Preface

The book *Biotechnology and Bioengineering* shows how biotechnology and bioengineering can be used to solve the demands of modern society. In these eleven chapters, the book explores several important topics of biotechnology and bioengineering such as bioinstrumentation, biomaterials and nanotechnology, cell and tissue engineering and design, and application of bioreactors. The chapters are intended to help provide a deeper and wider understanding of the promises and challenges for biotechnology and bioengineering. This book is primarily dedicated to scientists, academicians, industrial representatives, and innovative technology representatives, and also to any nonspecialist reader willing to learn about the dynamic field of biotechnology and bioengineering.

**II**

**Chapter 9 117**

**Chapter 10 137** Phytoremediation of Effluents Contaminated with Heavy Metals by Floating

**Chapter 11 155** Performance of Anoxic-Oxic Sequencing Batch Reactor for Nitrification and

Microbial Bioremediation and Different Bioreactors Designs Applied

*by Juan C. Alzate Marin, Alejandro H. Caravelli and Noemí E. Zaritzky*

*by Cleide Barbieri de Souza and Gabriel Rodrigues Silva*

*by Memory Tekere*

Aquatic Macrophytes Species

Aerobic Denitrification

**Eduardo Jacob-Lopes and Leila Queiroz Zepka** Food Science and Technology Department, Federal University of Santa Maria, Santa Maria, RS, Brazil

**1**

**Chapter 1**

Introductory Chapter:

*and Eduardo Jacob-Lopes*

*Rosangela Rodrigues Dias, Leila Queiroz Zepka* 

**1. A general overview on biotechnology and bioengineering**

biotechnology is more focused on the natural sciences [2].

scientific principles were not understood [3, 4].

ecology, computer science, and chemical engineering.

processes in favor of human benefit [6–8].

Biotechnology and Bioengineering

Biotechnology and bioengineering can be defined as "the integration of natural

These fields, today considered priority, are the fruit of strategies from various areas that aim to benefit humanity and its environment. Biotechnology and bioengineering, however, despite growing attention in recent decades, are not a new science. Humans have been developing them since the earliest beginnings, mainly in food production. Some ancestral examples include the preparation of fermented beverages from cereals in Babylon and Egypt (8000 to 6000 years BC); the production of bread, using ferment in Egypt (4000 years BC); and wine production in Greece (2000 years BC). Historically, the use of these traditional techniques in this period of history is called discoveries, and not development, once the underlying

Indeed today, the biotechnology and bioengineering based on scientific progress find applications in several areas, including agriculture, livestock, human health, preservation of the environment, and manufacturing industry [5]. This wide applicability was only possible due to the combination of several fields of knowledge that include biochemistry, physiology, genetics, microbiology, virology, botany, zoology,

Therefore, this is a field of work typically multidisciplinary, which makes the effective collaboration and integration of professionals from different areas of knowledge absolutely indispensable so that all potential of biotechnology and bioengineering can be exploited. The interface between these fields is now understood not only as a "science" to learn about nature but also as a "technology" of susceptible alteration. The intersection between biotechnology and bioengineering and its kindred disciplines proved their economic importance, being capable to expand and promote the manufacture of products and services, besides modifying

The current scenario points for biotechnology and bioengineering as being the main technology of the twenty-first century should be absorbed by the general public. Undoubtedly, knowledge of the principles of vital processes already achieved will proportionate changes significant in the society. Therefore, it is important to

sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services" [1]. Although these areas overlap, depending upon the use of techniques and their applications, both have peculiar characteristics. While the focus of bioengineering is the implementation of engineering principles and design concepts in biology,

#### **Chapter 1**

## Introductory Chapter: Biotechnology and Bioengineering

*Rosangela Rodrigues Dias, Leila Queiroz Zepka and Eduardo Jacob-Lopes*

#### **1. A general overview on biotechnology and bioengineering**

Biotechnology and bioengineering can be defined as "the integration of natural sciences and engineering sciences in order to achieve the application of organisms, cells, parts thereof and molecular analogues for products and services" [1]. Although these areas overlap, depending upon the use of techniques and their applications, both have peculiar characteristics. While the focus of bioengineering is the implementation of engineering principles and design concepts in biology, biotechnology is more focused on the natural sciences [2].

These fields, today considered priority, are the fruit of strategies from various areas that aim to benefit humanity and its environment. Biotechnology and bioengineering, however, despite growing attention in recent decades, are not a new science. Humans have been developing them since the earliest beginnings, mainly in food production. Some ancestral examples include the preparation of fermented beverages from cereals in Babylon and Egypt (8000 to 6000 years BC); the production of bread, using ferment in Egypt (4000 years BC); and wine production in Greece (2000 years BC). Historically, the use of these traditional techniques in this period of history is called discoveries, and not development, once the underlying scientific principles were not understood [3, 4].

Indeed today, the biotechnology and bioengineering based on scientific progress find applications in several areas, including agriculture, livestock, human health, preservation of the environment, and manufacturing industry [5]. This wide applicability was only possible due to the combination of several fields of knowledge that include biochemistry, physiology, genetics, microbiology, virology, botany, zoology, ecology, computer science, and chemical engineering.

Therefore, this is a field of work typically multidisciplinary, which makes the effective collaboration and integration of professionals from different areas of knowledge absolutely indispensable so that all potential of biotechnology and bioengineering can be exploited. The interface between these fields is now understood not only as a "science" to learn about nature but also as a "technology" of susceptible alteration. The intersection between biotechnology and bioengineering and its kindred disciplines proved their economic importance, being capable to expand and promote the manufacture of products and services, besides modifying processes in favor of human benefit [6–8].

The current scenario points for biotechnology and bioengineering as being the main technology of the twenty-first century should be absorbed by the general public. Undoubtedly, knowledge of the principles of vital processes already achieved will proportionate changes significant in the society. Therefore, it is important to

ensure a broad awareness of what these two fields of knowledge involve and what the consequences of accepting or rejecting the innovations [9].

Thus, the chapters presented in this book are intended to help provide a deeper understanding about the recent progresses on biotechnology and bioengineering contributing substantially to the consolidation of bio-based processes and products.

