikeng Campus, Mmabatho, 2Animal Health Programme, School of Agricultural Sciences, North-West University, Mafikeng Campus, Mmabatho South Africa* 

## **1. Introduction**

530 Polymerase Chain Reaction

[8] Yanagawa T., Hidaka Y., Guimaraes V., Soliman M. and DeGroot L.J. (1995) CTLA-4

[9] Yanagawa T., Taniyama M., Enomoto S., Gomi K., Maruyama H., Ban Y. and Saruta T.

[10] Wang P-W., Liu R-T., Jou S-H.H., Wang S-T., Hu Y-H., Hsieh C-J., Chen M-C., Chen I-Y.

[11] Veeramuthumari P, Isabel W, Kannan K. (2009) "A study on the level of serum T.Chol.,

[12] Veeramuthumari P, Isabel W, Kannan K. (2010) "A Study on the level of T3, T4,TSH

[13] Gunasekaran P, Sujatha K. (2010) UGC NRCBS Winter School on Gene Cloning and

[14] Satyanarayana U, Chakrapani U. ((2006) Biochemistry, Arunabha Sen Book and Allied

[15] de Vicente, C., T. Fulton (2003). *Molecular Marker Learning Modules – Vol. 1.*. IPGRI, Rome, Italy and Institute for Genetic Diversity, Ithaca, New York, USA. [16] de Vicente, C., T. Fulton (2004). *Molecular Marker Learning Modules – Vol. 2.*. IPGRI, Rome, Italy and Institute for Genetic Diversity, Ithaca, New York, USA.. [17] de Vicente, C., J-C. Glaszmann, editors (2006). *Molecular Markers for Allele Mining*. AMS

[18] Spooner, S., R van Treuren and M.C. de Vicente (2005). *Molecular markers for genebank* 

[19] Rodriguez S., Quinn F.B., Matthew W. and Ryan W. (2003) Benign thyroid disease. J.

[20] Constantinides R, Xenophoutos S, Neophyton P, Nomura S, Pierides A and Deltas CC:

Clin. Endocrinol. Metab. 80:41-45.

Endocrinol. Metab. 89(1): 169-173.

Indian J of Endocrinol and Met.

Expression in Bacteria, Lab Manual. 9-19.

Swaminathan Research Foundation.

disease 1 gene. Hum Genet 1997, 99:644-647.

*management*. CGN, IPGRI, USDA.

66-69.

(P) Ltd. 594-596.

Pathol. 33-34.

Japanese. Thyroid. 7:843-846.

gene polymorphism associated with Graves' disease in a Caucasian population. J.

(1997) CTLA-4 gene polymorphism confers susceptibility to Graves' disease in

and Wu C-L. (2004) Cytotoxic T lymphocyte associated molecule – 4 ploymophism and relapse of Graves' hypethyroidism after Antityroid withdrawal. J. Clin.

TGL, HDL, LDL in patients with Graves' hyperthyroid in Madurai population" in

and the association of A/G Polymorphism with CTLA-4 gene in Grave's Hyperthyroidism among South Indian Population" Indian J of Clin Biochem: 26,

(Bioversity's Regional Office for the Americas), CIRAD, GCP, IPGRI, M.S.

New amino acid polymorphism, Ala/Val4058, in exon 45 of the polycystic kidney

The aquatic environments receive a signicant number of human microbial pathogens from point and non-point sources of pollution. Point-source pollution enters the environment at different locations, through a direct route of discharge of treated or untreated domestic sewage, industrial effluent and acid mine drainage (State of the Environment Report [SER], 2002). Non-point (or diffuse) sources of pollution comprises up to 80 % of the pollution entering major river systems thus are of signicant concern with respect to the dissemination of pathogens and their indicators in water systems. They may be attributable to the run-off from urban and agricultural areas, leakage from sewers and septic systems, insecticides and herbicides from agricultural land, and sewer overows (Stewart et al., 2008). Although majority of pathogenic microbes can be eliminated by sewage treatment, many end up in the effluent which is then discharged into receiving bodies of water. These pathogenic microbes have been implicated in human diseases linked with the use of contaminated water and food. Adequate sanitation and clean water, being two critical factors in ensuring human health, protects against a wide range of water-related diseases. These include diarrhoea, cholera, trachoma, dysentery, typhoid, hepatitis, polio, malaria, and filariasis (United Nations Department of Public Information [UNDPI], 2005).

Water is a vital natural resource because of its basic role to life, quality of life, the environment, food production, hygiene, industry, and power generation (Meays et al., 2004). With the rapid increase in world population and increased urbanisation, there is a massive strain on the existing water supply and sanitation facilities (UNDPI, 2005). In the developing world, poor access to safe water and inadequate sanitation continues to be a danger to human health (World Health Organisation [WHO], 2004). The water situation, in the African continent, has attracted a lot of concern from all sectors of government as it is estimated that more than 300 million out of the 800 million people who live on the continent are in water-scarce environments (United Nations Educational, Scientific and Cultural Organisation [UNESCO], 2004). In Northern Africa, the present water supply is unstable as population growth and economic development have surpassed the traditional water

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 533

isolated from different environmental water sources including surface and ground water. Their presence has been correlated with that of faecal pollution indicators (Collado et al., 2008; Fong et al., 2007; Ho et al., 2006) as well as meat mainly from poultry, pork and beef (Collada et al., 2009; Houf, 2010; Wesley and Miller, 2010). Some members of the genus *Arcobacter*, like *A*. *butzleri*, *A*. *cryaerophilus*, and *A*. *skirrowii*, have been implicated in animal and human diarrhoeal cases, suggesting a faecal oral route of transmission to humans and animals (Gonzalez et al., 2007). *Helicobacter pylori* on the other hand, found to be present in surface water and wastewater has been implicated in gastritic, peptic, and duodenal ulcer

Biolms in drinking water distribution systems have been reported as possible reservoirs of H. p*ylori* and attempts to culture these cells from water samples have proven unsuccessful (Linke et al., 2010; Percival and Thomas, 2009). Due to the fastidious nature of this bacterium, the lack of standard culture methods for environmental samples, and the controversy in its ability to survive in an infectious state in the environment, very few quantitative studies have been reported (Percival and Thomas, 2009). *Legionella pneumophila* is a ubiquitous bacterium in natural aquatic environments that can also persist in humancontrolled systems containing water, such as air conditioning and plumbing infrastructures (Steinert et al., 2002). Furthermore, *Vibrio vulnicus*, an opportunistic human pathogen that cause gastroenteritis, severe necrotizing soft-tissue infections and primary septicaemia, has been found present in sh, shell sh, water, and wastewater. Infection generally, is associated with the ingestion of contaminated seafood and water (Harwood et al., 2004; Igbinosa et al., 2009). More so, the presence of enteric bacteria of the genera *Salmonella, Shigella, E.coli* and *Klebsiella* in water has been identified as a major threat to human health

*Salmonellae are* the most frequent agents of bacterial gastroenteritis and typhoid in humans and a prime example of a water- and shell fish-transmitted human pathogen. It is frequently isolated from the marine environment where it can remain viable for several hours (Malorny et al., 2008; Westrell et al., 2009). Contamination with *Salmonella* has been reported in surface water used for recreational purposes, source of drinking water (Till et al., 2008) and irrigation (Gannon et al. 2004) underlining the possible risk associated to the use of such contaminated water. The typhoid caused by *Salmonella enterica* serotype Typhi remains an important public health problem in developing countries and the burden of typhoid fever worldwide is further compounded by the spread of multiple drug resistant *S*. *typhi* (Kim 2010; Lynch et al., 2009; Srikantiah et al., 2006). The runoff from elds with animal husbandry, and untreated sewage disposal contribute to the presence of *Salmonella* in natural water resources (Jenkins et al., 2008; Moganedi et al., 2007). Low numbers of *Salmonella* in food, recreational, surface and potable water sources may pose a public health risk given that their infective dose can be as low as 15–100 CFU (Cobbold et al., 2006; Seo et

Species of *Shigella* and enteroinvasive *Escherichia coli* (EIEC) are important human pathogens identified as the major cause of bacillary dysentery (Wanger et al., 1988; Szakál et al., 2003). The infective dose of *Shigella* cells is very low (101-104 organisms), whereas EIEC strains require a larger infectious dose (between 106 and 1010 organisms) (Rowe and Gross, 1984). Both *Shigella* spp. and EIEC carry a large invasion plasmid and express a similar set of

diseases (Linke et al., 2010; Queralt et al., 2005).

and causative agents for many diseases (Leclerc et al., 2001).

al., 2006).

management practices, leading to water scarcity and pollution to a varying degree (UNESCO, 2004). According to Beukman and Uitenweerde (2002), Southern Africa faces very serious water challenges with an estimated half of the population lacking access to portable water and sanitation facilities. They further hinted that, by 2025, countries like Mozambique, Namibia, Tanzania and Zimbabwe will face more water pressures.

The scarcity of water does not only threaten food security, but also the production of energy and environmental integrity. This often results in water usage conflicts between different communities, and water contamination when humans and animals share the same source of water (Kusiluka et al., 2005). According to the Department of Water Affairs – DWA (2000), South Africa is a water scarce region, with 450mm rainfall per annum. This is lower than the world's 860mm average rainfall. Of the forty-four million people who live in South Africa, 12 million people were without access to portable water supply prior to 1994 (Momba et al., 2006). Although the South African government is making significant progress in ensuring the supply of potable water to all communities, 3.3 and 15.3 million inhabitants of South Africa are still identified to be living without access to potable water and adequate sanitation facilities (Council for Scientific and Industrial Research [CSIR], 2008). A total of 80% of the population live in the rural areas with the unavailability of potable basic water supplies and proper sanitation facilities (Kasrils, 2004; Reitveld et al., 2009).

Due to the scarcity of water in South Africa, extensive exploitation of water resources such as dams, pools, unprotected rivers and springs for domestic and other water uses, is common, particularly in the rural communities where access to potable water supply is limited (Younes and Bartram, 2001). In many developing countries with inadequate sanitation, faecal contaminations of environmental waters by enteric pathogens are very common and river water is major source of microbial pathogens (Sharma et al., 2010). In this study, we report the use of conventional identification, and multiplex PCR (m-PCR) method that permits the simultaneous detection of water-borne *Salmonella*, *Shigella*, *E*. *coli*, and *Klebsiella* bacteria spp. from rivers in the North West province of South Africa. The major rivers in the province include the Molopo, Groot Marico, Elands, Hex, and Crocodile Vaal, Skoonspruit, Harts and Mooi. These rivers are grouped into five catchment areas, which include the Crocodile and Elands, Marico and Hex, Marico and Molopo, Mooi and Vaal, and the Harts (SER, 2002; Department of Water Affairs [DWA], 2007). The water quality in these rivers has been impaired partly due to the frequent contamination of water sources with a number of pathogenic microorganisms from human as well as animal activities, which result in the spread of diarrhoeal diseases (Meays et al., 2004).

#### **1.1 Bacterial pathogens in the aquatic environment**

Microbial pathogens in water include viruses, bacteria, and protozoa (Girones et al., 2010). Currently, pathogenic bacteria have been identied as the major etiological agent in the majority of the waterborne outbreaks worldwide (WHO 2003; Liang et al., 2006). Bacillary dysentery caused by *Shigella* bacteria alone is responsible for approximately 165 million cases of bacterial diarrhoeal diseases annually. Of this, 163 million are in developing countries and 1.5 million in industrialized ones accounting for an estimated 1.1 million death cases each year (Sharma et al., 2010). Most members of the genus *Arcobacter* have been

management practices, leading to water scarcity and pollution to a varying degree (UNESCO, 2004). According to Beukman and Uitenweerde (2002), Southern Africa faces very serious water challenges with an estimated half of the population lacking access to portable water and sanitation facilities. They further hinted that, by 2025, countries like

The scarcity of water does not only threaten food security, but also the production of energy and environmental integrity. This often results in water usage conflicts between different communities, and water contamination when humans and animals share the same source of water (Kusiluka et al., 2005). According to the Department of Water Affairs – DWA (2000), South Africa is a water scarce region, with 450mm rainfall per annum. This is lower than the world's 860mm average rainfall. Of the forty-four million people who live in South Africa, 12 million people were without access to portable water supply prior to 1994 (Momba et al., 2006). Although the South African government is making significant progress in ensuring the supply of potable water to all communities, 3.3 and 15.3 million inhabitants of South Africa are still identified to be living without access to potable water and adequate sanitation facilities (Council for Scientific and Industrial Research [CSIR], 2008). A total of 80% of the population live in the rural areas with the unavailability of potable basic water supplies and proper sanitation facilities (Kasrils, 2004; Reitveld et al.,

Due to the scarcity of water in South Africa, extensive exploitation of water resources such as dams, pools, unprotected rivers and springs for domestic and other water uses, is common, particularly in the rural communities where access to potable water supply is limited (Younes and Bartram, 2001). In many developing countries with inadequate sanitation, faecal contaminations of environmental waters by enteric pathogens are very common and river water is major source of microbial pathogens (Sharma et al., 2010). In this study, we report the use of conventional identification, and multiplex PCR (m-PCR) method that permits the simultaneous detection of water-borne *Salmonella*, *Shigella*, *E*. *coli*, and *Klebsiella* bacteria spp. from rivers in the North West province of South Africa. The major rivers in the province include the Molopo, Groot Marico, Elands, Hex, and Crocodile Vaal, Skoonspruit, Harts and Mooi. These rivers are grouped into five catchment areas, which include the Crocodile and Elands, Marico and Hex, Marico and Molopo, Mooi and Vaal, and the Harts (SER, 2002; Department of Water Affairs [DWA], 2007). The water quality in these rivers has been impaired partly due to the frequent contamination of water sources with a number of pathogenic microorganisms from human as well as animal activities, which

Microbial pathogens in water include viruses, bacteria, and protozoa (Girones et al., 2010). Currently, pathogenic bacteria have been identied as the major etiological agent in the majority of the waterborne outbreaks worldwide (WHO 2003; Liang et al., 2006). Bacillary dysentery caused by *Shigella* bacteria alone is responsible for approximately 165 million cases of bacterial diarrhoeal diseases annually. Of this, 163 million are in developing countries and 1.5 million in industrialized ones accounting for an estimated 1.1 million death cases each year (Sharma et al., 2010). Most members of the genus *Arcobacter* have been

result in the spread of diarrhoeal diseases (Meays et al., 2004).

**1.1 Bacterial pathogens in the aquatic environment** 

Mozambique, Namibia, Tanzania and Zimbabwe will face more water pressures.

2009).

isolated from different environmental water sources including surface and ground water. Their presence has been correlated with that of faecal pollution indicators (Collado et al., 2008; Fong et al., 2007; Ho et al., 2006) as well as meat mainly from poultry, pork and beef (Collada et al., 2009; Houf, 2010; Wesley and Miller, 2010). Some members of the genus *Arcobacter*, like *A*. *butzleri*, *A*. *cryaerophilus*, and *A*. *skirrowii*, have been implicated in animal and human diarrhoeal cases, suggesting a faecal oral route of transmission to humans and animals (Gonzalez et al., 2007). *Helicobacter pylori* on the other hand, found to be present in surface water and wastewater has been implicated in gastritic, peptic, and duodenal ulcer diseases (Linke et al., 2010; Queralt et al., 2005).

