**4. Polymerase chain reaction**

Due to its high sensitivity, specificity, and rapid results, PCR is an efficient alternative to conventional microbiological culture methods to detect specific types of microorganisms in foods, water, and environmental samples (Moganedi et al., 2007; Glynn et al., 2006; Piknova´et al., 2002). The International Standardization Organization (ISO) recently published standards which address the PCR methodology for the detection of food-borne pathogens (Tomás et al., 2009).

Detection of *Salmonella* spp. Presence in Food 401

PCR – polymerase chain reaction – is an enzymatic reaction replicating a DNA molecule. A critical condition of the PCR course is the presence of an enzyme, thermostable DNA polymerase. Substrates of the reaction are deoxyribonucleotides (building blocks of newly synthesized DNA molecules) and oligonucleotides (starters), which on a complementary basis attach to the replicated DNA strand. Polymerase chain reaction is a thermal process, with cyclically changing temperatures. One cycle leading to synthesis of one copy of DNA from one matrix molecule is composed of the following stages: DNA denaturation (90-95°C), attachment of starters (the so-called annealing, 50-65°C) and strand elongation. Reaction under laboratory conditions is carried out in a device known as thermocycler. Usually, one PCR reaction is made of 35-40 cycles, which result in creating billions of copies of an

individual DNA molecule (with a logarithmic increase of the product in each cycle).

each cycle (a logarithmic increase of the amount of product results in a logarithmic

time from the collection of a sample to the final results does not exceed 24 hours.

A forty-cycle real-time PCR reaction lasts about 1.5 hours. After adding the time needed to isolate DNA from the analysed sample (up to 30 minutes), the entire determination of the presence or the lack of pathogen lasts 2 hours (while using the Applied Biosystems TaqMan® Pathogen Detection System). Samples for determination are taken from preenrichment cultures on buffered peptone water after 18 h incubation at 37°C, so the total

Chen *et al*. (2000) evaluated the TaqMan system for the detection of *Salmonella* that utilizes primers and probes developed from a novel target sequence (*inv*A). The detection limit was below 3 CFU/25 g or 25 ml when raw milk, ground beef and ground pork inoculated with *Salmonella* were pre-enriched overnight. Malorny *et al*. (2004) used specifically designed primers and a probe target within the ttrRSBCA locus, and included internal amplification control, which is coamplified with the same primers as the *Salmonella* DNA in the assay. The diagnostic accuracy was shown to be 100% compared to the traditional culture method when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for *Salmonella* by real-time PCR including a pre-enrichment step in buffered

A very frequent target of species-specific *Salmonella* PCR assays is the invasion protein *inv*A gene, and several *inv*A-based PCR assays have been already developed and validated

Real-time PCR is a polymerase chain reaction observed in real time, is an improvement of the classical PCR method (Al-Soud et al., 2005; Bansal et al., 2006). A real-time PCR thermocycler is equipped with an optical system which collects information about the course of reaction, cycle by cycle. TaqMan® probes labelled with fluorescent dyes make an additional component of PCR real time reactions. A probe is an oligonucleotide designed to bind highly specifically to a replicated fragment of DNA, therefore – in case of the analysed application in food microbiology – to "detect" a sequence which is characteristic for the required pathogen. The probe should bind specifically and strongly, which can be ensured by special protein molecules (a MGB molecule in TaqMan® Applied Biosystems probes). Polymerase, while synthesising a new strand, moves along the matrix, encountering a probe on its way. Because of its exonucleolytic activity, the enzyme starts to "unstick" the probe from the matrix and afterwards to destroy it, releasing a fluorescent dye. An optical system of the thermocycler triggers and then receives dye glow, which becomes more intense with

**4.1 Conventional PCR** 

**4.2 Real–time PCR** 

peptone water.

(Malorny et al. 2003a; b).


Table 3. Detection of *Salmonella* using real-time PCR.increase of the released dye concentration). Consequently, we are able to monitor, in real time, whether the product of reaction has been obtained.