### **Author details**

Rosangela Rodrigues Dias, Leila Queiroz Zepka and Eduardo Jacob-Lopes\* Food Science and Technology Department, Federal University of Santa Maria, UFSM, Santa Maria, RS, Brazil

\*Address all correspondence to: ejacoblopes@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**3**

*Introductory Chapter: Biotechnology and Bioengineering DOI: http://dx.doi.org/10.5772/intechopen.86380*

Elsevier; 2019. pp. 1-442. DOI: 10.1016/

[9] Farouki N. Biotechnologies: The ideal victim? Advances in Botanical Research.

2018;**86**:325-336. DOI: 10.1016/

C2017-0-01925-0

bs.abr.2017.11.008

[1] IUPAC. Compendium of Chemical Terminology. Oxford, UK: Blackwell Scientific Publications; 2014. DOI:

10.1351/goldbook.B00666

[2] Munshi A, Sharma V. Safety and ethics in biotechnology and bioengineering: What to follow and what not to. In: Omics Technologies and Bio-Engineering. London, UK: Academic Press; 2018. pp. 577-590. DOI: 10.1016/ B978-0-12-804659-3.00025-7

[3] Amarakoon II, Hamilton C-L, Mitchell SA, Tennant PF, Roye ME. Biotechnology. In: Pharmacognosy: Fundamentals, Applications and Strategies. London, UK: Academic Press; 2017. pp. 549-563. DOI: 10.1016/

B978-0-12-802104-0.00028-7

Essential for industry. In: Omics Technologies and Bio-Engineering. London, UK: Academic Press; 2018. pp. 233-249. DOI: 10.1016/ B978-0-12-815870-8.00013-9

[5] Crawford CA. Principles of

Salem Press; 2018. pp. 1-466

[6] Gartland KM, Gartland JS.

10.1016/j.jbiotec.2018.06.303

Biotechnology. Ipswich, Massachusetts:

Opportunities in biotechnology. Journal of Biotechnology. 2018;**282**:38-45. DOI:

[7] Tylecote A. Biotechnology as a new techno-economic paradigm that will help drive the world economy and mitigate climate change. Research Policy. 2019;**48**(4):858-868. DOI: 10.1016/j.respol.2018.10.001

[8] Singh SP, Pandey A, Du G, Kumar S. Current Developments in Biotechnology and Bioengineering: Synthetic Biology, Cell Engineering and Bioprocessing Technologies. Amsterdam, Netherlands:

[4] Mustafa MG, Khan MGM, Nguyen D, Iqbal S. Techniques in biotechnology:

**References**

*Introductory Chapter: Biotechnology and Bioengineering DOI: http://dx.doi.org/10.5772/intechopen.86380*

#### **References**

*Biotechnology and Bioengineering*

**2**

**Author details**

UFSM, Santa Maria, RS, Brazil

provided the original work is properly cited.

\*Address all correspondence to: ejacoblopes@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

Rosangela Rodrigues Dias, Leila Queiroz Zepka and Eduardo Jacob-Lopes\* Food Science and Technology Department, Federal University of Santa Maria,

ensure a broad awareness of what these two fields of knowledge involve and what

Thus, the chapters presented in this book are intended to help provide a deeper understanding about the recent progresses on biotechnology and bioengineering contributing substantially to the consolidation of bio-based processes and products.

the consequences of accepting or rejecting the innovations [9].

[1] IUPAC. Compendium of Chemical Terminology. Oxford, UK: Blackwell Scientific Publications; 2014. DOI: 10.1351/goldbook.B00666

[2] Munshi A, Sharma V. Safety and ethics in biotechnology and bioengineering: What to follow and what not to. In: Omics Technologies and Bio-Engineering. London, UK: Academic Press; 2018. pp. 577-590. DOI: 10.1016/ B978-0-12-804659-3.00025-7

[3] Amarakoon II, Hamilton C-L, Mitchell SA, Tennant PF, Roye ME. Biotechnology. In: Pharmacognosy: Fundamentals, Applications and Strategies. London, UK: Academic Press; 2017. pp. 549-563. DOI: 10.1016/ B978-0-12-802104-0.00028-7

[4] Mustafa MG, Khan MGM, Nguyen D, Iqbal S. Techniques in biotechnology: Essential for industry. In: Omics Technologies and Bio-Engineering. London, UK: Academic Press; 2018. pp. 233-249. DOI: 10.1016/ B978-0-12-815870-8.00013-9

[5] Crawford CA. Principles of Biotechnology. Ipswich, Massachusetts: Salem Press; 2018. pp. 1-466

[6] Gartland KM, Gartland JS. Opportunities in biotechnology. Journal of Biotechnology. 2018;**282**:38-45. DOI: 10.1016/j.jbiotec.2018.06.303

[7] Tylecote A. Biotechnology as a new techno-economic paradigm that will help drive the world economy and mitigate climate change. Research Policy. 2019;**48**(4):858-868. DOI: 10.1016/j.respol.2018.10.001

[8] Singh SP, Pandey A, Du G, Kumar S. Current Developments in Biotechnology and Bioengineering: Synthetic Biology, Cell Engineering and Bioprocessing Technologies. Amsterdam, Netherlands:

Elsevier; 2019. pp. 1-442. DOI: 10.1016/ C2017-0-01925-0

[9] Farouki N. Biotechnologies: The ideal victim? Advances in Botanical Research. 2018;**86**:325-336. DOI: 10.1016/ bs.abr.2017.11.008

**5**

**Chapter 2**

**Abstract**

technique standardization.

**1. Introduction**

Steps and Tools for PCR-Based

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for

*Nelson Enrique Arenas and Luz Mary Salazar*

**Keywords:** PCR, molecular biology, diagnosis, molecular typing, DNA fingerprinting, bacterial identification, DNA amplification

specific genes in a clinical sample to identify a pathogen [2].

challenge was addressed on serial steps to reach the expected aim.