Biolms in drinking water distribution systems have been reported as possible reservoirs of H. p*ylori* and attempts to culture these cells from water samples have proven unsuccessful (Linke et al., 2010; Percival and Thomas, 2009). Due to the fastidious nature of this bacterium, the lack of standard culture methods for environmental samples, and the controversy in its ability to survive in an infectious state in the environment, very few quantitative studies have been reported (Percival and Thomas, 2009). *Legionella pneumophila* is a ubiquitous bacterium in natural aquatic environments that can also persist in humancontrolled systems containing water, such as air conditioning and plumbing infrastructures (Steinert et al., 2002). Furthermore, *Vibrio vulnicus*, an opportunistic human pathogen that cause gastroenteritis, severe necrotizing soft-tissue infections and primary septicaemia, has been found present in sh, shell sh, water, and wastewater. Infection generally, is associated with the ingestion of contaminated seafood and water (Harwood et al., 2004; Igbinosa et al., 2009). More so, the presence of enteric bacteria of the genera *Salmonella, Shigella, E.coli* and *Klebsiella* in water has been identified as a major threat to human health and causative agents for many diseases (Leclerc et al., 2001).

*Salmonellae are* the most frequent agents of bacterial gastroenteritis and typhoid in humans and a prime example of a water- and shell fish-transmitted human pathogen. It is frequently isolated from the marine environment where it can remain viable for several hours (Malorny et al., 2008; Westrell et al., 2009). Contamination with *Salmonella* has been reported in surface water used for recreational purposes, source of drinking water (Till et al., 2008) and irrigation (Gannon et al. 2004) underlining the possible risk associated to the use of such contaminated water. The typhoid caused by *Salmonella enterica* serotype Typhi remains an important public health problem in developing countries and the burden of typhoid fever worldwide is further compounded by the spread of multiple drug resistant *S*. *typhi* (Kim 2010; Lynch et al., 2009; Srikantiah et al., 2006). The runoff from elds with animal husbandry, and untreated sewage disposal contribute to the presence of *Salmonella* in natural water resources (Jenkins et al., 2008; Moganedi et al., 2007). Low numbers of *Salmonella* in food, recreational, surface and potable water sources may pose a public health risk given that their infective dose can be as low as 15–100 CFU (Cobbold et al., 2006; Seo et al., 2006).

Species of *Shigella* and enteroinvasive *Escherichia coli* (EIEC) are important human pathogens identified as the major cause of bacillary dysentery (Wanger et al., 1988; Szakál et al., 2003). The infective dose of *Shigella* cells is very low (101-104 organisms), whereas EIEC strains require a larger infectious dose (between 106 and 1010 organisms) (Rowe and Gross, 1984). Both *Shigella* spp. and EIEC carry a large invasion plasmid and express a similar set of

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 535

the detection of bacterial pathogens in water and the assessment of their virulence potential. Therefore, a molecular detection method is needed, since such methods are

Molecular methods used are typically based on the detection and quantication of specic segments of the pathogen's genome (DNA or RNA). To achieve this, the specic segments are subjected to *in vitro* amplication. These methods allow researchers to speedily and specically detect microorganisms of public health concern (Girones et al., 2010). Recently, molecular techniques, specically nucleic acid amplication procedures, immunocapturing, uorescence *in*-*situ* hybridization (FISH), and polymerase chain reaction (PCR) have provided highly sensitive, rapid and quantitative analytical tools for detecting specic pathogens in environmental samples (Watson et al., 2004). These techniques are used to evaluate the microbiological quality of food and water, as well as microbial source-tracking (Albinana-Gimenez et al., 2009; Field et al., 2003; Hundesa et al., 2006). Most applied molecular techniques are based on protocols of nucleic acid amplication, of which the

PCR is a molecular tool that allows for the amplification of target DNA fragments using oligonucleotide primers in a chain of replication cycles catalysed by DNA polymerase (*Taq* polymerase) (Rompré et al., 2002). This tool is used for microbial identification and surveillance with high sensitivity and specificity (Watterworth et al., 2005). It has successfully been applied for the detection and identification of pathogenic bacteria in clinical and environmental samples, as well as for the investigation of food and waterborne disease outbreaks (Harakeh et al., 2006; Haryani et al., 2007; Hsu and Tsen, 2001; Riyaz-Ul-Hassan et al., 2004; Shabarinath et al., 2007). The use of quantitative PCR (qPCR) is rapidly becoming established in the environmental sector since it has been shown, in many cases, to be more sensitive than either the bacterial culture method or the viral plaque assay (He and Jiang, 2005). However, molecular protocols, unlike traditional culture-based methods, do not distinguish between viable and non-viable organisms hence the need for more information before replacing the current conventional methods

Molecular techniques for the specic detection and quantication of bacterial pathogens also offer several advantages over conventional methods: high sensitivity and specicity, speed, ease of standardization and automation. As with the viruses, direct PCR amplication of some bacterial pathogens from water samples is difcult due to the presence of only low numbers of these bacteria in environmental sources. Therefore, an enrichment step is usually required prior to performing a PCR (Noble and Weisberg, 2005). Improved detection of pathogenic *E*. *coli* (Ogunjimi and Choudary, 1999) by immuno-capture PCR, and the sensitive detection of *Salmonella* (Hoorfar et al., 2000) and *Campylobacter* (Nogva et al., 2000) by real-time PCR have also been developed; but these procedures are all monospecic and are either laborious or very expensive for routine use in water testing laboratories. More recent improvements have allowed simultaneous detection of several microorganisms in a single assay (Maynard et al., 2005; Straub et al., 2005; Marcelino et al., 2006). The use of such multiplex polymerase chain reaction (m-PCR) has provided rapid and highly sensitive methods for the specic detection of pathogenic microbes in the aquatic

polymerase chain reaction (PCR) is the most commonly used.

highly specic and sensitive.

by molecular ones.

environment (Kong et al., 2002).

proteins. Both of them are transmitted by direct contact from human to human or via contaminated food and water (Parsot, 1994; Rowe and Gross, 1984). Clinical features of bacillary dysentery caused by EIEC that resemble shigellosis include fever, severe abdominal cramps, malaise, toxemia, and watery diarrhea. The serotype *E*. *coli* O157:H7, an emergent pathogen of faecal origin frequently isolated from waters, has been implicated in food and water-borne disease outbreaks (Bavaro, 2009).

Bacteria of the genus *Klebsiella* are ubiquitous in nature and are a frequent cause of nosocomial infections (Horan et al., 1988). Their non-clinical habitats encompass the gastrointestinal tract of mammals as well as environmental sources such as soil, surface waters, and plants (Bagley, 1985). Environmental isolates have been described as being indistinguishable from human clinical isolates with respect to their biochemical reactions and virulence (Matsen et al., 1974). While the medical signicance of *Klebsiella* obtained in the natural environment is far from clear, such habitats are thought to be potential reservoirs for the growth and spread of these bacteria which may colonize animals and humans (Knittel et al., 1977). Of the five identified *Klebsiella* species, *K*. *oxytoca* and *K*. *Pneumonia*, remain the most clinically important opportunistic pathogen, implicated in communityacquired pyogenic liver abscess and bacterial meningitis in adults (Casolari et al., 2005; Haryani et al., 2007; Keynan and Rubinstein, 2007), has been reported to be present in water (Syposs et al., 2005).

## **1.2 Methods used in detection of bacterial pathogens in water**

Detection, differentiation, and identification of bacteria can be performed by numerous methods, including phenotypic, biochemical and immunological assays, and molecular techniques. These traditional methods for the detection and enumeration of bacterial pathogens have largely depended on the use of selective culture and standard biochemical methods. This classical microbiological methodology relies on the cultivation of specic bacteria, for example plate counts of coliforms. Drawbacks of these methods include firstly, pathogenic bacteria, which normally occur in low numbers, tend to incur large errors in sampling and enumeration (Fleischer, 1990). Secondly, culture-based methods are timeconsuming, tedious; detect only one type of pathogen, and no valid identication of the pathogen (Szewzyk et al., 2000). Thirdly, many pathogenic microbes in the environment, although viable, are either difficult to culture or are non-culturable (Roszak and Colwell, 1987). Sometimes too, it is often difcult to achieve appropriate enrichment, which makes the work even more tedious.

Moreover, concentrations may be too low for cultural detection but still be high enough to cause infection. These limitations therefore make routine examination of water samples for pathogens like *Vibrio cholerae*, *Shigella dysenteriae*, *Aeromonas* spp. and *Campylobacter* spp., difficult. Instead, bacterial indicator species like *Escherichia* coli, which is a normal flora present in very high numbers in the gut of warm-blooded animals, is widely used as an indicator of faecal pollution, to estimate the risk of exposure to other pathogenic microbes present in animal or human wastes (Lund, 1994). However, *Escherichia coli* as well as some bacterial species like *Enterococcus faecalis*, once released into freshwater bodies, enter a viable but non-culturable (VBNC) state and express different set of activities, including virulence traits (Lleo et al., 2005). As a result, the current methodology is unsuitable for

proteins. Both of them are transmitted by direct contact from human to human or via contaminated food and water (Parsot, 1994; Rowe and Gross, 1984). Clinical features of bacillary dysentery caused by EIEC that resemble shigellosis include fever, severe abdominal cramps, malaise, toxemia, and watery diarrhea. The serotype *E*. *coli* O157:H7, an emergent pathogen of faecal origin frequently isolated from waters, has been implicated in

Bacteria of the genus *Klebsiella* are ubiquitous in nature and are a frequent cause of nosocomial infections (Horan et al., 1988). Their non-clinical habitats encompass the gastrointestinal tract of mammals as well as environmental sources such as soil, surface waters, and plants (Bagley, 1985). Environmental isolates have been described as being indistinguishable from human clinical isolates with respect to their biochemical reactions and virulence (Matsen et al., 1974). While the medical signicance of *Klebsiella* obtained in the natural environment is far from clear, such habitats are thought to be potential reservoirs for the growth and spread of these bacteria which may colonize animals and humans (Knittel et al., 1977). Of the five identified *Klebsiella* species, *K*. *oxytoca* and *K*. *Pneumonia*, remain the most clinically important opportunistic pathogen, implicated in communityacquired pyogenic liver abscess and bacterial meningitis in adults (Casolari et al., 2005; Haryani et al., 2007; Keynan and Rubinstein, 2007), has been reported to be present in water

Detection, differentiation, and identification of bacteria can be performed by numerous methods, including phenotypic, biochemical and immunological assays, and molecular techniques. These traditional methods for the detection and enumeration of bacterial pathogens have largely depended on the use of selective culture and standard biochemical methods. This classical microbiological methodology relies on the cultivation of specic bacteria, for example plate counts of coliforms. Drawbacks of these methods include firstly, pathogenic bacteria, which normally occur in low numbers, tend to incur large errors in sampling and enumeration (Fleischer, 1990). Secondly, culture-based methods are timeconsuming, tedious; detect only one type of pathogen, and no valid identication of the pathogen (Szewzyk et al., 2000). Thirdly, many pathogenic microbes in the environment, although viable, are either difficult to culture or are non-culturable (Roszak and Colwell, 1987). Sometimes too, it is often difcult to achieve appropriate enrichment, which makes

Moreover, concentrations may be too low for cultural detection but still be high enough to cause infection. These limitations therefore make routine examination of water samples for pathogens like *Vibrio cholerae*, *Shigella dysenteriae*, *Aeromonas* spp. and *Campylobacter* spp., difficult. Instead, bacterial indicator species like *Escherichia* coli, which is a normal flora present in very high numbers in the gut of warm-blooded animals, is widely used as an indicator of faecal pollution, to estimate the risk of exposure to other pathogenic microbes present in animal or human wastes (Lund, 1994). However, *Escherichia coli* as well as some bacterial species like *Enterococcus faecalis*, once released into freshwater bodies, enter a viable but non-culturable (VBNC) state and express different set of activities, including virulence traits (Lleo et al., 2005). As a result, the current methodology is unsuitable for

food and water-borne disease outbreaks (Bavaro, 2009).

**1.2 Methods used in detection of bacterial pathogens in water** 

(Syposs et al., 2005).

the work even more tedious.

the detection of bacterial pathogens in water and the assessment of their virulence potential. Therefore, a molecular detection method is needed, since such methods are highly specic and sensitive.

Molecular methods used are typically based on the detection and quantication of specic segments of the pathogen's genome (DNA or RNA). To achieve this, the specic segments are subjected to *in vitro* amplication. These methods allow researchers to speedily and specically detect microorganisms of public health concern (Girones et al., 2010). Recently, molecular techniques, specically nucleic acid amplication procedures, immunocapturing, uorescence *in*-*situ* hybridization (FISH), and polymerase chain reaction (PCR) have provided highly sensitive, rapid and quantitative analytical tools for detecting specic pathogens in environmental samples (Watson et al., 2004). These techniques are used to evaluate the microbiological quality of food and water, as well as microbial source-tracking (Albinana-Gimenez et al., 2009; Field et al., 2003; Hundesa et al., 2006). Most applied molecular techniques are based on protocols of nucleic acid amplication, of which the polymerase chain reaction (PCR) is the most commonly used.

PCR is a molecular tool that allows for the amplification of target DNA fragments using oligonucleotide primers in a chain of replication cycles catalysed by DNA polymerase (*Taq* polymerase) (Rompré et al., 2002). This tool is used for microbial identification and surveillance with high sensitivity and specificity (Watterworth et al., 2005). It has successfully been applied for the detection and identification of pathogenic bacteria in clinical and environmental samples, as well as for the investigation of food and waterborne disease outbreaks (Harakeh et al., 2006; Haryani et al., 2007; Hsu and Tsen, 2001; Riyaz-Ul-Hassan et al., 2004; Shabarinath et al., 2007). The use of quantitative PCR (qPCR) is rapidly becoming established in the environmental sector since it has been shown, in many cases, to be more sensitive than either the bacterial culture method or the viral plaque assay (He and Jiang, 2005). However, molecular protocols, unlike traditional culture-based methods, do not distinguish between viable and non-viable organisms hence the need for more information before replacing the current conventional methods by molecular ones.

Molecular techniques for the specic detection and quantication of bacterial pathogens also offer several advantages over conventional methods: high sensitivity and specicity, speed, ease of standardization and automation. As with the viruses, direct PCR amplication of some bacterial pathogens from water samples is difcult due to the presence of only low numbers of these bacteria in environmental sources. Therefore, an enrichment step is usually required prior to performing a PCR (Noble and Weisberg, 2005). Improved detection of pathogenic *E*. *coli* (Ogunjimi and Choudary, 1999) by immuno-capture PCR, and the sensitive detection of *Salmonella* (Hoorfar et al., 2000) and *Campylobacter* (Nogva et al., 2000) by real-time PCR have also been developed; but these procedures are all monospecic and are either laborious or very expensive for routine use in water testing laboratories. More recent improvements have allowed simultaneous detection of several microorganisms in a single assay (Maynard et al., 2005; Straub et al., 2005; Marcelino et al., 2006). The use of such multiplex polymerase chain reaction (m-PCR) has provided rapid and highly sensitive methods for the specic detection of pathogenic microbes in the aquatic environment (Kong et al., 2002).

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 537

Fig. 1. A cross-section of the North West province map showing the rivers and dams

Bacterial strains used for the experimental work (Table 1) were American Type Culture Collection (ATCC) cultures. The strains were grown on Nutrient Agar (Biolab, Merck, South

**Bacterial Strains Source Reference** *Mdh IpaH IpaB GapA* 

*Salmonella paratyphi* ATCC 9150 This study − − + −

*Salmonella typhi* ATCC 14028 Hsu and Tsen, 2001 − − + −

*Shigella boydii* ATCC 9207 Wang et al., 1997 − + − −

*Shigella sonnei* ATCC 25931 Wang et al., 1997 − + − −

*K. oxytoca* ATCC 43086 This study − − − +

*Escherichia coli* ATCC 25922 Lu et al., 2000 − − − −

Table 1. Bacterial strains used in the study for evaluation of primer specificity

*Klebsiella pneumonia* ATCC 15611 Lu et al., 2000 − − − +

sampled

**2.1 Bacterial reference strains** 

Africa) under aerobic conditions at 37°C for 24 hours.