#### **4.1 Conventional PCR**

400 Salmonella – A Dangerous Foodborne Pathogen

GG

TC fish 3: CAC CGA CGG CGA GAC CGA CTT T

ice cream without 2: TGG TAT CGA CGC CTT TAT CTG AGA

GGC TGG G

GGC AA

CGT AA

TGG GTT TTG TTG

(pre-) 1: AAC GTG TTT CCG TGC GTA AT

shrimp (selective) 3: TGG AAG CGC TCG CAT TGT GG

(Set I)

meat 1: CATTGATGCCATGGGTGACART

 3: TACACGAGTCACTAAATCCTTCAGT (Set II)

concentration). Consequently, we are able to monitor, in real time, whether the product of

Table 3. Detection of *Salmonella* using real-time PCR.increase of the released dye

cheddar 2: TGAGCATCGCCATCGGCAT

6,1 x 101 CFU/ml

et al., 2008 chili

CFU/g 2: TCC ATC AAA TTA GCG GAG GC

1: TCACCTGCGACAGCCATGA

3: ATTCCAGCAGTCGGCCATAGCTG

2: CGTGACGATAATCCGTGTAC

**Primers (1-forward; 2-reverse;** 

1: CTC ACC AGG AGA TTA CAA CAT

2: AGC TCA GAC CAA AAG TGA CCA

1: ATA AAT CCG GCG GCC TGA TG

3: TTA CAC CGG AGT GGA TTA AAC

1: GTG AAA TAA TCG CCA CGT TCG

2: TCA TCG CAC CGT CAA AGG AAC

3: TTA TTG GCG ATA GCC TGG CGG

**3-probe) References** 

Malorny et al., 2004

Seo et al., 2006

Hein et al., 2006

Cheng

McCarthy et al., 2009

**detection**

CFU/ml

CFU/ml

2,5-5 CFU/25g

(salmon, chicken)

5 CFU/25 ml (milk)

20 h < 3

**Gene** 

*ttrBCA*

*invA* 

*invA* 

*oriC* 

chicken

minced meat

salmon

chicken meat

milk

turkey meat

cooked turkey

reaction has been obtained.

**Target Matrices Enrichment Limit of** 

*fimC* <sup>103</sup>

16h

35°C-24h

powder 41°C-24h 0,04

48 h (selective) PCR – polymerase chain reaction – is an enzymatic reaction replicating a DNA molecule. A critical condition of the PCR course is the presence of an enzyme, thermostable DNA polymerase. Substrates of the reaction are deoxyribonucleotides (building blocks of newly synthesized DNA molecules) and oligonucleotides (starters), which on a complementary basis attach to the replicated DNA strand. Polymerase chain reaction is a thermal process, with cyclically changing temperatures. One cycle leading to synthesis of one copy of DNA from one matrix molecule is composed of the following stages: DNA denaturation (90-95°C), attachment of starters (the so-called annealing, 50-65°C) and strand elongation. Reaction under laboratory conditions is carried out in a device known as thermocycler. Usually, one PCR reaction is made of 35-40 cycles, which result in creating billions of copies of an individual DNA molecule (with a logarithmic increase of the product in each cycle).

#### **4.2 Real–time PCR**

Real-time PCR is a polymerase chain reaction observed in real time, is an improvement of the classical PCR method (Al-Soud et al., 2005; Bansal et al., 2006). A real-time PCR thermocycler is equipped with an optical system which collects information about the course of reaction, cycle by cycle. TaqMan® probes labelled with fluorescent dyes make an additional component of PCR real time reactions. A probe is an oligonucleotide designed to bind highly specifically to a replicated fragment of DNA, therefore – in case of the analysed application in food microbiology – to "detect" a sequence which is characteristic for the required pathogen. The probe should bind specifically and strongly, which can be ensured by special protein molecules (a MGB molecule in TaqMan® Applied Biosystems probes). Polymerase, while synthesising a new strand, moves along the matrix, encountering a probe on its way. Because of its exonucleolytic activity, the enzyme starts to "unstick" the probe from the matrix and afterwards to destroy it, releasing a fluorescent dye. An optical system of the thermocycler triggers and then receives dye glow, which becomes more intense with each cycle (a logarithmic increase of the amount of product results in a logarithmic

A forty-cycle real-time PCR reaction lasts about 1.5 hours. After adding the time needed to isolate DNA from the analysed sample (up to 30 minutes), the entire determination of the presence or the lack of pathogen lasts 2 hours (while using the Applied Biosystems TaqMan® Pathogen Detection System). Samples for determination are taken from preenrichment cultures on buffered peptone water after 18 h incubation at 37°C, so the total time from the collection of a sample to the final results does not exceed 24 hours.