Bacterial culture is the conventional test to identify a microorganism which is based on the isolation and growth of live specimens [1]. Currently, these methods have been considered the gold standard method for assessing the validity of new diagnostic methods. However, phenotypic-based methods are time-consuming tests, difficult to interpret, low reproducible between laboratories, expensive, and laborious. Several commercial polymerase chain reactions or PCR-based methods are available nowadays, and like *in-house* PCR assays, they use different target

Bacterial identification in many cases is performed through a fingerprint comparison against some reference genotyping databases. Thus, many organisms can be taxonomically classified and specifically differentiated according to several conserved genes which are recognized as "molecular clocks" [3]. One well-known example is the ribosomal RNA (rRNA) gene, which is a good candidate due to its universal distribution and reasonably well conservation in sequence across evolution [4]. Thus, a good method is feasible, rapid, and cheap and can be implemented in local settings from highly endemic areas of a certain infectious disease [5]. Based on designing a proper PCR-based method, the

Technique Design

#### **Chapter 2**

## Steps and Tools for PCR-Based Technique Design

*Nelson Enrique Arenas and Luz Mary Salazar*

#### **Abstract**

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for technique standardization.

**Keywords:** PCR, molecular biology, diagnosis, molecular typing, DNA fingerprinting, bacterial identification, DNA amplification

#### **1. Introduction**

Bacterial culture is the conventional test to identify a microorganism which is based on the isolation and growth of live specimens [1]. Currently, these methods have been considered the gold standard method for assessing the validity of new diagnostic methods. However, phenotypic-based methods are time-consuming tests, difficult to interpret, low reproducible between laboratories, expensive, and laborious. Several commercial polymerase chain reactions or PCR-based methods are available nowadays, and like *in-house* PCR assays, they use different target specific genes in a clinical sample to identify a pathogen [2].

Bacterial identification in many cases is performed through a fingerprint comparison against some reference genotyping databases. Thus, many organisms can be taxonomically classified and specifically differentiated according to several conserved genes which are recognized as "molecular clocks" [3]. One well-known example is the ribosomal RNA (rRNA) gene, which is a good candidate due to its universal distribution and reasonably well conservation in sequence across evolution [4]. Thus, a good method is feasible, rapid, and cheap and can be implemented in local settings from highly endemic areas of a certain infectious disease [5]. Based on designing a proper PCR-based method, the challenge was addressed on serial steps to reach the expected aim.

#### **2. Step one: choosing the PCR-based method**

The first step is to select an accurate technique that provides enough genetic information about your model organism. Therefore, it is pivotal to outline a molecular method with high reproducibility, specificity, sensibility, and discrimination power. Some nucleic acid-based techniques with similar taxonomic ranges as other fingerprinting techniques for strain characterization include: restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), multilocus sequence typing (MLST), arbitrarily primed PCR (AP-PCR), repetitive sequence-based PCR (rep-PCR), and internal transcribed spacer-PCR (ITS-PCR) (**Table 1**). These techniques are regularly used for classification at genus, species, and subspecies level and even for strain characterization [6].

Molecular biologists are privileged to have a repertoire of tools which provide good molecular distinction which can resolve questions in the clinical settings and infectious disease control. Those methods are regularly performed for pathogen characterization and gave higher positive detection of target species than conventional methods [7]. Therefore, interlaboratory reproducibility is an important feature to produce highly valuable information [8, 9]. Thus, genotyping would provide accurate data that allow the implementation of multi-user international libraries, for example, MLST for multiple bacterial species and spoligotyping for mycobacteria [10–12]. Surveillance studies have identified certain species through DNA fingerprinting which can acquire drug-resistance determinants or clones prone to global dissemination [13, 14]. Tracking certain genotypes is a key to control them in specific geographic areas [15].

Techniques based on DNA amplification include two main approaches: the first involves amplification of one or few fragments of specific regions followed by specific restriction (e.g., ARDRA). The new fragments can be resolved by gel electrophoresis which provides specific patterns for each strain. Besides, differences in the DNA sequences or genomic regions allow its identification or polymorphisms specific at genus, species, or strains level [6]. On the other hand, unspecific probes or oligonucleotide arrays can be used for multiple amplification into the chromosomal DNA. Fragments are also separated by gel electrophoresis resulting in a band pattern or fingerprint that represents a specific genotype. Some available protocols included: arbitrary primers (e.g., RAPDs), double digest selective label (DDSL), extended primers in combination with low annealing temperatures (e.g., AP-PCR), or those based on DNA repetitive sequences from several bacteria genus (e.g., rep-PCR).


*ARDRA, amplified rDNA (ribosomal DNA) restriction analysis; tRNA-PCR, PCR-amplified length polymorphisms in tRNA intergenic spacers; RFLP, restriction fragment length polymorphism; PFGE, pulse-field gel electrophoresis; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA; MLST, multilocus sequence typing; AP-PCR, arbitrarily primed PCR; rep-PCR, repetitive sequence-based PCR; and ITS-PCR, internal transcribed spacer-PCR.*

**7**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

**3. Step two: defining a gene target**

tion in step ten).

to bacterial definition species [17].

med.miyazaki-u.ac.jp) [26].

**4. Step three: primer design for PCR-based methods**

with target DNA at the annealing temperature.

features have to be taken into consideration for accurate primer design:

To analyze the genetic variability, it is necessary to select an appropriate target which supports the required hierarchical level. Important insights remain regarding the choice of a "molecular clock" including traces of the evolutionary record from microbial diversity [16]. Ribosomal genes become ancient molecules that harbor information with high phylogenetic value and can differentiate organisms at genus and species level [17]. However, alternative core genes also defined as housekeeping have been proposed such as RNA polymerase beta subunit (*rpoB*), DNA gyrase alpha subunit (*gyrA*), glyceraldehyde 3-phosphate dehydrogenase A (*gapA*), GroEL genes (*groE, groL*), outer membrane protein A (*ompA*), and glucose-6-phosphate isomerase (*pgi*) ensuring the success for bacterial species definition [18–20]. Some of these genes are included in a multi-locus sequence typing (see further informa-

Typing methods based on the 16S rRNA genes represent an accurate strategy for strain characterization because these genes harbor both conserved and variable regions that might delineate changes on a specific position on the bacterial ribosome leading to strain differentiation [4, 17]. Although, it is important to consider that multiple copies of the 16S rRNA genes are present in all bacterial genomes. 16S rRNA gene has been subjected to many phylogenetic studies including those related