### **1.3 Multiplex polymerase chain reaction (m-PCR)**

Following the application of PCR in the simultaneous amplification of multiple loci in the human dystrophin gene (Chamberlain et al., 1998), multiplex PCR has been firmly established as a general technique. To date, the application of multiplex PCR in pathogen identification, gender screening, linkage analysis, template quantitation, and genetic disease diagnosis is widely established (Chehab and Wall, 1992; Kong et al., 2002; Serre et al., 1991; Shuber et al., 1993). For pathogen identification, PCR analysis of bacteria is advantageous, as the culturing and typing of some pathogens has proven difficult or lengthy. Bacterial multiplexes indicate a particular pathogen among others, or distinguish species or strains of the same genus. An amplicon of sequence conserved among several groups is often included in the reaction to indicate the presence of phylogenetically or epidemiologically similar, or environmentally associated, bacteria and to signal a functioning PCR. Multiplex assays of this set-up distinguish species of Le*gionella* (Bej et al., 1990)*, Mycobacterium* (Wilton and Cousin, 1992), *Salmonella* (Chamberlain et al., 1998), *Escherichia coli,* and *Shigella* (Bej et al., 1991) and major groups of *Chlamydia* (Kaltenboek et al., 1992) from other genus members or associated bacteria. It has also been shown that multiplex PCR remains the ideal technique for DNA typing because the probability of identical alleles in two individuals decreases with the number of polymorphic loci examined. Reactions have been developed with potential applications in paternity testing, forensic identification, and population genetics (Edwards et al., 1991, 1992; Klimpton et al., 1993). Multiplex PCR can be a twoamplicon system or it can amplify 13 or more separate regions of DNA. It may be the end point of analysis, or preliminary to further analyses such as sequencing or hybridization. The steps for developing a multiplex PCR and the benefits of having multiple fragments amplified simultaneously, however, are similar in each system (Edwards and Gibbs, 1994).

#### **1.4 Aim/Objectives of the study**

To detect the presence of pathogenic *Escherichia coli*, *Klebsiella, Salmonella*, and *Shigella* species in water samples obtained from rivers in the North-West Province of South Africa, conventional typing and multiplex PCR methods were applied to enriched cultures. The objectives of the study were to use conventional methods to check for the presence and molecular tools to confirm the identity of *Escherichia*, *Klebsiella, Salmonella*, and *Shigella* species in river water. Our prognosis is that the results will emphasize the need for a rapid and accurate detection method for water-borne disease outbreaks and bacterial pathogens in water to protect human health.

#### **2. Materials and methods**

A total of 54 water samples were collected using sterile 500mL McCartney bottles, downstream, midstream, and upstream of the Crocodile, Elands, Hex, Mooi, Vaal, Molopo, Groot Marico, Harts and Skoonspruit rivers between November 2007 and March 2008 (Fig. 1). These rivers form the five major catchments in the province, which are the Crocodile and Eland, Marico and Hex, Marcio and Molopo, Mooi and Vaal, and Harts catchments. Samples collected were transported on ice to the laboratory for analysis.

Following the application of PCR in the simultaneous amplification of multiple loci in the human dystrophin gene (Chamberlain et al., 1998), multiplex PCR has been firmly established as a general technique. To date, the application of multiplex PCR in pathogen identification, gender screening, linkage analysis, template quantitation, and genetic disease diagnosis is widely established (Chehab and Wall, 1992; Kong et al., 2002; Serre et al., 1991; Shuber et al., 1993). For pathogen identification, PCR analysis of bacteria is advantageous, as the culturing and typing of some pathogens has proven difficult or lengthy. Bacterial multiplexes indicate a particular pathogen among others, or distinguish species or strains of the same genus. An amplicon of sequence conserved among several groups is often included in the reaction to indicate the presence of phylogenetically or epidemiologically similar, or environmentally associated, bacteria and to signal a functioning PCR. Multiplex assays of this set-up distinguish species of Le*gionella* (Bej et al., 1990)*, Mycobacterium* (Wilton and Cousin, 1992), *Salmonella* (Chamberlain et al., 1998), *Escherichia coli,* and *Shigella* (Bej et al., 1991) and major groups of *Chlamydia* (Kaltenboek et al., 1992) from other genus members or associated bacteria. It has also been shown that multiplex PCR remains the ideal technique for DNA typing because the probability of identical alleles in two individuals decreases with the number of polymorphic loci examined. Reactions have been developed with potential applications in paternity testing, forensic identification, and population genetics (Edwards et al., 1991, 1992; Klimpton et al., 1993). Multiplex PCR can be a twoamplicon system or it can amplify 13 or more separate regions of DNA. It may be the end point of analysis, or preliminary to further analyses such as sequencing or hybridization. The steps for developing a multiplex PCR and the benefits of having multiple fragments amplified simultaneously, however, are similar in each system (Edwards and Gibbs,

To detect the presence of pathogenic *Escherichia coli*, *Klebsiella, Salmonella*, and *Shigella* species in water samples obtained from rivers in the North-West Province of South Africa, conventional typing and multiplex PCR methods were applied to enriched cultures. The objectives of the study were to use conventional methods to check for the presence and molecular tools to confirm the identity of *Escherichia*, *Klebsiella, Salmonella*, and *Shigella* species in river water. Our prognosis is that the results will emphasize the need for a rapid and accurate detection method for water-borne disease outbreaks and bacterial pathogens in

A total of 54 water samples were collected using sterile 500mL McCartney bottles, downstream, midstream, and upstream of the Crocodile, Elands, Hex, Mooi, Vaal, Molopo, Groot Marico, Harts and Skoonspruit rivers between November 2007 and March 2008 (Fig. 1). These rivers form the five major catchments in the province, which are the Crocodile and Eland, Marico and Hex, Marcio and Molopo, Mooi and Vaal, and Harts catchments. Samples

collected were transported on ice to the laboratory for analysis.

**1.3 Multiplex polymerase chain reaction (m-PCR)** 

1994).

**1.4 Aim/Objectives of the study** 

water to protect human health.

**2. Materials and methods** 

Fig. 1. A cross-section of the North West province map showing the rivers and dams sampled

#### **2.1 Bacterial reference strains**

Bacterial strains used for the experimental work (Table 1) were American Type Culture Collection (ATCC) cultures. The strains were grown on Nutrient Agar (Biolab, Merck, South Africa) under aerobic conditions at 37°C for 24 hours.


Table 1. Bacterial strains used in the study for evaluation of primer specificity

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 539

Oligonucleotide primers used in the study were synthesized by Inqaba Biotech, South Africa. Sequences of the four PCR primer pairs for m-PCR, their corresponding gene targets and size of the expected amplifications are as shown (Table 2). The malate dehyrogenase gene (*Mdh*) of *E*. *coli* (Hsu and Tsen, 2001; Wose Kinge and Mbewe, 2011), the invasive pasmid antigen B gene (*IpaB*) of *Salmonella* spp. (Kong et al., 2002), the invasive plasmid antigen gene H (*IpaH*) of *Shigella* spp. (Kong et al., 2002; Wose Kinge and Mbewe, 2010), and the glyceralehye-3-phospahate dehydrogenase gene (G*apA*) genes for *Klebsiella* spp*.* (Diancourt et al., 2005; Wose Kinge and Mbewe, 2011) were simultaneously detected by multiplex polymerase chain reaction (m-PCR) assays. DNA from 50µL extract from enriched cultures was used for PCR amplification in a final volume of 25µL. The reaction mixture consisted of 2X PCR Master mix (0.05µL Taq DNA polymerase, 4mM MgCl2, 0.4mM dNTPs) (Fermentas, Inqaba Biotechnical Industries (Pty) Ltd, South Africa), 0.3µM of IpaB, 0.2µM of IpaH and 1.0µM each of Mdh and GapA genes. PCR amplification was performed in a Peltier Thermal Cycler (model-PTC-220 DYADTM DNA ENGINE; MJ Research Inc. USA) under the following conditions: heat denaturation at 94°C for 3 min, followed by 34 cycles of denaturation at 94°C for 30 s; annealing at 60°C for 60 s and extension at 72°C for 1 min. This was followed by a final extension step at 72°C for 7 min and 4°C hold. To create a negative

**Primer Primer sequence (5′→3′) Expected size** 

CGTTCTGTTCAAATGGCCTCAGG ACTGAAAGGCAAACAGCCAAG

GGACTTTTTAAAGCGGCGG GCCTCTCCCAGAGCCGTCTGG

CCTTGACCGCCTTTCCGATA CAGCCACCCTCTGAGGTACT

GTTTTCCCAGTCACGACGTTGTATGAA ATATGACTCCACTCACG TTGTGAGCGGATAACAATTTCCTTCAG AAGCGGCTTTGATGGCT

Following amplification, 10μL of each sample was electrophoresed in a horizontal agarose (LONZA, South Africa) 1% w/v slab gel containing ethidium bromide (0.1μg/mL) in 1X TAE buffer (40 mM tris-acetate; 2 mM EDTA, pH8.3). The agarose gel was electrophoresed for six hours at 60 V. The gel was visualized with UV light (Gene Genius Bio Imaging System, SYNGENE model GBOX CHEMI HR). The relative molecular sizes of the PCR products were estimated by comparing their electrophoretic mobility with 100bp marker

**(bp)** 

392

314

606

700

**2.6 Oligonucleotide primers and multiplex PCR method** 

control template DNA was excluded.

**gene** 

*E*. *coli Mdh* Mdh F

*Salmonella IpaB* IpaB F

*Shigella IpaH* IpaH F

*Klebsiella GapA* GapA F

Mdh R

IpaB R

IpaH R

GapA R

**2.7 Electrophoresis and visualization of PCR products** 

Table 2. Oligonucleotide primers used in this study

(Fermentas O' GeneRuler DNA ladder; Canada).

**Organism Target** 

### **2.2 Selective isolation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella*

Water analysis for *Salmonella, Shigella*, *E*. *coli* and *Klebsiella* bacteria, was done using the spread plate method (American Public Health Association [APHA], 1998). In brief, 1mL of each water sample was enriched in 9mL of 2% buffered-peptone water (Biolab, Merck Diagnostics, South Africa) and serial dilutions performed. Aliquots of 0.1mL of each dilution were plated out on Eosin Methylene Blue (EMB) agar plates (Biolab, Merck Diagnostics, South Africa) for the presumptive isolation of *E*. *coli* and *Klebsiella*, and on Salmonella-Shigella agar for *Salmonella* and *Shigella* isolation. All plates were incubated at 37oC for 24 hours. Presumptive isolates were sub-cultured on fresh media plates incubated at 37oC for 24hours and then preserved on 2.3% w/v Nutrient agar plates for further analysis.

## **2.3 Bacterial Identification using triple sugar iron (TSI) agar test**

All 2992 and 1180 presumptive isolates on EMBA and SSA plates, respectively were Gram stained using the method of Cruikshank et al., (1975) to confirm their morphology as Gram negative rod-shaped bacteria. All Gram negative isolates were subjected to the TSI test, a biochemical test, which distinguishes the *Enterobacteriaceae* from other intestinal Gramnegative bacilli by the ability of the organisms to catabolise the sugars glucose, lactose and sucrose present at different concentrations in the medium, and the production of acid and gas (Prescott, 2002). The test was performed as previously recommended (United States Pharmacopeia Convention; Inc., 2001). Briefly, isolates were streak-plated on TSI agar slopes and incubated at 37oC for 24hours. The results were interpreted as previously determined by Forbes and Weissfeld (1998).

#### **2.4 Differentiation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella* **using conventional serological assay**

All *Salmonella, Shigella*, *E*. *coli* and *Klebsiella* candidate isolates obtained from culture plates and identified by Triple Sugar Iron [TSI] agar test, were differentiated by conventional serotyping (Ballmer et al., 2007). To test for surface antigens, *E*. *coli* Poly D1–D8; *Shigella boydii* Poly C, C1, C2 and C3, *Shigella dysenteriae* Poly A Types 1, 2, 3, 4, 5, 6, 7, *Shigella sonnie* Poly D Phase I and II, *Shigella flexneri* Poly B Types I, II, III, IV, V, VI; *Salmonella* O Poly O (Factors A–G, O2, O4, O7, O8, O9, O9, 46, O3, 10, O1, 3, 4) and O1 (Factors O11, O13, O6, 14, O16, O18, O21, O35), *Salmonella* H Poly Phase 1 and 2; and *Klebsiella* Capsular Types 1, 2, 3, 4, 5, 6 antisera (Inqaba Biotech, South Africa) were used.

#### **2.5 DNA extraction**

Genomic DNA was extracted from all positive bacteria cells inoculated in 5mL Luria Bertani (LB) broth (Merck, South Africa) following overnight incubation at 37oC in a shaker (Doyle and Doyle, 1990). The pellets obtained were re-suspended in 50µL of sterile distilled water. The concentration of the extracted DNA in solution was determined spectrophotometrically (UV Visible spectrophotometer model S-22, Boeco, Germany) by measuring the absorbance at 260 nm. The DNA in solution was used as a template for multiplex PCR.

## **2.6 Oligonucleotide primers and multiplex PCR method**

538 Polymerase Chain Reaction

Water analysis for *Salmonella, Shigella*, *E*. *coli* and *Klebsiella* bacteria, was done using the spread plate method (American Public Health Association [APHA], 1998). In brief, 1mL of each water sample was enriched in 9mL of 2% buffered-peptone water (Biolab, Merck Diagnostics, South Africa) and serial dilutions performed. Aliquots of 0.1mL of each dilution were plated out on Eosin Methylene Blue (EMB) agar plates (Biolab, Merck Diagnostics, South Africa) for the presumptive isolation of *E*. *coli* and *Klebsiella*, and on Salmonella-Shigella agar for *Salmonella* and *Shigella* isolation. All plates were incubated at 37oC for 24 hours. Presumptive isolates were sub-cultured on fresh media plates incubated at 37oC for 24hours and then preserved on 2.3% w/v Nutrient agar plates for

All 2992 and 1180 presumptive isolates on EMBA and SSA plates, respectively were Gram stained using the method of Cruikshank et al., (1975) to confirm their morphology as Gram negative rod-shaped bacteria. All Gram negative isolates were subjected to the TSI test, a biochemical test, which distinguishes the *Enterobacteriaceae* from other intestinal Gramnegative bacilli by the ability of the organisms to catabolise the sugars glucose, lactose and sucrose present at different concentrations in the medium, and the production of acid and gas (Prescott, 2002). The test was performed as previously recommended (United States Pharmacopeia Convention; Inc., 2001). Briefly, isolates were streak-plated on TSI agar slopes and incubated at 37oC for 24hours. The results were interpreted as previously determined

**2.4 Differentiation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella* **using conventional** 

All *Salmonella, Shigella*, *E*. *coli* and *Klebsiella* candidate isolates obtained from culture plates and identified by Triple Sugar Iron [TSI] agar test, were differentiated by conventional serotyping (Ballmer et al., 2007). To test for surface antigens, *E*. *coli* Poly D1–D8; *Shigella boydii* Poly C, C1, C2 and C3, *Shigella dysenteriae* Poly A Types 1, 2, 3, 4, 5, 6, 7, *Shigella sonnie* Poly D Phase I and II, *Shigella flexneri* Poly B Types I, II, III, IV, V, VI; *Salmonella* O Poly O (Factors A–G, O2, O4, O7, O8, O9, O9, 46, O3, 10, O1, 3, 4) and O1 (Factors O11, O13, O6, 14, O16, O18, O21, O35), *Salmonella* H Poly Phase 1 and 2; and *Klebsiella* Capsular Types 1, 2, 3,

Genomic DNA was extracted from all positive bacteria cells inoculated in 5mL Luria Bertani (LB) broth (Merck, South Africa) following overnight incubation at 37oC in a shaker (Doyle and Doyle, 1990). The pellets obtained were re-suspended in 50µL of sterile distilled water. The concentration of the extracted DNA in solution was determined spectrophotometrically (UV Visible spectrophotometer model S-22, Boeco, Germany) by measuring the absorbance at 260 nm. The DNA in solution was used as a template for

**2.2 Selective isolation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella*

**2.3 Bacterial Identification using triple sugar iron (TSI) agar test** 

further analysis.

by Forbes and Weissfeld (1998).