Chen *et al*. (2000) evaluated the TaqMan system for the detection of *Salmonella* that utilizes primers and probes developed from a novel target sequence (*inv*A). The detection limit was below 3 CFU/25 g or 25 ml when raw milk, ground beef and ground pork inoculated with *Salmonella* were pre-enriched overnight. Malorny *et al*. (2004) used specifically designed primers and a probe target within the ttrRSBCA locus, and included internal amplification control, which is coamplified with the same primers as the *Salmonella* DNA in the assay. The diagnostic accuracy was shown to be 100% compared to the traditional culture method when 110 various food samples (chicken rinses, minced meat, fish, and raw milk) were investigated for *Salmonella* by real-time PCR including a pre-enrichment step in buffered peptone water.

A very frequent target of species-specific *Salmonella* PCR assays is the invasion protein *inv*A gene, and several *inv*A-based PCR assays have been already developed and validated (Malorny et al. 2003a; b).

Detection of *Salmonella* spp. Presence in Food 403

The FISH technique consists in hybridization of the rRNA sequence of immobilized cells by a fluorescently-labelled 16S rRNA oligonucleotide probe (Zwirglmaier, 2005; Baudart et al., 2005). Oligonucleotide probes are short fragments of deoxyribonucleic acid which hybridize or are paired with complementary sequences of DNA or RNA extracted from the analysed microorganisms. They are paired in the same way as double-stranded DNA forms (adenine with thymine and guanine with cytosine). If the sequence of bases on the DNA probe is complementary to the sequence characteristic for the determined microorganism, the probe binds only with the DNA of the identified microorganism. Probes are most often marked on one or on both ends with a fluorescent dye. Molecular probes bind specifically to rRNA in ribosomes of the target cells, identifying them on various taxonomic levels. Such a solution significantly increases the sensitivity of determination – since rRNA is an integral part of bacterial ribosome, it is found in the cell in large number of copies (between 1,000 and 10,000). Another advantage of this solution is the availability of vast information concerning rRNA sequences originating from various microorganisms which are often very closely genetically related, which allows probes to have very high specificity (Sakai et al., 2004; Ercoloni et al., 2005) Due to the range of probe specificity, the following probes can be distinguished: universal, e.g. EUB338 (GCTGCCTCCCGTAGGAGT), specific for *Bacteria* domain, except for the *Planctomycetales* order, antisense, e.g. NON388 (CGACGGAGGGCATCCTCA) designed to detect non-specific probe binding, and specific

probes, e.g. for *Salmonella* sp.: Sal3 (5'-AATCACTTCACCTACGTG-3').

The FISH method with the application of fluorescently labelled 16S rRNA oligonucleotide probes is used for determining only the number of physiologically active cells, since rRNA

Fig. 1. Flow chart of a typical FISH procedure.

Validated PCR methods are available from Bio-Rad, Roche, Qualicon/Oxoid, Genesystems, AES Chemunex, Applied BioSystems, Idaho Technology Inc., Lantmännen, IEH Laboratries and Consulting Group, ADNucleis and BioControl systems. Validation is an important step in the process of standardizing a method because it provides evidence that the new method gives similar results and is in agreement with the currently used reference method (Patel et al., 2006).

One major difficulty with PCR is the presence of compounds that inhibit the PCR reaction. These compounds can contaminate the DNA templates extracted from food samples and may in turn generate false-negative results (Elizaquivel et al., 2008). Therefore, evaluation and elimination of PCR inhibitory compounds are important steps in the development of PCR and real-time PCR assays (Abu Al-Soud et al., 2000). The PCR procedure is sufficiently sensitive such that, in theory, only a few template molecules are required to initiate the synthesis reactions (Uyttendaele et al., 2003). However, an enrichment step is still required to detect small numbers of *Salmonella* in food samples. This step may consist of non-selective enrichment with buffered peptone water (BPW) and selective enrichment with Rappaport-Vassiliadis. These enrichment broth have been directly utilized for *Salmonella* DNA template preparation. However, limited research has been conducted to quantitatively evaluate the effects of the enrichment broths using conventional PCR assays and even less using a realtime PCR protocol. Therefore, identifying and eliminating the PCR inhibitory effects of the enrichment broths is key to enhancing the performance of PCR assays in detecting *Salmonella* in foods.