Sequencing of rRNA genes is the preferential method for phylogenetic reconstruction, nucleic acid-based detection, and quantification of microbial diversity [21]. Thus, many genotyping approaches remain based on 16S rRNA gene analysis or ribosomal gene sequencing which still constitutes a gold standard for bacterial taxonomy [22]. Therefore, it is possible to explore a sequence by searching against the Ribosomal Gene Database (RGD) release 11.5 which contains 3.356.809 16S rRNAs and 125.525 fungal 28S rRNA gene sequences by November on 2018 (https://rdp.cme.msu.edu/) [23, 24]. A similar resource containing information regarding the ribosomal genes is the SILVA databases (https://www.arb-silva.de/). It includes over 6,800,000 small (16S/18S, SSU) and large (23S/28S, LSU) subunit rRNA sequences [25]. Another useful database provides detailed information about the ribosomal protein gene database (RPG). This tool contains information from some eukarya, archaea, and bacteria organisms including sequences (genomic, cDNA, and amino acid sequences), intron/exon structures, genome locations, small nucleolar RNAs (snoRNAs), and ortholog data (http://ribosome.

Once we have chosen a gene target, the next step is the design of specific primers to detect it in the DNA sample. In the last decades, PCR-based techniques have been successfully employed for the genetic characterization of many taxa of many pathogens [2, 27]. Primers for DNA amplification are short synthetic oligonucleotides which may be complementary to target sites on the template DNA. PCR is performed at different temperatures (denaturation, annealing, and extension) where efficiency is determined based on primer annealing [28]. Some essential

1.Primers should contain guanine or cytosine, or both at the 3′-ends to increase the efficiency of oligonucleotide binding. Primers must form a stable duplex

#### **Table 1.**

*Techniques commonly used for bacterial genotyping and identification [6].*

*Biotechnology and Bioengineering*

characterization [6].

them in specific geographic areas [15].

**2. Step one: choosing the PCR-based method**

The first step is to select an accurate technique that provides enough genetic information about your model organism. Therefore, it is pivotal to outline a molecular method with high reproducibility, specificity, sensibility, and discrimination power. Some nucleic acid-based techniques with similar taxonomic ranges as other fingerprinting techniques for strain characterization include: restriction fragment length polymorphism (RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD), multilocus sequence typing (MLST), arbitrarily primed PCR (AP-PCR), repetitive sequence-based PCR (rep-PCR), and internal transcribed spacer-PCR (ITS-PCR) (**Table 1**). These techniques are regularly used for classification at genus, species, and subspecies level and even for strain

Molecular biologists are privileged to have a repertoire of tools which provide good molecular distinction which can resolve questions in the clinical settings and infectious disease control. Those methods are regularly performed for pathogen characterization and gave higher positive detection of target species than conventional methods [7]. Therefore, interlaboratory reproducibility is an important feature to produce highly valuable information [8, 9]. Thus, genotyping would provide accurate data that allow the implementation of multi-user international libraries, for example, MLST for multiple bacterial species and spoligotyping for mycobacteria [10–12]. Surveillance studies have identified certain species through DNA fingerprinting which can acquire drug-resistance determinants or clones prone to global dissemination [13, 14]. Tracking certain genotypes is a key to control

Techniques based on DNA amplification include two main approaches: the first involves amplification of one or few fragments of specific regions followed by specific restriction (e.g., ARDRA). The new fragments can be resolved by gel electrophoresis which provides specific patterns for each strain. Besides, differences in the DNA sequences or genomic regions allow its identification or polymorphisms specific at genus, species, or strains level [6]. On the other hand, unspecific probes or oligonucleotide arrays can be used for multiple amplification into the chromosomal DNA. Fragments are also separated by gel electrophoresis resulting in a band pattern or fingerprint that represents a specific genotype. Some available protocols included: arbitrary primers (e.g., RAPDs), double digest selective label (DDSL), extended primers in combination with low annealing temperatures (e.g., AP-PCR), or those based on DNA repetitive sequences from several bacteria genus (e.g., rep-PCR).

**Family Genus Species Subspecies Strain**

ARDRA, tRNA-PCR, DNA-DNA reassociation

Sequencing of 16S rRNA genes

*Techniques commonly used for bacterial genotyping and identification [6].*

Whole-genome sequencing

*ARDRA, amplified rDNA (ribosomal DNA) restriction analysis; tRNA-PCR, PCR-amplified length polymorphisms in tRNA intergenic spacers; RFLP, restriction fragment length polymorphism; PFGE, pulse-field gel electrophoresis; AFLP, amplified fragment length polymorphism; RAPD, random amplification of polymorphic DNA; MLST, multilocus sequence typing; AP-PCR, arbitrarily primed PCR; rep-PCR, repetitive sequence-based PCR; and ITS-*

RFLP, PFGE, AFLP, RAPD, MLST, AP-PCR, rep-PCR, ITS-PCR

**6**

**Table 1.**

*PCR, internal transcribed spacer-PCR.*

#### **3. Step two: defining a gene target**

To analyze the genetic variability, it is necessary to select an appropriate target which supports the required hierarchical level. Important insights remain regarding the choice of a "molecular clock" including traces of the evolutionary record from microbial diversity [16]. Ribosomal genes become ancient molecules that harbor information with high phylogenetic value and can differentiate organisms at genus and species level [17]. However, alternative core genes also defined as housekeeping have been proposed such as RNA polymerase beta subunit (*rpoB*), DNA gyrase alpha subunit (*gyrA*), glyceraldehyde 3-phosphate dehydrogenase A (*gapA*), GroEL genes (*groE, groL*), outer membrane protein A (*ompA*), and glucose-6-phosphate isomerase (*pgi*) ensuring the success for bacterial species definition [18–20]. Some of these genes are included in a multi-locus sequence typing (see further information in step ten).

Typing methods based on the 16S rRNA genes represent an accurate strategy for strain characterization because these genes harbor both conserved and variable regions that might delineate changes on a specific position on the bacterial ribosome leading to strain differentiation [4, 17]. Although, it is important to consider that multiple copies of the 16S rRNA genes are present in all bacterial genomes. 16S rRNA gene has been subjected to many phylogenetic studies including those related to bacterial definition species [17].