4, 5, 6 antisera (Inqaba Biotech, South Africa) were used.

**serological assay** 

**2.5 DNA extraction** 

multiplex PCR.

Oligonucleotide primers used in the study were synthesized by Inqaba Biotech, South Africa. Sequences of the four PCR primer pairs for m-PCR, their corresponding gene targets and size of the expected amplifications are as shown (Table 2). The malate dehyrogenase gene (*Mdh*) of *E*. *coli* (Hsu and Tsen, 2001; Wose Kinge and Mbewe, 2011), the invasive pasmid antigen B gene (*IpaB*) of *Salmonella* spp. (Kong et al., 2002), the invasive plasmid antigen gene H (*IpaH*) of *Shigella* spp. (Kong et al., 2002; Wose Kinge and Mbewe, 2010), and the glyceralehye-3-phospahate dehydrogenase gene (G*apA*) genes for *Klebsiella* spp*.* (Diancourt et al., 2005; Wose Kinge and Mbewe, 2011) were simultaneously detected by multiplex polymerase chain reaction (m-PCR) assays. DNA from 50µL extract from enriched cultures was used for PCR amplification in a final volume of 25µL. The reaction mixture consisted of 2X PCR Master mix (0.05µL Taq DNA polymerase, 4mM MgCl2, 0.4mM dNTPs) (Fermentas, Inqaba Biotechnical Industries (Pty) Ltd, South Africa), 0.3µM of IpaB, 0.2µM of IpaH and 1.0µM each of Mdh and GapA genes. PCR amplification was performed in a Peltier Thermal Cycler (model-PTC-220 DYADTM DNA ENGINE; MJ Research Inc. USA) under the following conditions: heat denaturation at 94°C for 3 min, followed by 34 cycles of denaturation at 94°C for 30 s; annealing at 60°C for 60 s and extension at 72°C for 1 min. This was followed by a final extension step at 72°C for 7 min and 4°C hold. To create a negative control template DNA was excluded.


Table 2. Oligonucleotide primers used in this study

#### **2.7 Electrophoresis and visualization of PCR products**

Following amplification, 10μL of each sample was electrophoresed in a horizontal agarose (LONZA, South Africa) 1% w/v slab gel containing ethidium bromide (0.1μg/mL) in 1X TAE buffer (40 mM tris-acetate; 2 mM EDTA, pH8.3). The agarose gel was electrophoresed for six hours at 60 V. The gel was visualized with UV light (Gene Genius Bio Imaging System, SYNGENE model GBOX CHEMI HR). The relative molecular sizes of the PCR products were estimated by comparing their electrophoretic mobility with 100bp marker (Fermentas O' GeneRuler DNA ladder; Canada).

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 541

ltration of the sample on membrane filters, bacteria retained on lters can then be detected by culturing in or on selective media. Additional steps, such as biochemical tests, serological assays, and molecular methods, are necessary for conrmation. The isolation and identication of *Shigella* spp. and *E*. *coli* are straightforward and well established (Echeverria et al., 1991, 1992). However, *Shigella* spp. and entero-invasive *E*. *coli* [EIEC] are genetically close and exhibit considerable antigenic cross-reactivity, thus differentiating between them using a single method can be difcult (Cheasty and Rowe, 1983; Lan et al., 2001; Kingombe

The O and H antigen serotyping method provide important epidemiological information. However it is not appropriate for routine diagnostic use because of its high cost and the labour-intensive requirements (Ballmer et al., 2007). There is, therefore, an urgent need for an accurate and simple detection, identication, and differentiation technique for *Shigella* spp. and EIEC, especially for epidemiological studies. On the contrary, serotyping is currently the most widely used technique for typing *Klebsiella* species. It is based mainly on a division according to the K (capsule) antigens (Ørskov and Ørskov, 1984) and shows good reproducibility and capability in differentiating most clinical isolates (Ayling-Smith and Pitt,

**River Catchments** *E. coli* **%** *Klebsiella* **%** *Shigella* **%** *Salmonella* **%** 

Crocodile and Elands 29 4 37 6

Marico and Hex 9 7 18 4

Marico and Molopo 9 4 12 1

Mooi and Vaal 24 19 15 8

Harts 7 11 41 6

The m-PCR was designed to target genes specific to the four entero-pathogenic bacteria selected for this study. Results obtained showed the presence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* contamination in the five catchment areas (Table 4). A total of 39% of *E*. *coli* was recorded for the Crocodile and Elands catchment and up to 45% of *Shigella* spp. was recovered from the Marico and Hex catchment. The presence of *Klebsiella* and *Salmonella* spp. was also observed with 10% and 11% in the Mooi and Vaal catchment, respectively. Of these bacteria species, contamination with *Shigella* was widespread in all catchments. Detection of the *IpaH* gene, which is present on both the chromosome and the *inv* plasmid of all *Shigella*  spp., confirmed the presence of this bacterium in water (Hsu and Tsen, 2001). Understanding the ecology of *Shigella* had been limited mainly due to the lack of suitable techniques to detect the presence of *Shigella* in environment samples (Faruque et al., 2002).

Table 3. Prevalence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* bacteria obtained by

et al., 2005; Yang et al., 2005).

1990).

serotyping

**3.2 Multiplex PCR** 

## **2.8 Specificity of primers**

The specificity of the primers used for multiplex-PCR was confirmed against related enteric bacterial DNA. The DNA was extracted from 5mL of overnight bacterial suspensions cultured in Luria Bertani broth as described under section 2.5. The extracted DNA was then stored at -20°C for use in m-PCR.

## **3. Results and discussion**

#### **3.1 Differentiation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella* **using conventional serotyping assay**

In order to differentiate the bacterial isolates using surface antigens present, conventional serotyping by slide agglutination was performed using polyvalent antisera. The commercially available typing antisera are not sufficient to recognize all prevalent serotypes of *Salmonella*, *E*. *coli* and *Klebsiella* spp. In our study, the antisera assay was not used to identify these serotypes, but rather to determine if a given isolate was a member of the genera of interest or not. The percentages of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* isolates obtained, showing a positive agglutination to antisera, were calculated for each catchment area and results recorded as contained in Table 3. The results indicate a presence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* spp. in all five catchments areas. According to Table 3, *E*. *coli* (the main indicator for faecal contamination) was present in all five catchment samples. The highest was 29% in the Crocodile and Elands catchment, followed by the Mooi and Vaal catchment with 24% agglutination with surface antigen specific antisera. The other three catchments were not free of *E*. *coli* although at lesser levels, comparably.

According to DWA and WHO standards, water meant for irrigation (DWA, 1996) and human consumption (WHO, 2001) should contain no *E*. *coli* bacteria. The use of such contaminated water for irrigation as well as direct consumption as it is before treatment would result in the transmission of potentially pathogenic bacteria to humans through contaminated vegetables and other crops eaten raw, as well as milk from grazing cattle. *Klebsiella* was highest in the Mooi and Vaal followed by Harts catchments with 19% and 11%, respectively. Podschun et al. (2001) also reported a high percentage (53%) distribution of *Klebsiella* spp. from surface water samples, the most common species being *K. pneumoniae*. Bacteria species of the genera *Escherichia* and *Klebsiella* are amongst the group of faecal coliforms. Generally, faecal coliform bacteria inhabit the gastrointestinal tract of all warm and some cold-blooded animals as normal commensals, hence their presence in any given water body is a clear indication of faecal contamination. Although their presence in water cannot be pinpointed to a specific source of faecal contamination, faecal material from human and animal sources can be regarded as high risk due to the possible presence of pathogenic bacteria (Harwood et al., 2000).

High levels of *Shigella* contamination were also seen in all catchments with 31% and 41% in the Crocodile and Elands catchment and Harts catchment, respectively. In general, there was lesser contamination with *Salmonella* compared to other faecal coliforms in all catchments with a maximum of 8% in Mooi and Vaal catchment. Water-borne pathogens often occur in reasonably low concentrations in environmental waters. Therefore, some form of ltration and proliferation are needed for pathogen detection (Hsu et al., 2010). Following ltration of the sample on membrane filters, bacteria retained on lters can then be detected by culturing in or on selective media. Additional steps, such as biochemical tests, serological assays, and molecular methods, are necessary for conrmation. The isolation and identication of *Shigella* spp. and *E*. *coli* are straightforward and well established (Echeverria et al., 1991, 1992). However, *Shigella* spp. and entero-invasive *E*. *coli* [EIEC] are genetically close and exhibit considerable antigenic cross-reactivity, thus differentiating between them using a single method can be difcult (Cheasty and Rowe, 1983; Lan et al., 2001; Kingombe et al., 2005; Yang et al., 2005).

The O and H antigen serotyping method provide important epidemiological information. However it is not appropriate for routine diagnostic use because of its high cost and the labour-intensive requirements (Ballmer et al., 2007). There is, therefore, an urgent need for an accurate and simple detection, identication, and differentiation technique for *Shigella* spp. and EIEC, especially for epidemiological studies. On the contrary, serotyping is currently the most widely used technique for typing *Klebsiella* species. It is based mainly on a division according to the K (capsule) antigens (Ørskov and Ørskov, 1984) and shows good reproducibility and capability in differentiating most clinical isolates (Ayling-Smith and Pitt, 1990).


Table 3. Prevalence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* bacteria obtained by serotyping

#### **3.2 Multiplex PCR**

540 Polymerase Chain Reaction

The specificity of the primers used for multiplex-PCR was confirmed against related enteric bacterial DNA. The DNA was extracted from 5mL of overnight bacterial suspensions cultured in Luria Bertani broth as described under section 2.5. The extracted DNA was then

**3.1 Differentiation of** *Salmonella***,** *Shigella***,** *E***.** *coli* **and** *Klebsiella* **using conventional** 

catchments were not free of *E*. *coli* although at lesser levels, comparably.

pathogenic bacteria (Harwood et al., 2000).

In order to differentiate the bacterial isolates using surface antigens present, conventional serotyping by slide agglutination was performed using polyvalent antisera. The commercially available typing antisera are not sufficient to recognize all prevalent serotypes of *Salmonella*, *E*. *coli* and *Klebsiella* spp. In our study, the antisera assay was not used to identify these serotypes, but rather to determine if a given isolate was a member of the genera of interest or not. The percentages of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* isolates obtained, showing a positive agglutination to antisera, were calculated for each catchment area and results recorded as contained in Table 3. The results indicate a presence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* spp. in all five catchments areas. According to Table 3, *E*. *coli* (the main indicator for faecal contamination) was present in all five catchment samples. The highest was 29% in the Crocodile and Elands catchment, followed by the Mooi and Vaal catchment with 24% agglutination with surface antigen specific antisera. The other three

According to DWA and WHO standards, water meant for irrigation (DWA, 1996) and human consumption (WHO, 2001) should contain no *E*. *coli* bacteria. The use of such contaminated water for irrigation as well as direct consumption as it is before treatment would result in the transmission of potentially pathogenic bacteria to humans through contaminated vegetables and other crops eaten raw, as well as milk from grazing cattle. *Klebsiella* was highest in the Mooi and Vaal followed by Harts catchments with 19% and 11%, respectively. Podschun et al. (2001) also reported a high percentage (53%) distribution of *Klebsiella* spp. from surface water samples, the most common species being *K. pneumoniae*. Bacteria species of the genera *Escherichia* and *Klebsiella* are amongst the group of faecal coliforms. Generally, faecal coliform bacteria inhabit the gastrointestinal tract of all warm and some cold-blooded animals as normal commensals, hence their presence in any given water body is a clear indication of faecal contamination. Although their presence in water cannot be pinpointed to a specific source of faecal contamination, faecal material from human and animal sources can be regarded as high risk due to the possible presence of

High levels of *Shigella* contamination were also seen in all catchments with 31% and 41% in the Crocodile and Elands catchment and Harts catchment, respectively. In general, there was lesser contamination with *Salmonella* compared to other faecal coliforms in all catchments with a maximum of 8% in Mooi and Vaal catchment. Water-borne pathogens often occur in reasonably low concentrations in environmental waters. Therefore, some form of ltration and proliferation are needed for pathogen detection (Hsu et al., 2010). Following

**2.8 Specificity of primers** 

stored at -20°C for use in m-PCR.

**3. Results and discussion** 

**serotyping assay** 

The m-PCR was designed to target genes specific to the four entero-pathogenic bacteria selected for this study. Results obtained showed the presence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* contamination in the five catchment areas (Table 4). A total of 39% of *E*. *coli* was recorded for the Crocodile and Elands catchment and up to 45% of *Shigella* spp. was recovered from the Marico and Hex catchment. The presence of *Klebsiella* and *Salmonella* spp. was also observed with 10% and 11% in the Mooi and Vaal catchment, respectively. Of these bacteria species, contamination with *Shigella* was widespread in all catchments. Detection of the *IpaH* gene, which is present on both the chromosome and the *inv* plasmid of all *Shigella*  spp., confirmed the presence of this bacterium in water (Hsu and Tsen, 2001). Understanding the ecology of *Shigella* had been limited mainly due to the lack of suitable techniques to detect the presence of *Shigella* in environment samples (Faruque et al., 2002).

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 543

Although direct consumption of water by humans from these rivers was minimal throughout the study, indirect consumption through fishing was common. This was particularly evident in the Crocodile and Elands, Marico and Molopo, and the Mooi and Vaal catchment areas. This may be a cause for concern because fish in water bodies contaminated with human and animal waste, harbour a considerable number of bacteria such as *Salmonella*, *Clostridium botulinum*, *Vibrio cholerae*, *E*. *coli* and other coliforms, which could be transmitted to humans if eaten raw or under-cooked (Jayasinghe and Rajakaruna, 2005). Fish and shellfish accounts for 5% of individual cases and 10% of all food-borne illness outbreaks in the United States (Flick, 2008) and not only does fish constitute potential sources of bacteria, they also harbour antibiotic resistant bacteria that could be transmitted to humans resulting in the spread of a pool of antibiotic resistant genes into the environment (Miranda and Zemelman, 2001; Pathak and Gopal, 2005). This also might be compounded by the presence of opportunistic pathogens like *Klebsiella* species in water with serious health implications for consumers that utilize water directly or indirectly from the rivers, especially high risk patients with impaired immune systems such as the elderly or young, patients with burns or excessive wounds, those undergoing immunosuppressive therapy or those with HIV/AIDS infection. Colonization may lead to invasive infections and on very rare occasions, *Klebsiella* spp., notably *K. pneumoniae* and *K. oxytoca*, may cause serious infections, such as destructive pneumonia (Bartram et al.,

**River Catchments** *E. coli* **%** *Klebsiella* **%** *Shigella* **%** *Salmonella* **%**  Crocodile and Elands 39 0 11 6

Marico and Hex 4 6 45 0

Marico and Molopo 0 6 5 1

Mooi and Vaal 15 10 5 11

Harts 0 0 23 9

Table 4. Prevalence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* bacteria obtained by m-PCR

In order to evaluate and verify the specicity of the primers in this study, each primer pair was tested by PCR on DNA templates prepared from a panel of seven different bacterial control strains. The analysis indicated that all primer pairs showed specicities only for their corresponding target organisms (Table 1) and all four sets of PCR primers were targeted at a virulence-associated gene. The Mdh primers specically amplied a 392bp malic acid dehydrogenase gene fragment from *E*. *coli* strain obtained from the American Type Culture Collection (Table 1) and 4-39% of isolates obtained from the different river catchments. The IpaH primers produced a specic 606bp amplimer in all *Shigella* spp. examined in this study (Table 1; Fig 2. lane 3), which included two species of the genus, viz., *S*. *sonnei* and *S*. *boydii*, which are known to be pathogenic to humans. In a previously reported study, Kong et al.