Sequencing of rRNA genes is the preferential method for phylogenetic reconstruction, nucleic acid-based detection, and quantification of microbial diversity [21]. Thus, many genotyping approaches remain based on 16S rRNA gene analysis or ribosomal gene sequencing which still constitutes a gold standard for bacterial taxonomy [22]. Therefore, it is possible to explore a sequence by searching against the Ribosomal Gene Database (RGD) release 11.5 which contains 3.356.809 16S rRNAs and 125.525 fungal 28S rRNA gene sequences by November on 2018 (https://rdp.cme.msu.edu/) [23, 24]. A similar resource containing information regarding the ribosomal genes is the SILVA databases (https://www.arb-silva.de/). It includes over 6,800,000 small (16S/18S, SSU) and large (23S/28S, LSU) subunit rRNA sequences [25]. Another useful database provides detailed information about the ribosomal protein gene database (RPG). This tool contains information from some eukarya, archaea, and bacteria organisms including sequences (genomic, cDNA, and amino acid sequences), intron/exon structures, genome locations, small nucleolar RNAs (snoRNAs), and ortholog data (http://ribosome. med.miyazaki-u.ac.jp) [26].

#### **4. Step three: primer design for PCR-based methods**

Once we have chosen a gene target, the next step is the design of specific primers to detect it in the DNA sample. In the last decades, PCR-based techniques have been successfully employed for the genetic characterization of many taxa of many pathogens [2, 27]. Primers for DNA amplification are short synthetic oligonucleotides which may be complementary to target sites on the template DNA. PCR is performed at different temperatures (denaturation, annealing, and extension) where efficiency is determined based on primer annealing [28]. Some essential features have to be taken into consideration for accurate primer design:

1.Primers should contain guanine or cytosine, or both at the 3′-ends to increase the efficiency of oligonucleotide binding. Primers must form a stable duplex with target DNA at the annealing temperature.


Many resources are available for primer and probe design to optimize the PCR method; therefore, the researcher might consider parameters including molecular weight, millimolar extinction coefficient, Tm, and prediction of secondary structure formation and magnesium chloride (MgCl2) concentration (**Table 2**). For some molecular biology procedures, it is recommended to design the forward primer less 35 pb downstream from the start site of the coding gene and also it applies for the reverse primer regarding the stop site. For example, in a sequencing protocol, the fragment size should not be large due to artifacts introduced on lecture sequence. Likewise, unclear results might increase concomitantly with larger fragments [29].

#### **5. Step four:** *in silico* **simulation of molecular biology experiments**

Nowadays, many resources (Web servers and programs) are available to simulate PCR results, predicting expected bands and successful primer annealing [30, 31]. Although, *in silico* simulation of several PCR-based methods is possible by using tools to obtain theoretical PCR results with many bacterial species sequenced up to date [32]. The list of target genomes is updated according to their availability at NCBI. Many experiments against prokaryotic genomes can be performed such as PCR amplification, restriction digest, and PFGE, PCR-RFLP, double digestion fingerprinting, AFLP-PCR, and other DNA fingerprinting techniques (http://insilico. ehu.es/) [33]. The PCR simulation is also possible if the researcher already knows the target sequence and can test it by using certain resources already mentioned in **Table 2**.

**9**

**6. Step five: guidelines for standardization of PCR mix**

Once, you have your DNA target and PCR primer sequences, you should set experimental conditions including water (HPLC-grade), 10× reaction buffer, MgCl2,

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

clc-main-workbench/

premierbiosoft.com/primerdesign/

free.fr/Serial\_Cloner.html

amplification (LAMP) method. https://primerexplorer.jp/e/

(OD/μmol, μg/OD) for oligos:

Oligo Calculation Tool

ervlet?TYPE=ANALYSIS

northwestern.edu/OligoCalc.html

tools-and-resources/interactive-tools.

custom-dna-oligos/oligo-design-tools.html Eurofins offers a PCR Primer Design Tool at:

https://www.idtdna.com/calc/analyzer

tools/primer-blast/

Oligo®.net This primer analysis software is useful to design sequencing and PCR primers. Also,

Primer Premier It is a PCR Primer Design Software from Premier Biosoft company. Primer Premier's

Gene Runner It is a free program for multiple sequence analysis including PCR, restriction analysis,

at: https://bibiserv.cebitec.uni-bielefeld.de/genefisher2/

https://www.genscript.com/tools/oligo-primer-calculation

PrimerExplorer This software is specifically for designing the primer sets for loop-mediated isothermal

Serial Cloner Serial Cloner 2.5 (free) lets the user to find restriction sites, define ORF, calculate Tm of

homology. Available at https://www.oligo.net/

user can check DNA and RNA secondary structure, dimer formation, false priming, and

It is commercial software from Qiagen. It provides basic and advanced sequence analysis in a user-friendly platform. Access at: https://www.qiagenbioinformatics.com/products/

search algorithm finds optimal PCR, multiplex and SNP genotyping primers with the most accurate melting temperature using the nearest neighbor algorithm. Available at: www.

sequencing primers, and hybridization probes. Available at: http://www.generunner.net/

selected fragments, %GC, and scan sequence features. Download at: http://serialbasics.

A tool for finding specific primers on a PCR template developed by the National Center for Biotechnology Information (NCBI), U.S. Available at: https://www.ncbi.nlm.nih.gov/

Since the first release, this popular Web interface has assisted many researchers with PCR

The following tools calculate Tm, molecular weight, and millimolar extinction coefficient

Thermo Fisher scientific provides the Oligo Design Tools which can be found at: https:// www.thermofisher.com/co/en/home/life-science/oligonucleotides-primers-probes-genes/

Sigma-Aldrich provides the OligoEvaluator™ http://www.oligoevaluator.com/ShowToolS

This directory contains links for: calculation of oligonucleotide physicochemical parameters, PCR primers based upon protein sequence, PCR and cloning, PCR primers based upon multi-alignments, genomic scale primers, overlapping primer sets, short interfering RNA (siRNA) design, real-time PCR primer design, introduction of mutations, and primer presentation on the DNA sequence. Available at: https://molbiol-tools.ca/PCR.htm

Oligo Calc: Oligonucleotide Properties Calculator available at http://biotools.nubic.