2003; Genthe and Steyn, 2006).

**3.3 Specificity of primers** 

In the present study, we used molecular techniques as well as conventional serotyping method to detect *Shigella* as well as *E*. *coli*, *Salmonella* and *Klebsiella* spp. in river waters with special reference to virulence genes. We standardized the assay by culturing the environmental water samples and simultaneously conducting m-PCR tests. In a similar study by Faruque et al. (2002) and Sharma et al. (2010), the *IpaH* gene was used as an indicator tool to detect the presence of *Shigella* in environmental waters. Fresh contamination of surface water by faecal material of dysentery patients is a possibility in developing countries where sanitation is poor resulting in the presence of *Shigella* in surface water. Several previous studies have also detected *Shigella* in surface waters or sewage samples and have indicated that *Shigella* strains can possibly be transported by surface waters (Alamanos et al., 2000; Faruque et al., 2002; Obi et al., 2004a; Pergram et al., 1998).

Similarly, amplification of the *Mdh* gene, which codes for malic acid dehydrogenase, a housekeeping enzyme of the citric acid cycle, and reportedly found in all *E. coli* strains (Hsu and Tsen, 2001), confirmed the presence of both commensal and pathogenic *E. coli* in the water samples. Although *E*. *coli* is usually present as harmless commensals of the human and animal intestinal tracts, pathogenic strains possess virulent factors that enable them to cause diseases and hence, constitute a potential risk to the health of consumers (Kuhnert et al., 2000). For the detection of *Salmonella* spp. the *IpaB* gene, which is a virulence gene found on the invasion plasmid of *Salmonella* spp., was selected for the PCR as it is reportedly present in most *Salmonell*a strains (Kong et al., 2002). *Salmonella* is isolated from water in lower numbers than indicator bacteria such as faecal coliforms, faecal streptococci and enterococci, which are several orders of magnitude higher (Sidhu and Toze, 2009).

However, low numbers (15-100 colony-forming units [CFU]) of *Salmonella* in water may pose a public health risk (Jyoti et al., 2009). In the aquatic environment this pathogen has been repeatedly detected in various types of natural waters such as rivers, lakes, coastal waters, estuarine as well as contaminated ground water (Haley et al., 2009; Levantesi et al., 2010; Lin and Biyela, 2005; Moganedi et al., 2007; Theron et al., 2001; Wilkes et al., 2009). Their presence has been attributable to runoff from elds with animal husbandry, addition of untreated sewage from nearby civilization contribute *Salmonella* in natural water resources (Moganedi et al., 2007; Jenkins et al., 2008). *Salmonella* contaminated waters might contribute through direct ingestion of the water or via indirect contamination of fresh food to the transmission of this microorganism. *Salmonella* prevalence in surface water and drinking water has not been uniformly investigated in different countries in recent papers.

Surveys of *Salmonella* in fresh surface water environment were mainly performed in industrialized nations, particularly in Canada and North America. Reports of *Salmonella* prevalence in drinking water were instead more frequent from developing nations reecting the higher concern relating to the use of low quality drinking water in these countries. Overall, the scientic community has mainly recently focused on the prevalence of is microorganism in impacted and non-impacted watersheds (Haley et al., 2009; Jokinen et al., 2011; Patchanee et al., 2010), on the identication of the routes of salmonellae contamination (Gorski et al., 2011; Jokinen et al., 2010, 2011; Obi et al., 2004b; Patchanee et al., 2010), and on the inuence of environmental factors on the spread of *Salmonella* in water (Haley et al., 2009; Jokinen et al., 2010; Meinersmann et al., 2008; Wilkes et al., 2009).

In the present study, we used molecular techniques as well as conventional serotyping method to detect *Shigella* as well as *E*. *coli*, *Salmonella* and *Klebsiella* spp. in river waters with special reference to virulence genes. We standardized the assay by culturing the environmental water samples and simultaneously conducting m-PCR tests. In a similar study by Faruque et al. (2002) and Sharma et al. (2010), the *IpaH* gene was used as an indicator tool to detect the presence of *Shigella* in environmental waters. Fresh contamination of surface water by faecal material of dysentery patients is a possibility in developing countries where sanitation is poor resulting in the presence of *Shigella* in surface water. Several previous studies have also detected *Shigella* in surface waters or sewage samples and have indicated that *Shigella* strains can possibly be transported by surface waters (Alamanos et al., 2000; Faruque et al., 2002; Obi et al., 2004a; Pergram et al., 1998).

Similarly, amplification of the *Mdh* gene, which codes for malic acid dehydrogenase, a housekeeping enzyme of the citric acid cycle, and reportedly found in all *E. coli* strains (Hsu and Tsen, 2001), confirmed the presence of both commensal and pathogenic *E. coli* in the water samples. Although *E*. *coli* is usually present as harmless commensals of the human and animal intestinal tracts, pathogenic strains possess virulent factors that enable them to cause diseases and hence, constitute a potential risk to the health of consumers (Kuhnert et al., 2000). For the detection of *Salmonella* spp. the *IpaB* gene, which is a virulence gene found on the invasion plasmid of *Salmonella* spp., was selected for the PCR as it is reportedly present in most *Salmonell*a strains (Kong et al., 2002). *Salmonella* is isolated from water in lower numbers than indicator bacteria such as faecal coliforms, faecal streptococci and

enterococci, which are several orders of magnitude higher (Sidhu and Toze, 2009).

recent papers.

However, low numbers (15-100 colony-forming units [CFU]) of *Salmonella* in water may pose a public health risk (Jyoti et al., 2009). In the aquatic environment this pathogen has been repeatedly detected in various types of natural waters such as rivers, lakes, coastal waters, estuarine as well as contaminated ground water (Haley et al., 2009; Levantesi et al., 2010; Lin and Biyela, 2005; Moganedi et al., 2007; Theron et al., 2001; Wilkes et al., 2009). Their presence has been attributable to runoff from elds with animal husbandry, addition of untreated sewage from nearby civilization contribute *Salmonella* in natural water resources (Moganedi et al., 2007; Jenkins et al., 2008). *Salmonella* contaminated waters might contribute through direct ingestion of the water or via indirect contamination of fresh food to the transmission of this microorganism. *Salmonella* prevalence in surface water and drinking water has not been uniformly investigated in different countries in

Surveys of *Salmonella* in fresh surface water environment were mainly performed in industrialized nations, particularly in Canada and North America. Reports of *Salmonella* prevalence in drinking water were instead more frequent from developing nations reecting the higher concern relating to the use of low quality drinking water in these countries. Overall, the scientic community has mainly recently focused on the prevalence of is microorganism in impacted and non-impacted watersheds (Haley et al., 2009; Jokinen et al., 2011; Patchanee et al., 2010), on the identication of the routes of salmonellae contamination (Gorski et al., 2011; Jokinen et al., 2010, 2011; Obi et al., 2004b; Patchanee et al., 2010), and on the inuence of environmental factors on the spread of *Salmonella* in water (Haley et al.,

2009; Jokinen et al., 2010; Meinersmann et al., 2008; Wilkes et al., 2009).

Although direct consumption of water by humans from these rivers was minimal throughout the study, indirect consumption through fishing was common. This was particularly evident in the Crocodile and Elands, Marico and Molopo, and the Mooi and Vaal catchment areas. This may be a cause for concern because fish in water bodies contaminated with human and animal waste, harbour a considerable number of bacteria such as *Salmonella*, *Clostridium botulinum*, *Vibrio cholerae*, *E*. *coli* and other coliforms, which could be transmitted to humans if eaten raw or under-cooked (Jayasinghe and Rajakaruna, 2005). Fish and shellfish accounts for 5% of individual cases and 10% of all food-borne illness outbreaks in the United States (Flick, 2008) and not only does fish constitute potential sources of bacteria, they also harbour antibiotic resistant bacteria that could be transmitted to humans resulting in the spread of a pool of antibiotic resistant genes into the environment (Miranda and Zemelman, 2001; Pathak and Gopal, 2005). This also might be compounded by the presence of opportunistic pathogens like *Klebsiella* species in water with serious health implications for consumers that utilize water directly or indirectly from the rivers, especially high risk patients with impaired immune systems such as the elderly or young, patients with burns or excessive wounds, those undergoing immunosuppressive therapy or those with HIV/AIDS infection. Colonization may lead to invasive infections and on very rare occasions, *Klebsiella* spp., notably *K. pneumoniae* and *K. oxytoca*, may cause serious infections, such as destructive pneumonia (Bartram et al., 2003; Genthe and Steyn, 2006).


Table 4. Prevalence of *E*. *coli*, *Klebsiella*, *Shigella* and *Salmonella* bacteria obtained by m-PCR

#### **3.3 Specificity of primers**

In order to evaluate and verify the specicity of the primers in this study, each primer pair was tested by PCR on DNA templates prepared from a panel of seven different bacterial control strains. The analysis indicated that all primer pairs showed specicities only for their corresponding target organisms (Table 1) and all four sets of PCR primers were targeted at a virulence-associated gene. The Mdh primers specically amplied a 392bp malic acid dehydrogenase gene fragment from *E*. *coli* strain obtained from the American Type Culture Collection (Table 1) and 4-39% of isolates obtained from the different river catchments. The IpaH primers produced a specic 606bp amplimer in all *Shigella* spp. examined in this study (Table 1; Fig 2. lane 3), which included two species of the genus, viz., *S*. *sonnei* and *S*. *boydii*, which are known to be pathogenic to humans. In a previously reported study, Kong et al.

Detection of Bacterial Pathogens in River Water Using Multiplex-PCR 545

presence of potentially pathogenic *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* bacteria in river water. The water quality is affected by human activities around the areas, which include industrial processes, mining, agriculture and domestic usage. Thus, the main source of *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* in these rivers may be discharge from wastewater effluent as well as domestic sewage around the catchment areas. Our results indicate that the water-borne and food-borne spread of these pathogens is possible due to drinking water contamination, recreational activities, and sheries. Since the aquatic environment is implicated as the reservoir for these microorganisms, and consequently responsible for their transmission in humans, it is obvious that detailed studies on the pathogenic potential of the environmental strains will certainly contribute to understanding the virulence properties of these bacteria and to establish the importance of these signicant pathogens of aquatic systems. The results thus emphasize the need for the implementation of a rapid and accurate detection method in cases of water-borne disease outbreaks and the need for more rapid detection of bacterial pathogens in water to protect human health. The ability to rapidly monitor for various types of microbial pathogens would be extremely useful not only for routine assessment of water quality to protect public health, but also allow effective assessments of water treatment processes to be made by permitting pre- and post-treatment

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**5. References** 

(2002) tested two virulence genes of *Shigella*, the *virA* gene and the *IpaH* gene and obtained more positive amplifications with the *IpaH* gene when compared with the *virA* gene.

Although the *virA* gene was previously reported by Villalobo and Torres (1998) to be specic for virulent *Shigella* spp., the *IpaH* gene was found to be more reliable in detecting *Shigella* spp. in environmental isolates (Kong et al., 2002; Wose Kinge and Mbewe, 2010). The IpaB primers were found to produce a specic 314bp amplimer, in all *Salmonella* spp. examined, which included *S*. *paratyphi*, and *S*. *typhimurium* (Table 1; Fig 2. Lanes 7 and 8) as well as 1-11% of the isolates tested. Similar results were obtained with the GapA primers which generated a 700bp amplimer specific to *Klebsiella*. The amplimers were conrmed by sequencing (Inqaba Biotech, South Africa) all showed a high percentage of sequence similarity (>90%) with published malic acid dehydrogenase, invasive plasmid antigen H and B, and glyceraldehydes-3-phosphate gene sequences in the GenBank database. Our results therefore, indicated that this particular set of primers were suitable for the specic detection of most general strains of *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* from water samples.

Fig. 2. Electrophoretic analysis of PCR-amplied target genes from six different bacterial pathogens. Mobilities of the different target gene amplicons are indicated on the right. Lane M, 100bp DNA ladder (size marker); lanes 1 and 2, Mdh amplicon of *Escherichia coli* ATCC 25922; lane 3, IpaH amplicon of *Shigella boydii* ATCC 9207; lane 4, GapA amplicon of *Klebsiella oxytoca* ATCC 43086; lanes 5 and 6, GapA amplicon of *K*. *pneumoniae* ATCC 15611; lane 7, IpaB amplicon of *Salmonella paratyphi* ATCC 9150, lane 8, IpaB amplicon of *S*. *typhimurium* ATCC 14028

## **4. Conclusion**

Both conventional and molecular methods successfully identified bacteria of interest, however, the multiplex-PCR assays were sensitive and faster than conventional serotyping methods for detecting *E. coli*, *Salmonella*, *Shigella,* and *Klebsiella* spp. from river water samples. The 392bp *Mdh*, 314bp *IpaB*, 606bp *IpaH* and 700bp *GapA* genes were found to be specific and present in the control strains analyzed. Therefore, m-PCR screening of these strains for *Mdh*, *IpaB*, *IpaH* and *GapA* genes should provide a better indicator of possible presence of potentially pathogenic *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* bacteria in river water. The water quality is affected by human activities around the areas, which include industrial processes, mining, agriculture and domestic usage. Thus, the main source of *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* in these rivers may be discharge from wastewater effluent as well as domestic sewage around the catchment areas. Our results indicate that the water-borne and food-borne spread of these pathogens is possible due to drinking water contamination, recreational activities, and sheries. Since the aquatic environment is implicated as the reservoir for these microorganisms, and consequently responsible for their transmission in humans, it is obvious that detailed studies on the pathogenic potential of the environmental strains will certainly contribute to understanding the virulence properties of these bacteria and to establish the importance of these signicant pathogens of aquatic systems. The results thus emphasize the need for the implementation of a rapid and accurate detection method in cases of water-borne disease outbreaks and the need for more rapid detection of bacterial pathogens in water to protect human health. The ability to rapidly monitor for various types of microbial pathogens would be extremely useful not only for routine assessment of water quality to protect public health, but also allow effective assessments of water treatment processes to be made by permitting pre- and post-treatment waters to be rapidly analyzed.

#### **5. References**

544 Polymerase Chain Reaction

(2002) tested two virulence genes of *Shigella*, the *virA* gene and the *IpaH* gene and obtained

Although the *virA* gene was previously reported by Villalobo and Torres (1998) to be specic for virulent *Shigella* spp., the *IpaH* gene was found to be more reliable in detecting *Shigella* spp. in environmental isolates (Kong et al., 2002; Wose Kinge and Mbewe, 2010). The IpaB primers were found to produce a specic 314bp amplimer, in all *Salmonella* spp. examined, which included *S*. *paratyphi*, and *S*. *typhimurium* (Table 1; Fig 2. Lanes 7 and 8) as well as 1-11% of the isolates tested. Similar results were obtained with the GapA primers which generated a 700bp amplimer specific to *Klebsiella*. The amplimers were conrmed by sequencing (Inqaba Biotech, South Africa) all showed a high percentage of sequence similarity (>90%) with published malic acid dehydrogenase, invasive plasmid antigen H and B, and glyceraldehydes-3-phosphate gene sequences in the GenBank database. Our results therefore, indicated that this particular set of primers were suitable for the specic detection of most general strains of *E. coli*, *Salmonella*, *Shigella* and *Klebsiella* from water

Fig. 2. Electrophoretic analysis of PCR-amplied target genes from six different bacterial pathogens. Mobilities of the different target gene amplicons are indicated on the right. Lane M, 100bp DNA ladder (size marker); lanes 1 and 2, Mdh amplicon of *Escherichia coli* ATCC 25922; lane 3, IpaH amplicon of *Shigella boydii* ATCC 9207; lane 4, GapA amplicon of *Klebsiella oxytoca* ATCC 43086; lanes 5 and 6, GapA amplicon of *K*. *pneumoniae* ATCC 15611; lane 7, IpaB amplicon of *Salmonella paratyphi* ATCC 9150, lane 8, IpaB amplicon of *S*.

Both conventional and molecular methods successfully identified bacteria of interest, however, the multiplex-PCR assays were sensitive and faster than conventional serotyping methods for detecting *E. coli*, *Salmonella*, *Shigella,* and *Klebsiella* spp. from river water samples. The 392bp *Mdh*, 314bp *IpaB*, 606bp *IpaH* and 700bp *GapA* genes were found to be specific and present in the control strains analyzed. Therefore, m-PCR screening of these strains for *Mdh*, *IpaB*, *IpaH* and *GapA* genes should provide a better indicator of possible

more positive amplifications with the *IpaH* gene when compared with the *virA* gene.

samples.

*typhimurium* ATCC 14028

**4. Conclusion** 


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**27** 

*Turkey* 

**PCR-RFLP and Real-Time PCR Techniques in** 

The polymerase chain reaction (PCR) is a rapid scientific method for generating a 106-107 fold increase in the number of copies of discrete DNA or RNA sequences (Boehm,1989; Imboden et al,1993). The use of PCR technology has greatly increased the ability to study on genetic material. PCR is a rapid and reliable molecular biology technique that allows quick replication of mainly DNA, the starting material can be a single molecule of rRNA or mRNA. It was developed by Kary Mullis in 1983, and he was awarded the Nobel Prize in 1993. PCR method is useful in situations of limited amount of DNA sample as in forensics, prenatal testing, because it amplifies a single or a few copies of DNA creating millions of copies of the region(1). The ability to quickly produce large quantities of genetic material has enabled significant scientific advances including DNA fingerprinting and sequencing of the human genome. As PCR technology allows taking specimen of genetic material even from just one cell, copy its genetic material several times, this facilitates genetic studies. Currently, besides research purposes, PCR technology is heavily used in diagnosis and patient management especially for viral diseases such as AIDS and hepatitis. Other than detection of infectious organisms, this technology is also useful for determination of genetic

The method relies on thermal cycles of repeated heating and cooling of the reaction for DNA melting. Double stranded DNA can be disrupted by heat or high pH, giving rise to single stranded DNA. The single stranded DNA serves as a template for synthesis of a complementary strand by replicating enzymes, DNA polymerases. In order to imitate the accelerated form of DNA replication for a gene region, a special form of DNA polymerase is used. This DNA polymerase should be resistant to the thermal denaturation. Most of the PCR applications employ Taq polymerase, an enzyme isolated from the bacterium Thermus aquaticus, but there are some other heat-stable DNA polymerases used by the same purpose. Most polymerases require short regions of double stranded nucleic acid to initiate synthesis. For in vitro PCR reactions, this can be provided by synthetic oligonucleotides of about 21-25 bp that are complementary to the negatice strand of main DNA molecule. Those

**1. Introduction** 

**1.1 Polymerase Chain Reaction (PCR)** 

polymorphisms or mutations of individuals (Stahlberg,2011).

**Molecular Cancer Investigations** 

*2Ege University, Department of Medical Biology* 

*3Istanbul University, Department of Molecular Medicine* 

Uzay Gormus1, Nur Selvi2 and Ilhan Yaylim-Eraltan3 *1Istanbul Bilim University, Department of Medical Biochemistry,* 


## **PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations**

Uzay Gormus1, Nur Selvi2 and Ilhan Yaylim-Eraltan3 *1Istanbul Bilim University, Department of Medical Biochemistry,* 

*2Ege University, Department of Medical Biology 3Istanbul University, Department of Molecular Medicine Turkey* 

## **1. Introduction**

554 Polymerase Chain Reaction

Yang, F., Yang, J., Zhang, X., Chen, L., Jiang, Y., Yan, Y., Tang, X., Wang, J., Xiong, Z., Dong,

Younes, M., & Bartram, J. (2001). Waterborne health risks and the WHO perspectives. *Int. J.* 

dysentery. *Nucleic Acids Res*. 33, 19, pp. (6445–6458)

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J., Xue, Y., Zhu, Y., Xu, X., Sun, L., Chen, S., Nie, H., Peng, J., Xu, J., Wang, Y., Yuan, Z., Wen, Y., Yao, Z., Shen, Y., Qiang, B., Hou, Y., Yu, J., Jin, Q. (2005). Genome dynamics and diversity of *Shigella* species, the etiologic agents of bacillary

#### **1.1 Polymerase Chain Reaction (PCR)**

The polymerase chain reaction (PCR) is a rapid scientific method for generating a 106-107 fold increase in the number of copies of discrete DNA or RNA sequences (Boehm,1989; Imboden et al,1993). The use of PCR technology has greatly increased the ability to study on genetic material. PCR is a rapid and reliable molecular biology technique that allows quick replication of mainly DNA, the starting material can be a single molecule of rRNA or mRNA. It was developed by Kary Mullis in 1983, and he was awarded the Nobel Prize in 1993. PCR method is useful in situations of limited amount of DNA sample as in forensics, prenatal testing, because it amplifies a single or a few copies of DNA creating millions of copies of the region(1). The ability to quickly produce large quantities of genetic material has enabled significant scientific advances including DNA fingerprinting and sequencing of the human genome. As PCR technology allows taking specimen of genetic material even from just one cell, copy its genetic material several times, this facilitates genetic studies. Currently, besides research purposes, PCR technology is heavily used in diagnosis and patient management especially for viral diseases such as AIDS and hepatitis. Other than detection of infectious organisms, this technology is also useful for determination of genetic polymorphisms or mutations of individuals (Stahlberg,2011).

The method relies on thermal cycles of repeated heating and cooling of the reaction for DNA melting. Double stranded DNA can be disrupted by heat or high pH, giving rise to single stranded DNA. The single stranded DNA serves as a template for synthesis of a complementary strand by replicating enzymes, DNA polymerases. In order to imitate the accelerated form of DNA replication for a gene region, a special form of DNA polymerase is used. This DNA polymerase should be resistant to the thermal denaturation. Most of the PCR applications employ Taq polymerase, an enzyme isolated from the bacterium Thermus aquaticus, but there are some other heat-stable DNA polymerases used by the same purpose. Most polymerases require short regions of double stranded nucleic acid to initiate synthesis. For in vitro PCR reactions, this can be provided by synthetic oligonucleotides of about 21-25 bp that are complementary to the negatice strand of main DNA molecule. Those

PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations 557

Fig. 1. An example of RFLP results from our laboratories. As shown in the figure, there is a 50 bp marker to compare with our own results and detect the basepair (bp) length. After

• The uncut homozygote cases (having the same alleles) were expected to be having only

• The cut homozygote cases (having the same alleles) were expected to be having three

• The heterozygote cases (having two separate alleles) were expected to be having four

• **Conventional PCR:** This is the DNA-based PCR, primers target specific sequences on DNA and amplification follows the usual steps of denaturation, annealing and elongation. • **Reverse transcription-PCR:** mRNA or rRNA can be the main material to be amplified. The first step is the enzymatic 'reverse transcriptase' reaction to transcribe RNA to cDNA. Subsequent steps are similar to conventional PCR steps (Tania et al, 2006). • **Asymmetric PCR:** It can be used for generation of single strand for sequencing studies. This can be done by adjusting primer concentrations to favor one strand; by this way after first cycles, only the strand complementary to the first strand continues to be copied. • **Nested PCR:** In this type of PCR, there are two stages of the procedure; in the first part, by using a set of primers, a fragment is amplified. After this, by using another primer set, a sub-region of the previously amplified region is re-amplified. Main aim is to

Real-time PCR (PCR with real time) is also known as kinetic PCR, QPCR, QRT- PCR. Automated thermal cycling devices have been improved by using Taq DNA polymerase which is thermostable and continued to be developed by fluorescence luminescence techniques( Higuchi et al,1992; Logan j et al, 2009.). Real-time PCR is easy to perform, providing reliable results with high accuracy as well as rapid quantification. Quantification of polymorphic DNA regions and genotyping single nucleotide polymorphisms are detected by using the real-time PCR reaction. For gene expression analyses, the mRNA

treatment and incubation with specific restriction enzyme:

bands of 217, 62, 35 bp (as in number 3 and 6)

bands of 252, 217, 62, 35 bp (as in number 1,4 and 5)

one 314 bp band (as in number 2).

increase sensitivity and specificity.

**1.3 Types of PCR** 

• **Real-time PCR.** 

**2. Real-time PCR** 

oligonucleotide sequences are known as 'primer' and chosen due to the DNA region that we want to amplify. In PCR, two synthetic primers that flank the region of interest are used; one primer is complementary to the negative strand of DNA and second primer to the positive strand. The primers must be oriented that DNA synthesis proceeds across the regions defined by the primers. By this way, only a single region of giant DNA molecule can be amplified. As only one amplification is not enough, PCR is a cyclic process to generate 106- 107-fold increase in a gene region; each PCR cycle contains three steps. Those thermal cycling steps are necessary separate two strands in the DNA double helix at a high temperature by a process called DNA melting. There are three main sequentially repeating steps of PCR:


In the *denaturation* step, the purpose is to separate strand to be ready for replication, denaturation temperature is higher than the other steps. In the *annealing* step, at a lower temperature, each strand is used as templates for DNA synthesis. The selectivity of PCR results from this step by the usage of primers complementary to the targeted DNA region under specific thermal cycling conditions. After this, there is *extention* step continuing by the heat-stable DNA polymerase to amplify the target DNA region (Boehm,1989).

After 20 cycles of amplification, a million copies of DNA can be generated from a single copy. After several rounds of amplification (about 40 times), the PCR product is analysed on an agarose gel an sis abundant enough to be detected with an ethidium bromide stain.

After this stage, to detect the changes on the DNA sequence, the classical PCR-RFLP method (the next heading) can be used. But also specific DNA sequences can be detected without opening the reaction tube (Higuchi, 1992). Recently, after first preliminary studies the technique developed to get both structural and quantitative informations about the amplified DNA region by real-time PCR devices using flourescent dyes, as we will mention in following headings.

#### **1.2 PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) method**

In contemporary, there are several forms of PCR that are extensively modified to perform a wide array of genetic manipulations. PCR-RFLP (PCR-restriction fragment length polymorphism) is one of those that was preliminary to most of classified PCR methods. RFLP is a technique referring to a difference between restriction enzyme sites on DNA samples, broken into pieces (digested) by those restriction enzymes and the resulting fragments are separated according to their lengths by gel electrophoresis.

Restriction endonucleases are specific enzymes that can cleave specific nucleotide sequences; because of that property, it is possible for them to discriminate nucleotide changes in DNA. Sometimes they can effect the loci other than the target one, but the important part of the procedure is the possible polymorphism or mutation loci to be detected whether the cleavage site is intact or not. If there is a change in the cleavage site of restriction endonuclease, it will not cleave the site, or by addition of the mutation, there may ocur a previously not existing cleavage site.

Fig. 1. An example of RFLP results from our laboratories. As shown in the figure, there is a 50 bp marker to compare with our own results and detect the basepair (bp) length. After treatment and incubation with specific restriction enzyme:


#### **1.3 Types of PCR**

556 Polymerase Chain Reaction

oligonucleotide sequences are known as 'primer' and chosen due to the DNA region that we want to amplify. In PCR, two synthetic primers that flank the region of interest are used; one primer is complementary to the negative strand of DNA and second primer to the positive strand. The primers must be oriented that DNA synthesis proceeds across the regions defined by the primers. By this way, only a single region of giant DNA molecule can be amplified. As only one amplification is not enough, PCR is a cyclic process to generate 106- 107-fold increase in a gene region; each PCR cycle contains three steps. Those thermal cycling steps are necessary separate two strands in the DNA double helix at a high temperature by a process called DNA melting. There are three main sequentially repeating

In the *denaturation* step, the purpose is to separate strand to be ready for replication, denaturation temperature is higher than the other steps. In the *annealing* step, at a lower temperature, each strand is used as templates for DNA synthesis. The selectivity of PCR results from this step by the usage of primers complementary to the targeted DNA region under specific thermal cycling conditions. After this, there is *extention* step continuing by the

After 20 cycles of amplification, a million copies of DNA can be generated from a single copy. After several rounds of amplification (about 40 times), the PCR product is analysed on an agarose gel an sis abundant enough to be detected with an ethidium bromide stain.

After this stage, to detect the changes on the DNA sequence, the classical PCR-RFLP method (the next heading) can be used. But also specific DNA sequences can be detected without opening the reaction tube (Higuchi, 1992). Recently, after first preliminary studies the technique developed to get both structural and quantitative informations about the amplified DNA region by real-time PCR devices using flourescent dyes, as we will mention

In contemporary, there are several forms of PCR that are extensively modified to perform a wide array of genetic manipulations. PCR-RFLP (PCR-restriction fragment length polymorphism) is one of those that was preliminary to most of classified PCR methods. RFLP is a technique referring to a difference between restriction enzyme sites on DNA samples, broken into pieces (digested) by those restriction enzymes and the resulting

Restriction endonucleases are specific enzymes that can cleave specific nucleotide sequences; because of that property, it is possible for them to discriminate nucleotide changes in DNA. Sometimes they can effect the loci other than the target one, but the important part of the procedure is the possible polymorphism or mutation loci to be detected whether the cleavage site is intact or not. If there is a change in the cleavage site of restriction endonuclease, it will not cleave the site, or by addition of the mutation, there may

steps of PCR:

in following headings.

• *Denaturation* of DNA duplex (94-98°C),

• *Extention* (elongation) of primers by polymerase reaction (~72°C)

heat-stable DNA polymerase to amplify the target DNA region (Boehm,1989).

**1.2 PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) method** 

fragments are separated according to their lengths by gel electrophoresis.

ocur a previously not existing cleavage site.