Oligo Calculator http://mcb.berkeley.edu/labs/krantz/tools/oligocalc.html

New England Biolabs with multiple tools available at https://www.neb.com/

https://www.eurofinsgenomics.eu/en/ecom/tools/pcr-primer-design/

https://www.genscript.com/tools/real-time-pcr-tagman-primer-design-tool

OligoAnalyzer 3.0 from Integrated DNA Technology (IDT):

GenScript Real-time PCR (TaqMan) Primer Design

primer design. The newer version of Primer3 available at http://primer3.ut.ee Genefisher2 Genefisher2 is an interactive Web-based program for designing degenerate primers. Access

**Resource Description**

CLC Main Workbench

Primer designing tool

Primer3web version 4.1.0

Tools for calculation of oligo properties

Interactive tools provided by suppliers

Directory of different tools

*Available tools for primer design.*

**Table 2.**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

*Biotechnology and Bioengineering*

above.

secondary structures such as hairpins loops.

(usually between 18 and 30 bases).

extension step (1 min/kb of PCR product).

the primers (40–60%).

to check for the specificity.

larger fragments [29].

3.No complementarity between forward and reverse primers.

2.Oligonucleotide should not be self-complementary to avoid the generation of

4.Melting temperature (Tm) defines the balance between the unbounded primer and free template compared with primer bound to the target DNA (50%). Tm is recommended to be among 42–65°C with an ideal temperature of 62°C. This parameter is a key because too high Tm can affect specificity and decrease the amount of PCR product (amplicon). If Tm is too low, unspecific products can be produced due to mismatch base pairing. Usually, Tm is set, 5°C below or 5°C

5.The optimal length of primers should be long enough to increase specificity

6.It is recommended to ensure a similar distribution of G/C and A/T content in

7.For standard PCR, length of the amplified product should be between 200 and 1000 bp and for quantitative PCR in the range from 75 to 150 bp. Nevertheless, PCR products larger than 1000 bp may require additional time during the

8.Primers must be designed specifically in the target gene to avoid cross contamination with unwanted DNA sequences in the PCR. Typically, primers are designed, and sequences are analyzed *in silico* using BLAST analysis or others,

Many resources are available for primer and probe design to optimize the PCR method; therefore, the researcher might consider parameters including molecular weight, millimolar extinction coefficient, Tm, and prediction of secondary structure formation and magnesium chloride (MgCl2) concentration (**Table 2**). For some molecular biology procedures, it is recommended to design the forward primer less 35 pb downstream from the start site of the coding gene and also it applies for the reverse primer regarding the stop site. For example, in a sequencing protocol, the fragment size should not be large due to artifacts introduced on lecture sequence. Likewise, unclear results might increase concomitantly with

**5. Step four:** *in silico* **simulation of molecular biology experiments**

Nowadays, many resources (Web servers and programs) are available to simulate PCR results, predicting expected bands and successful primer annealing [30, 31]. Although, *in silico* simulation of several PCR-based methods is possible by using tools to obtain theoretical PCR results with many bacterial species sequenced up to date [32]. The list of target genomes is updated according to their availability at NCBI. Many experiments against prokaryotic genomes can be performed such as PCR amplification, restriction digest, and PFGE, PCR-RFLP, double digestion fingerprinting, AFLP-PCR, and other DNA fingerprinting techniques (http://insilico. ehu.es/) [33]. The PCR simulation is also possible if the researcher already knows the target sequence and can test it by using certain resources already mentioned in

**8**

**Table 2**.


#### **Table 2.**

*Available tools for primer design.*

#### **6. Step five: guidelines for standardization of PCR mix**

Once, you have your DNA target and PCR primer sequences, you should set experimental conditions including water (HPLC-grade), 10× reaction buffer, MgCl2, dNTPs, primers (forward and reverse), sample DNA, and DNA polymerase [29]. The 10× reaction buffer includes magnesium, thus it is optional to use separately as MgCl2. If so, typical MgCl2 concentration in a standard PCR should be between 1.5 and 2.0 millimolar (mM). When magnesium is too low, no amplicon might appear; but if it is too high, undesired amplicons would be observed as extra bands in the agarose gel. For dNTP, concentration should be 200 μM of each nucleotide. Regarding the Taq (obtained from *Thermus aquaticus*) DNA polymerase (further information in step 6), it is recommended the addition between 0.5 and 2.0 units per 50 μL mix (preferably 1.25 units). Primers work well at the default concentration (50 nM), but concentrations between 0.1 μM and 1 μM of each primer are recommended. Last, DNA sample should be used between 1 ng and 1 μg of genomic templates because higher amounts might reduce PCR product specificity [28].

#### **7. Step six: choosing the DNA polymerase enzyme**

DNA polymerase is an enzyme which synthesizes the new DNA strands. DNA polymerase was first isolated from *T. aquaticus* in 1976 [34]. This enzyme has an optimum temperature between 75 and 80°C, which possess a half-life until 97.5°C during 9 min and can polymerize 150 nucleotides per second [35]. When choosing a DNA polymerase, the researcher must consider key aspects such as specificity, thermostability, fidelity, and processivity. First, if specificity is low, low-quality amplicon would affect the yield product, sensitivity, and possible problems in downstream applications (e.g., cloning or protein expression). Second, regarding thermostability, consider using enzymes with a half-life above 90°C because of the denaturing step. Third, if the researcher needs amplicons with 100% similarity to DNA target, consider using high-fidelity DNA polymerases with significantly proof-reading activity. Last, processivity reflects the rate and speed of the reaction from the enzyme. Thus, processivity should be considered in case of long templates, self-complementary targets, high G/C content, and samples containing PCR inhibitors including if the amplicon is accumulating during later PCR cycles [29, 36].

#### **8. Step seven: setting the PCR conditions**

The PCR runs in cycles composed of three called steps: denaturation, annealing, and extension. For default, PCR includes between 25 and 35 cycles per reaction. The denaturation step produces single-stranded DNA and usually is performed initially at 95°C for 2 min [37]. The following step is the primer annealing which pair-base primer with the complementary DNA template and generally is carried out considering the primer's Tm of both PCR oligos. Third, for extension step is recommended to set 1 min per 1000 bp (or 30 s per 500 bp) of the amplicon. Larger PCR products (>3 kb) may require longer extension times. The extension is usually performed at 72°C which is considered the optimum temperature for thermostable DNA polymerases. The standard PCR protocol for a 500 bp amplicon includes: an initial denaturation at 95°C for 2 min, followed by 25 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 68°C for 45 s. One additional cycle of final extension is at 68°C for 5 min [28, 29].