• *Annealing* of primers (37-60°C),


## **2. Real-time PCR**

Real-time PCR (PCR with real time) is also known as kinetic PCR, QPCR, QRT- PCR. Automated thermal cycling devices have been improved by using Taq DNA polymerase which is thermostable and continued to be developed by fluorescence luminescence techniques( Higuchi et al,1992; Logan j et al, 2009.). Real-time PCR is easy to perform, providing reliable results with high accuracy as well as rapid quantification. Quantification of polymorphic DNA regions and genotyping single nucleotide polymorphisms are detected by using the real-time PCR reaction. For gene expression analyses, the mRNA

PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations 559

Sequence-specific probes are based on oligonucleotides or their analogs that one or two

There are some types of probes with two dyes (Holland et al 1991; Tyagi et al, 1996; Tyagi et al, 1998; Caplin et al 1999): a) hydrolysis probes (TaqMan® probes), b) molecular beacons, c)

a. **Hydrolysis probes:** This probe is a single oligonucleotide labeled with two different fluorophores. The fluorophore near the 3' end(acceptor) acts as a fluorescence emission "quencher" of the other one near the 5' end(donor) (Holland et al 1991). As soon as Taq DNA polymerase hydrolyzes the probe via its 5' exonuclease activity during a combined annealing/extension step, the 5' fluorophore (donor) is liberated. Therefore, its emission can no longer be suppressed by the quencher and can be measured in the fluorimeter**.** TaqMan real-time PCR is one of the two types of quantitative PCR methods, and uses a fluorogenic probe which is a single stranded oligonucleotide of 20- 26 nucleotides and is designed to bind only the DNA sequence between two PCR primers. In this case, two primers with a preferred product size of 50-150 bp, a probe with a fluorescent reporter or fluorophore such as 6-carboxyfluorescein (FAM) and tetrachlorofluorescin (TET) and quencher such as tetramethylrhodamine (TAMRA) covalently attached to its 5' and 3' ends are required, respectively (Giller et al, 2011). b. **Hybridization probes**: In this case, there are two oligonucleotides that hybridize to adjacent internal sequences of the same amplicon (Witther et al, 2011). For instance, the 5' oligonucleotide (donor) has a fluorescence in label at its 3' end. The 3' oligonucleotide(acceptor) has either LightCycler-Red 640 or LightCycler-Red 705 at its 5' end. Only after hybridization to the template DNA, two probes come in close proximity, resulting in fluorescence resonance energy transfer (FRET) between the two fluorophores. During FRET, fluorescein, the donor fluorophore, is excited especially by the light source of the LightCycler Instrument, and part of the excitation energy is transferred to either LightCycler-Red 640 or LightCycler-Red 705, the acceptor fluorophores. Emitted fluorescence of these acceptor fluorophores are then measured by the LightCycler Instrument. Specific detections are performed with these probes. For example, the mutation detections are analysed via the external and internal standards . c. **Molecular Beacon Probes:** A molecular beacon is one oligonucleotide labeled with two different fluorophores, an acceptor and a donor. Due to the specific secondary structure formed by the oligonucleotide (beacon), acceptor (quencher) and donor dyes are in close proximity. A molecular beacon unfolds while binding to the growing PCR product, thereby separating the dyes and enhancing the fluorescence of the donor dye. Four different fluorophores can be designed to detect different point mutations

At the beginning of a melting curve analysis, the reaction temperature is low and the fluorescence signal is high. As the temperature steadily increases, the fluorescence will suddenly drop as the reaction reaches the melting point (Tm) of each DNA fragment. More specific analysis of PCR reactions can be performed with SYBR Green I because of its specific melting behaviour, identification/differentiation of multiple specific PCR products

**2.2.1 Hybridization probes (pair of sequence-specific, single-labeled probes)** 

fluorescent dyes are coupled.

simultaneously (Vincent et al, 2005 ).

**2.3 Melting curve analysis** 

hybridization probes.

levels can be done quantitatively by reverse transcriptase–PCR (RT-PCR) reaction (Tanie Eet al, 2006). By this way, it is possible to monitor gene outputs numerically in many different fields, from the drug-resistant tumor cells to the chemotherapy scanning and also to the molecular determination of tumor stages. The use of gene expression analysis is getting increased in many notable fields of biological research. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment.

This method has been preferred especially in the samples, the analysis of which cannot be possible, or in the samples, the cytogenetic analysis of which are turned out as auxiliary techniques to the molecular analysis. Therefore, it has became one of the indispensable methods. The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become a valuable tool for many scientist working in different disciplines. Especially in the field of molecular diagnosis, real-time PCR-based assays took their advantage (Pfaffl, 2004).

## **2.1 Real-time PCR protocols**

Real-time PCR has been preferred as one of the favored methods in molecular studies and in routine analyses, since the process takes short time as 20-30 minutes,it provides fast heating and cooling cycles of 30-40 times, in addition to these, it benefits the control of PCR reaction on a computer monitor (Wittwer, 1997). High sensitivity of real-time PCR makes the technique applicable to very small samples, such as fine needle aspirates. Real-time PCR instruments can simultaneously amplify and detect, eliminating the need to open tubes containing PCR products and therefore reducing the risk of future contamination (Lyon, 2009). Additionally nested PCR and touchdown PCR can be performed using real-time PCR Machine. There are various real-time PCR machines that are used mostly in laboratory experiments:


## **2.2 Probing techniques**

Today, fluorescence is exclusively used as the detection method in real-time PCR. The fluorescent reporters can be divided into two categories: nonspecific and sequence-specific labels (Wilhelm, 2003).

**Nonspecific labels**: These are DNA-binding dyes such as SYBR Green I ( Wittwer et al,1997; Zipper et al, 2004.) and BEBO (Bengtsson et al, 2003), which become strongly fluorescent when they are bound to double-stranded DNA. SYBR Green I binds all double-stranded DNA molecules regardless of their sequence. The Double-stranded DNA bindind dye SYBR Green I is proven to be effective. Maximum excitation of SYBR Green I dye occurs at 497 nm. Maximal emission of DNA stained with SYBR Green I occurs at 521 nm. The specifity and sensitivity of SYBR Green I detection can be monitored by performing a melting curve analysis after using the amplification reaction with external standard.

Differentiation of single point mutant alleles from wild type allele is not possible with SYBR Green I but it is possible to detect small deletions/insertions (10 to 20 bp).

levels can be done quantitatively by reverse transcriptase–PCR (RT-PCR) reaction (Tanie Eet al, 2006). By this way, it is possible to monitor gene outputs numerically in many different fields, from the drug-resistant tumor cells to the chemotherapy scanning and also to the molecular determination of tumor stages. The use of gene expression analysis is getting increased in many notable fields of biological research. Gene profiling opens new possibilities to classify the disease into subtypes and guide a differentiated treatment.

This method has been preferred especially in the samples, the analysis of which cannot be possible, or in the samples, the cytogenetic analysis of which are turned out as auxiliary techniques to the molecular analysis. Therefore, it has became one of the indispensable methods. The introduction of real-time PCR technology has significantly improved and simplified the quantification of nucleic acids, and this technology has become a valuable tool for many scientist working in different disciplines. Especially in the field of molecular

Real-time PCR has been preferred as one of the favored methods in molecular studies and in routine analyses, since the process takes short time as 20-30 minutes,it provides fast heating and cooling cycles of 30-40 times, in addition to these, it benefits the control of PCR reaction on a computer monitor (Wittwer, 1997). High sensitivity of real-time PCR makes the technique applicable to very small samples, such as fine needle aspirates. Real-time PCR instruments can simultaneously amplify and detect, eliminating the need to open tubes containing PCR products and therefore reducing the risk of future contamination (Lyon, 2009). Additionally nested PCR and touchdown PCR can be performed using real-time PCR Machine. There are various real-time PCR machines that are used mostly in laboratory

Today, fluorescence is exclusively used as the detection method in real-time PCR. The fluorescent reporters can be divided into two categories: nonspecific and sequence-specific

**Nonspecific labels**: These are DNA-binding dyes such as SYBR Green I ( Wittwer et al,1997; Zipper et al, 2004.) and BEBO (Bengtsson et al, 2003), which become strongly fluorescent when they are bound to double-stranded DNA. SYBR Green I binds all double-stranded DNA molecules regardless of their sequence. The Double-stranded DNA bindind dye SYBR Green I is proven to be effective. Maximum excitation of SYBR Green I dye occurs at 497 nm. Maximal emission of DNA stained with SYBR Green I occurs at 521 nm. The specifity and sensitivity of SYBR Green I detection can be monitored by performing a melting curve

Differentiation of single point mutant alleles from wild type allele is not possible with SYBR

diagnosis, real-time PCR-based assays took their advantage (Pfaffl, 2004).


analysis after using the amplification reaction with external standard.

Green I but it is possible to detect small deletions/insertions (10 to 20 bp).

**2.1 Real-time PCR protocols** 

experiments:



**2.2 Probing techniques** 

labels (Wilhelm, 2003).

#### **2.2.1 Hybridization probes (pair of sequence-specific, single-labeled probes)**

Sequence-specific probes are based on oligonucleotides or their analogs that one or two fluorescent dyes are coupled.

There are some types of probes with two dyes (Holland et al 1991; Tyagi et al, 1996; Tyagi et al, 1998; Caplin et al 1999): a) hydrolysis probes (TaqMan® probes), b) molecular beacons, c) hybridization probes.


#### **2.3 Melting curve analysis**

At the beginning of a melting curve analysis, the reaction temperature is low and the fluorescence signal is high. As the temperature steadily increases, the fluorescence will suddenly drop as the reaction reaches the melting point (Tm) of each DNA fragment. More specific analysis of PCR reactions can be performed with SYBR Green I because of its specific melting behaviour, identification/differentiation of multiple specific PCR products

PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations 561

electrophoresis, melting point analysis permits clear identification of the amplicon, since each PCR product possesses a characteristic melting point. Moreover, nonspecific products (primer dimers) can also be identified by this method. If performed with hybridization probes, melting point analysis can also detect point mutations. For instance, the acquired Janus Kinase 2(JAK2) V617F point mutation can be found in more than 90% patient with polycytaemia, and in 50% of patients with other chronic myeloproliferative diseases. For instance in the figures 2-4, our own laboratory results are given. Myeloproliferative neoplasms JAK2V617F-mutation analysis results are shown as melting curve analyses. The

genotype is identified by running a melting curve with specific melting points (Tm).

Fig. 4. Mutant result which indicate one melting curve (53.0 ⁰C)

High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex (Martin-Nunez et al, 2011). Melting analysis using new instruments have been designated for high-resolution melting curve analysis (HRM or HRMA) based on its ease of use, simplicity, flexibility, cost-effectivity, nondestructive nature, superb sensitivity, and specificity (Vossen et al, 2009). It enables researchers to rapidly detect and categorize genetic mutations and single nucleotide polymorphisms(SNPs), identify new genetic variants without sequencing (gene scanning) or determine the genetic variation in a population (e.g. viral diversity) prior to sequencing. SYBR®Green I is introduced into a sensitive conventional dye for PCR product melting analysis. High-resolution melting analysis have been used clinically to detect somatic

**2.4 High-Resolution Melting Analysis (HRMA)** 

(multiplex PCR ) with SYBR Green I, genotyping and mutation analyses with hybridization probes. Melting Curve Analysis has many advantages (Wittwer et al, 2009). Just like gel

Fig. 2. Heterozygote result indicating two melting curves (53.0 ⁰C and 62.0 ⁰C)

Fig. 3. Wild type result which indicate one melting curve (62.0 ⁰C)

(multiplex PCR ) with SYBR Green I, genotyping and mutation analyses with hybridization probes. Melting Curve Analysis has many advantages (Wittwer et al, 2009). Just like gel

Fig. 2. Heterozygote result indicating two melting curves (53.0 ⁰C and 62.0 ⁰C)

Fig. 3. Wild type result which indicate one melting curve (62.0 ⁰C)

electrophoresis, melting point analysis permits clear identification of the amplicon, since each PCR product possesses a characteristic melting point. Moreover, nonspecific products (primer dimers) can also be identified by this method. If performed with hybridization probes, melting point analysis can also detect point mutations. For instance, the acquired Janus Kinase 2(JAK2) V617F point mutation can be found in more than 90% patient with polycytaemia, and in 50% of patients with other chronic myeloproliferative diseases. For instance in the figures 2-4, our own laboratory results are given. Myeloproliferative neoplasms JAK2V617F-mutation analysis results are shown as melting curve analyses. The genotype is identified by running a melting curve with specific melting points (Tm).

Fig. 4. Mutant result which indicate one melting curve (53.0 ⁰C)

#### **2.4 High-Resolution Melting Analysis (HRMA)**

High resolution melting is a post-PCR-based method for detecting DNA sequence variation by measuring changes in the melting of a DNA duplex (Martin-Nunez et al, 2011). Melting analysis using new instruments have been designated for high-resolution melting curve analysis (HRM or HRMA) based on its ease of use, simplicity, flexibility, cost-effectivity, nondestructive nature, superb sensitivity, and specificity (Vossen et al, 2009). It enables researchers to rapidly detect and categorize genetic mutations and single nucleotide polymorphisms(SNPs), identify new genetic variants without sequencing (gene scanning) or determine the genetic variation in a population (e.g. viral diversity) prior to sequencing. SYBR®Green I is introduced into a sensitive conventional dye for PCR product melting analysis. High-resolution melting analysis have been used clinically to detect somatic

PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations 563

99% in appropriate conditions, but this method needs intense attention while chosing

The advances in molecular techniques provide new molecular targets for diagnosis and therapy of cancer. These advances can provide both researchers and clinicians with precious information concerning the behavior of tumors. Therefore, these tumors can detect at earlier stages when the tumor burden is smaller and be potentially more curable currently. After the human genome project has completed, the application of highthroughput technologies for polymorphism detection for explaining molecular mechanism for complex disease has

Single nucleotide polymorphisms (SNPs) offers widespread use in gene mapping of genetic disorders, in the delineation of genetic influences in multifactorial diseases such as cancer, cardiovascular disease, in haplotype mapping, and as genetic markers to predict responses to drugs (Riddick et al, 2005). However, for example, there are some inconsistent results regarding the relationship between the presence of polymorphic forms of genes encoded detoxifying enzymes and chemotherapeutic response. It has been reported that the genetic polymorphism analysis in peripheral blood may not be enough representative for the status in tumour tissue. For instance, Uchida et al reported that individuals heterozygous for the 28-bp polymorphism in thymidylate synthase (TS) gene may have increased risk for cancer that are homozygous for this polymorphism due to loss of one allele during carcinogenesis(Uchida et al, 2004). They also showed that the response to 5-FU-based chemotherapy in these cases was comparable to cases where the individual was homozygous. Therefore, it may be excellent to determine the genotype of polymorphisms in

Some data obtained from combined genotype studies have demonstrated that these data may have significance for models of cancer prognosis or treatment. But, many researchers suggest that larger studies will be needed also to investigate the effect of specific treatment modalities in cancer. While investigating the post-initiation stages of cancer, four basic parts can be dedicated to gene polymorphisms affecting: (a)growth control of cell (cell proliferation, differentiation and death); (b)factors involved in tumour invasion and metastasis (immune and inflammatory responses, extracellular matrix remodelling, angiogenesis and cell adhesion); (c)effects of hormones and vitamins on growing tumours; (d)outcome of cancer therapy (cancer pharmacogenetics) (Loktionov, 2004). Quantitation of gene expression in tumor or host cells has another an enormous importance for investigating the gene patterns responsible for cancer development, progression and

Analysis of transcriptional activity of tumor cells or detection of possible new tumor markers by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques have the potential to change cancer diagnosis and treatment (Mocellin, 2003). New molecular tecniques for diagnosis offers the promise of accurately matching patient with treatment. It has been shown that there is a resultant significant effect on improved disease outcome. Currently, the real-time reverse transcription polymerase chain reaction (qRT-PCR), has a

primers and arranging study conditions(Gulley et al.).

**3. PCR-based studies in cancer research** 

created very important opportunities (Khoury,1997).

tumour cells than in peripheral blood.

response or resistance to therapy.

changes in select exons of oncogenes such as *EGFR*,53 *KRAS*,54 *PDGFRA*,55 *KIT*,56 *BRAF*,57 and*TP53* ( Bastien et al, 2008).

#### **2.5 Gene expression analysis**

Conventional microarrays have limitations in flexibility, speed, cost, and sensitivity. Gene expression analysis by microarray techniques and real-time PCR offers new possibilities to classify malignant tumors, such as lymphomas, into more distinct subtypes for diagnosis and treatment (Schmit et al, 2010; Bagg et al, 1999; Stahlberg et al, 2005. ). The study of biological regulation usually involves gene expression assays and requires quantification of RNA frequently. In the past, conventional gel- or blot-based techniques were used for these assays. However, these techniques often have limitations in speed, sensitivity, dynamic range, and reproducibility required by current experimental systems. In contrast, real-time PCR methods, can easily meet these requirements. Reverse transcription PCR (RT-PCR) is a common and powerful tool for highly sensitive RNA expression profiling. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with that of a presumed constantly expressed reference gene (Stahlberg et al, 2005).

A flow- chart, represents the steps of Real-time PCR and its applications, is given in Figure 5.

Fig. 5. Real-time PCR Flow-chart

#### **2.6 Epigenetic studies with PCR**

Epigenetic information is usually lost during the PCR because of the insensitivity of DNA polymerase, it cannot distinguish between methylated and unmethylated cytosines. After PCR, any methylated allele will be extremely diluted. Therefore, something must be done to preserve methylated form of DNA. Treatment with sodium bisulfite will deaminate cytosine to uracil, the rate of deamination of 5-methylcytosine to thymine is slower than the conversion of cytosine to uracil, thus it is assumed that the only cytosines remaining after sodium bisulfite treatment are derived from 5-methylcytosines. By this way, during subsequent PCR cycles, uracil residues are replicated as thymine residues, and 5 methylcytosine residues are replicated as cytosines. The efficiency of the method is about 99% in appropriate conditions, but this method needs intense attention while chosing primers and arranging study conditions(Gulley et al.).

## **3. PCR-based studies in cancer research**

562 Polymerase Chain Reaction

changes in select exons of oncogenes such as *EGFR*,53 *KRAS*,54 *PDGFRA*,55 *KIT*,56 *BRAF*,57

Conventional microarrays have limitations in flexibility, speed, cost, and sensitivity. Gene expression analysis by microarray techniques and real-time PCR offers new possibilities to classify malignant tumors, such as lymphomas, into more distinct subtypes for diagnosis and treatment (Schmit et al, 2010; Bagg et al, 1999; Stahlberg et al, 2005. ). The study of biological regulation usually involves gene expression assays and requires quantification of RNA frequently. In the past, conventional gel- or blot-based techniques were used for these assays. However, these techniques often have limitations in speed, sensitivity, dynamic range, and reproducibility required by current experimental systems. In contrast, real-time PCR methods, can easily meet these requirements. Reverse transcription PCR (RT-PCR) is a common and powerful tool for highly sensitive RNA expression profiling. Quantification by real-time PCR may be performed as either absolute measurements using an external standard, or as relative measurements, comparing the expression of a reporter gene with

that of a presumed constantly expressed reference gene (Stahlberg et al, 2005).

cDNA Synthesis

A flow- chart, represents the steps of Real-time PCR and its applications, is given in Figure 5.

**Analysis**

Melting curve analysis

Genotyping SNP

Mutation

QRT-PCR Chromosomal

Translocation Gene Profiling

Epigenetic information is usually lost during the PCR because of the insensitivity of DNA polymerase, it cannot distinguish between methylated and unmethylated cytosines. After PCR, any methylated allele will be extremely diluted. Therefore, something must be done to preserve methylated form of DNA. Treatment with sodium bisulfite will deaminate cytosine to uracil, the rate of deamination of 5-methylcytosine to thymine is slower than the conversion of cytosine to uracil, thus it is assumed that the only cytosines remaining after sodium bisulfite treatment are derived from 5-methylcytosines. By this way, during subsequent PCR cycles, uracil residues are replicated as thymine residues, and 5 methylcytosine residues are replicated as cytosines. The efficiency of the method is about

and*TP53* ( Bastien et al, 2008).

**2.5 Gene expression analysis** 

Fig. 5. Real-time PCR Flow-chart

DNA Isolation

RNA Isolation

**SAMPLE Tissue Bone borrow Peripheral blood Cerebrospinal fluid (CSF)**

**2.6 Epigenetic studies with PCR** 

The advances in molecular techniques provide new molecular targets for diagnosis and therapy of cancer. These advances can provide both researchers and clinicians with precious information concerning the behavior of tumors. Therefore, these tumors can detect at earlier stages when the tumor burden is smaller and be potentially more curable currently. After the human genome project has completed, the application of highthroughput technologies for polymorphism detection for explaining molecular mechanism for complex disease has created very important opportunities (Khoury,1997).

Single nucleotide polymorphisms (SNPs) offers widespread use in gene mapping of genetic disorders, in the delineation of genetic influences in multifactorial diseases such as cancer, cardiovascular disease, in haplotype mapping, and as genetic markers to predict responses to drugs (Riddick et al, 2005). However, for example, there are some inconsistent results regarding the relationship between the presence of polymorphic forms of genes encoded detoxifying enzymes and chemotherapeutic response. It has been reported that the genetic polymorphism analysis in peripheral blood may not be enough representative for the status in tumour tissue. For instance, Uchida et al reported that individuals heterozygous for the 28-bp polymorphism in thymidylate synthase (TS) gene may have increased risk for cancer that are homozygous for this polymorphism due to loss of one allele during carcinogenesis(Uchida et al, 2004). They also showed that the response to 5-FU-based chemotherapy in these cases was comparable to cases where the individual was homozygous. Therefore, it may be excellent to determine the genotype of polymorphisms in tumour cells than in peripheral blood.

Some data obtained from combined genotype studies have demonstrated that these data may have significance for models of cancer prognosis or treatment. But, many researchers suggest that larger studies will be needed also to investigate the effect of specific treatment modalities in cancer. While investigating the post-initiation stages of cancer, four basic parts can be dedicated to gene polymorphisms affecting: (a)growth control of cell (cell proliferation, differentiation and death); (b)factors involved in tumour invasion and metastasis (immune and inflammatory responses, extracellular matrix remodelling, angiogenesis and cell adhesion); (c)effects of hormones and vitamins on growing tumours; (d)outcome of cancer therapy (cancer pharmacogenetics) (Loktionov, 2004). Quantitation of gene expression in tumor or host cells has another an enormous importance for investigating the gene patterns responsible for cancer development, progression and response or resistance to therapy.

Analysis of transcriptional activity of tumor cells or detection of possible new tumor markers by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques have the potential to change cancer diagnosis and treatment (Mocellin, 2003). New molecular tecniques for diagnosis offers the promise of accurately matching patient with treatment. It has been shown that there is a resultant significant effect on improved disease outcome. Currently, the real-time reverse transcription polymerase chain reaction (qRT-PCR), has a

PCR-RFLP and Real-Time PCR Techniques in Molecular Cancer Investigations 565

t(9;22) BCR-ABL Translocation t(15;17) PML-RARα Translocation t(1;19) E2A-PRL Translocation t(8,21) AML1-ETO Translocation t(12;21) TEL-AML1 Translocation inv (16) CBFβ- MYH11 Inversion t(4;11) MLL-AF4 Translocation **CRONIC MYELOID LEUKEMIA (KML)** 

Adam Bagg, Bhaskar Kallakury. Molecular pathology of Leukemia and Lymphpma.

Alexandre Loktionov. Common gene polymorphisms, cancer progression and prognosis

Anders Stahlberg†, Neven Zoric, Pierre Åman and Mikael Kubista Quantitative real-time PCR for cancer detection: the lymphoma case: *Expert Rev. Mol. Diagn.* 5(2), 2005. Bastien R, Lewis TB, Hawkes JE, Quackenbush JF, Robbins TC, Palazzo J, Perou CM,

Bengtsson M, Karlsson JH, Westman G, Kubista M. A new minor groove binding asymmetric cyanine reporter dye for realtime PCR. *Nucleic Acids Res.* 31(8), E45, 2003. Caplin BE, Rasmussen RP, Bernard PS, Wittwer CT. LightCyclerTM hybridization probes –

Corlnne D. Boehm. Use of Polymerase Chain Reaction for Diagnosis of Inherited Disorders.

Devita VT, Hellman S, Rosenberg SA. (2005). Cancer princibles and Practise of Oncology.7th

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Gillet JP, Gottesman MM. Advances in the molecular detection of ABC transporters involved in multidrug resistance in cancer. *Review.Curr Pharm Biotechnol*. 12(4):686-92, 2011 Gulley ML, Shea TC, Fedoriw Y. Genetic tests to evaluate prognosis and predict therapeutic

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circulating tumor cells in melanoma prostatic and breast carcinomas*. In Vivo.* 2000

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reaction product by utilizing the 5´–3´ exonuclease activity of Thermus aquaticus

Bernard PS: High-throughput amplicon scanning of the TP53 gene in breast cancer using high-resolution fluorescent melting curve analyses and automatic mutation

the most direct way to monitor PCR amplification and mutation detection.

Multidrug resistance 1 (MDR1) t(9;22) BCR-ABL Translocation Table 1. The chromosomal aberrations that can be detected by RNA quantification.

*Cancer Letters* Volume 208, Issue 1, 10 May 2004, Pages 1-33

**ACUTE NON-LYMPHOBLASTIC LEUKEMIA (ANLL)** 

**ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)** 

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**4. References** 

potential to become an important analytical tecnique for the mRNA detection in tissue biopsies or body fluids. qRT-PCR is especially promising in prognostic assays and monitoring response to treatment for cancer patients. It is known that histopathological staging in cancer defines patient prognosis. However, there are some limitations in the prognostic heterogeneity of patients within a given tumour stage. According to this view, not all patients with lymph node-negative are treated and not all patients with lymph nodepositive tumours die from their cancer. So, more accurate staging protocols are needed for detection clinical tumour staging by using molecular techniques.

Gene expression analysis is one of the most important parameter that utilises the qRT-PCR assay's potential for generating quantitative data (Skrzypski, 2008; Schuster et al, 2004). It is reported that the detection of disseminated tumor cells in peripheral blood obtained from colorectal cancer patients by RT-PCR could be an effective method for identifying patients for adjuvant therapy. It is known that the mRNA for prostate specific antigen (PSA) is expressed only by prostatic cells. RT-PCR are suggested as a potentially more sensitive assay for the detection of cells expressing PSA mRNA in peripheral blood or in extraprostatic tissues. Some studies suggest that the molecular detection of circulating tumor cells (CTC) and micrometastases may help develop new prognostic markers in patients with solid tumors (Ghossein et al, 2000). It has been reported that prostatic tissue specific markers and melanoma related transcripts were detected by RT PCR in the peripheral blood, bone marrow and lymph nodes of patients with localized and advanced tumors. Currently, many reliable methods emerged with fast and efficient mechanisms for screening and monitorizing large populations for genetically linked traits and for cancerrelated genes discovery.

In addition to gene expression profiling, real-time PCR is also useful to detect chromosomal aberrations. Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematologic malignancies and childhood sarkomas (Peter et al, 2006). For example t(15,17) translocation is found only in the leukemic cells. Only in patients with acute promyelocytic leukemia (APL) and the other forms of leukemia, t(1;19) translocation is found with B-cell precursor acute lympoblastic leukemia (ALL). Quantitative analysis provide small number of remaining malignant cells (minimal residual disease, MRD) in patients to be revealed whose disease is in a clinical remission. Therefore, quantitative results are very important in terms of detection in malignancies and MRD. For example, *BCR-ABL* quantification monitors MRD and therapy of chronic myelogenous leukemia (Lyon et al, 2009). Using the real-time PCR Instrument as a closed tube, rapid amplification and real-time fluorescence detection system, for example quantitative measurement of the BCR-ABL expression level can be performed with a minimum risk of cross contamination. Relative expression levels of different samples may be calculated by standardizing the amount of BCR-ABL transcripts in a sample to the amount of an endogenous expressed housekeeping gene. The values for BCR-ABL and housekeeping gene for each sample are calculated by the real-time PCR software by the comparing the crossing points to the standard curve. A normalized target value (the ratio of BCR-ABL/housekeeping ) is then derived by dividing the amount of BCR-ABL by the amount of housekeeping gene. The chromosomal aberration examples in various leukemia types can be detected by RNA quantification, shown in Table 1. On the other hand, melting analysis of the PCR product or the probe is used to confirm detection of the correct product.


Table 1. The chromosomal aberrations that can be detected by RNA quantification.

#### **4. References**

564 Polymerase Chain Reaction

potential to become an important analytical tecnique for the mRNA detection in tissue biopsies or body fluids. qRT-PCR is especially promising in prognostic assays and monitoring response to treatment for cancer patients. It is known that histopathological staging in cancer defines patient prognosis. However, there are some limitations in the prognostic heterogeneity of patients within a given tumour stage. According to this view, not all patients with lymph node-negative are treated and not all patients with lymph nodepositive tumours die from their cancer. So, more accurate staging protocols are needed for

Gene expression analysis is one of the most important parameter that utilises the qRT-PCR assay's potential for generating quantitative data (Skrzypski, 2008; Schuster et al, 2004). It is reported that the detection of disseminated tumor cells in peripheral blood obtained from colorectal cancer patients by RT-PCR could be an effective method for identifying patients for adjuvant therapy. It is known that the mRNA for prostate specific antigen (PSA) is expressed only by prostatic cells. RT-PCR are suggested as a potentially more sensitive assay for the detection of cells expressing PSA mRNA in peripheral blood or in extraprostatic tissues. Some studies suggest that the molecular detection of circulating tumor cells (CTC) and micrometastases may help develop new prognostic markers in patients with solid tumors (Ghossein et al, 2000). It has been reported that prostatic tissue specific markers and melanoma related transcripts were detected by RT PCR in the peripheral blood, bone marrow and lymph nodes of patients with localized and advanced tumors. Currently, many reliable methods emerged with fast and efficient mechanisms for screening and monitorizing large populations for genetically linked traits and for cancer-

In addition to gene expression profiling, real-time PCR is also useful to detect chromosomal aberrations. Non-random chromosomal translocations are frequently associated with a variety of cancers, particularly hematologic malignancies and childhood sarkomas (Peter et al, 2006). For example t(15,17) translocation is found only in the leukemic cells. Only in patients with acute promyelocytic leukemia (APL) and the other forms of leukemia, t(1;19) translocation is found with B-cell precursor acute lympoblastic leukemia (ALL). Quantitative analysis provide small number of remaining malignant cells (minimal residual disease, MRD) in patients to be revealed whose disease is in a clinical remission. Therefore, quantitative results are very important in terms of detection in malignancies and MRD. For example, *BCR-ABL* quantification monitors MRD and therapy of chronic myelogenous leukemia (Lyon et al, 2009). Using the real-time PCR Instrument as a closed tube, rapid amplification and real-time fluorescence detection system, for example quantitative measurement of the BCR-ABL expression level can be performed with a minimum risk of cross contamination. Relative expression levels of different samples may be calculated by standardizing the amount of BCR-ABL transcripts in a sample to the amount of an endogenous expressed housekeeping gene. The values for BCR-ABL and housekeeping gene for each sample are calculated by the real-time PCR software by the comparing the crossing points to the standard curve. A normalized target value (the ratio of BCR-ABL/housekeeping ) is then derived by dividing the amount of BCR-ABL by the amount of housekeeping gene. The chromosomal aberration examples in various leukemia types can be detected by RNA quantification, shown in Table 1. On the other hand, melting analysis

of the PCR product or the probe is used to confirm detection of the correct product.

detection clinical tumour staging by using molecular techniques.

related genes discovery.


Bengtsson M, Karlsson JH, Westman G, Kubista M. A new minor groove binding asymmetric cyanine reporter dye for realtime PCR. *Nucleic Acids Res.* 31(8), E45, 2003.


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*Edited by Patricia Hernández-Rodríguez and Arlen Patricia Ramirez Gómez*

This book is intended to present current concepts in molecular biology with the emphasis on the application to animal, plant and human pathology, in various aspects such as etiology, diagnosis, prognosis, treatment and prevention of diseases as well as the use of these methodologies in understanding the pathophysiology of various diseases that affect living beings.

Polymerase Chain Reaction

Polymerase Chain Reaction

*Edited by Patricia Hernández-Rodríguez* 

*and Arlen Patricia Ramirez Gómez*

Photo by yupiramos / Depositphotos