#### **9. Step eight: setting specificity and sensitivity of the PCR method**

To test assay specificity, it should be assessed against many related microorganisms. Potential cross-reactivity with DNA contaminants in the sample should also be investigated especially when the method applies to natural populations [38]. This issue is essential, particularly when the new method is compared with traditional techniques.

**11**

**Table 3.**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

**10. Step nine: evaluation of the amplicons**

**databases**

Specificity is first tested *in silico* using the BLAST tool [39]. Then, specificity can be assessed in vitro by the PCR amplification of genomic DNA purified from taxonomically related species. Regarding sensitivity, defines the detection limit of the minimum of DNA target in a sample. This issue is relevant when it is difficult to obtain cultures or when the low number of bacteria cannot be detected in other diagnostic technique [40]. Identifying the bacterial species related to clinical phenotypes requires a method

to cluster fingerprints into groups which are likely to share most genotypic and phenotypic traits [8]. Instead, species have been genotyped by measuring genetic variation in the number of repetitive genetic elements in the direct repeat region or detection of polymorphic sequence [41]. These techniques have identified clonal groups of isolates that each appears to be related through a common ancestor [42].

Experimental validation of PCR results entails two possibilities: the first option

is to load and run the PCR product on an agarose gel testing the expected sizes with the suitable molecular weight marker. In the second place, it is to sequence the amplicon evaluating its sequence identity compared to the DNA target [28, 29].

**11. Step ten: comparing a query band pattern or DNA sequence against** 

Genotyping programs rely on the collection and analysis of large quantities of data. Control infection programs are implementing genotyping programs for comparing against a database. Central databases for isolate tracking, laboratory

**Resource Description Reference**

BIGSdb software runs in PubMLST to store and analyze sequence data for

reads. It is brought by the Center for Genomic Epidemiology https://cge.

the typing of microbial isolates from whole-genome sequencing data.

quantification (real-time PCR). User can search by using GenBank Accession, NCBI protein accession, NCBI Gene ID, Gene Symbol, Primer-Bank ID or Keyword (gene description) or blast a gene sequence.

[10]

[45]

[47]

[15]

[50]

[48, 49]

[46]

PubMLST This database contains more than 140 MLST allelic profiles and sequences.

MLST 2.0 Multilocus sequence typing from an assembled genome or from a set of

LOCUST It is an automated classification program that allows users to customize

BacWGSTdb This database was designed for genotyping and source tracking bacterial

http://sourceforge.net/projects/locustyper

PrimerBank Databank of PCR primers for gene expression detection and

https://pga.mgh.harvard.edu/primerbank/

RTPrimerDB It is a public database for primer and probe sequences used in real-time

RUCS 1.0 Rapid identification of PCR primers pairs for unique core sequences.

*Web resources for genotyping based on MLST and WGS data. Primer databases were also included.*

bacterial isolates. https://pubmlst.org/

cbs.dtu.dk/services/MLST/

http://bacdb.org/BacWGSTdb/

http://www.rtprimerdb.org/

https://cge.cbs.dtu.dk/services/rucs/

pathogens.

PCR assays.

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

*Biotechnology and Bioengineering*

dNTPs, primers (forward and reverse), sample DNA, and DNA polymerase [29]. The 10× reaction buffer includes magnesium, thus it is optional to use separately as MgCl2. If so, typical MgCl2 concentration in a standard PCR should be between 1.5 and 2.0 millimolar (mM). When magnesium is too low, no amplicon might appear; but if it is too high, undesired amplicons would be observed as extra bands in the agarose gel. For dNTP, concentration should be 200 μM of each nucleotide. Regarding the Taq (obtained from *Thermus aquaticus*) DNA polymerase (further information in step 6), it is recommended the addition between 0.5 and 2.0 units per 50 μL mix (preferably 1.25 units). Primers work well at the default concentration (50 nM), but concentrations between 0.1 μM and 1 μM of each primer are recommended. Last, DNA sample should be used between 1 ng and 1 μg of genomic templates because higher amounts might reduce PCR product specificity [28].

DNA polymerase is an enzyme which synthesizes the new DNA strands. DNA polymerase was first isolated from *T. aquaticus* in 1976 [34]. This enzyme has an optimum temperature between 75 and 80°C, which possess a half-life until 97.5°C during 9 min and can polymerize 150 nucleotides per second [35]. When choosing a DNA polymerase, the researcher must consider key aspects such as specificity, thermostability, fidelity, and processivity. First, if specificity is low, low-quality amplicon would affect the yield product, sensitivity, and possible problems in downstream applications (e.g., cloning or protein expression). Second, regarding thermostability, consider using enzymes with a half-life above 90°C because of the denaturing step. Third, if the researcher needs amplicons with 100% similarity to DNA target, consider using high-fidelity DNA polymerases with significantly proof-reading activity. Last, processivity reflects the rate and speed of the reaction from the enzyme. Thus, processivity should be considered in case of long templates, self-complementary targets, high G/C content, and samples containing PCR inhibitors including if the amplicon is accumulating during later PCR cycles [29, 36].

The PCR runs in cycles composed of three called steps: denaturation, annealing, and extension. For default, PCR includes between 25 and 35 cycles per reaction. The denaturation step produces single-stranded DNA and usually is performed initially at 95°C for 2 min [37]. The following step is the primer annealing which pair-base primer with the complementary DNA template and generally is carried out considering the primer's Tm of both PCR oligos. Third, for extension step is recommended to set 1 min per 1000 bp (or 30 s per 500 bp) of the amplicon. Larger PCR products (>3 kb) may require longer extension times. The extension is usually performed at 72°C which is considered the optimum temperature for thermostable DNA polymerases. The standard PCR protocol for a 500 bp amplicon includes: an initial denaturation at 95°C for 2 min, followed by 25 cycles of denaturation at 95°C for 15 s, annealing at 55°C for 15 s, and extension at 68°C for 45 s. One additional cycle of final extension is at 68°C for 5 min [28, 29].

**9. Step eight: setting specificity and sensitivity of the PCR method**

To test assay specificity, it should be assessed against many related microorganisms. Potential cross-reactivity with DNA contaminants in the sample should also be investigated especially when the method applies to natural populations [38]. This issue is essential, particularly when the new method is compared with traditional techniques.

**7. Step six: choosing the DNA polymerase enzyme**

**8. Step seven: setting the PCR conditions**

**10**

Specificity is first tested *in silico* using the BLAST tool [39]. Then, specificity can be assessed in vitro by the PCR amplification of genomic DNA purified from taxonomically related species. Regarding sensitivity, defines the detection limit of the minimum of DNA target in a sample. This issue is relevant when it is difficult to obtain cultures or when the low number of bacteria cannot be detected in other diagnostic technique [40].

Identifying the bacterial species related to clinical phenotypes requires a method to cluster fingerprints into groups which are likely to share most genotypic and phenotypic traits [8]. Instead, species have been genotyped by measuring genetic variation in the number of repetitive genetic elements in the direct repeat region or detection of polymorphic sequence [41]. These techniques have identified clonal groups of isolates that each appears to be related through a common ancestor [42].

#### **10. Step nine: evaluation of the amplicons**

Experimental validation of PCR results entails two possibilities: the first option is to load and run the PCR product on an agarose gel testing the expected sizes with the suitable molecular weight marker. In the second place, it is to sequence the amplicon evaluating its sequence identity compared to the DNA target [28, 29].

#### **11. Step ten: comparing a query band pattern or DNA sequence against databases**

Genotyping programs rely on the collection and analysis of large quantities of data. Control infection programs are implementing genotyping programs for comparing against a database. Central databases for isolate tracking, laboratory


**Table 3.**

*Web resources for genotyping based on MLST and WGS data. Primer databases were also included.*

results, and epidemiologic data are essential. Because cluster investigations are an epidemiologic activity, the infectious disease programs should maintain the principal databases for spread analysis and control measurements [43]. The information in these databases can enable infectious disease programs to identify easily patients with matching genotypes and epidemiologic links [44]. Today, information available regarding bacterial genotyping at both traditional MLST and whole-genome sequencing (WGS) are available (**Table 3**) [10, 15, 45–47]. For more applications, public resources also store primer information for quantitative gene expression analysis or comparing with previous reports [48–50].

### **12. Variants of the PCR methods**

Beyond PCR use in the lab, availability of an improved version of thermocyclers, dyes, primers, probes, and DNA polymerases have extended applications.


**13**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

**13. Educational tools for beginners**

exercises based on available bacterial genomes [32].

conventional tests [68].

**14. Case study: identification at species and subspecies level**

PCR-based detection based on the conserved regions of the 16S rRNA sequence of bacterial pathogens is currently performed by several groups [4, 18, 21]. The rRNA at SSU contains segments that are conserved in species, genus, and kingdom level. In this case, *Klebsiella pneumoniae* is divided into three subspecies: *K. pneumoniae pneumoniae*, *K. pneumoniae rhinoscleromatis*, and *K. pneumoniae ozaenae*. All together are phenotypically closely related and difficult to differentiate based on

The 16S rRNA gene sequences from *K. pneumoniae* subspecies were retrieved in FASTA format and aligned by using MACAW program (download link http://en.biosoft.net/format/MACAW.html). Subsequently, sequences were virtually cleaved according to restriction enzyme database obtained from Rebase (GCG format) implemented in GeneDoc program (available in https://www.softpedia.com/get/ Science-CAD/GeneDoc.shtml). Restriction patterns were predicted by testing each enzyme and considering specific patterns. Primers were custom-designed using Gene Runner program targeting 16S rRNA gene (**Table 2**). Experimental validation was performed by harvesting the reference strains and extracting their genomic DNA. After PCR, amplification products and restriction enzyme digests were resolved by using agarose gel electrophoresis. Restriction enzyme combinations generated fragments that allowed easy identification of subspecies after separation of digested DNA. Primer design entailed a challenge since 16S rRNA genes are highly conserved in all bacterial genomes and may be present in multiple copies [69, 70]. PCR-RFLP protocol was designed specifically to detect single nucleotide

antibiotics [65].

Many PCR variants allow quantify gene expression, improving diagnostic sensitivity and genotyping without further procedures as restriction analysis (**Table 4**) [51–64]. Advanced technique such as digital PCR (dPCR) has showed to improve sensitivity and reliability until single-cell applications and tolerance to PCR inhibitors such as chelating agents [64]. Finally, another common application of several PCR-based techniques (conventional PCR, RT-PCR, qPCR, and LAMP assays) is the detection of antimicrobial resistance against first-line drugs or even last-resort

Biotechnology students face challenges understanding molecular biology principles and techniques. E-learning resources simulate a real environment where students create a real experience when running protocols in the lab [66, 67]. The genetic science learning center provides a flash-simulation of PCR principles useful for beginners (https://learn.genetics.utah.edu/content/labs/pcr/). This virtual lab explains step-by-step concepts behind the technique principles and concept background. Another educational tool to train students not only in the method but also regarding interpretation results is the virtual application for bacterial identification (available on http://www.hhmi.org/biointeractive/vlabs/bacterial\_id/index.html). The experiment's purpose is to use molecular tools to identify different bacterial species based mainly on their 16S rRNA genes. This virtual lab also requires that students prepare a patient's sample, isolate the whole bacterial DNA, perform a DNA sequencing analysis, run the query sequence in the BLAST tool, and identify the pathogen. Finally, students can practice PCR-RFLP simulations through online

#### **Table 4.**

*Comparison of several PCR-based methods.*

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

*Biotechnology and Bioengineering*

analysis or comparing with previous reports [48–50].

Nested PCR Two pairs of primers are used to amplify a

the first fragment

Multiplex PCR

Real-time PCR or quantitative PCR

Arbitrary Primed PCR (AP-PCR)

Reverse transcriptase PCR

Loop Mediated Isothermal Amplification (LAMP)

fragment. First pair amplifies similar to a conventional PCR. Second pair is nested within

This PCR variation enables the simultaneous amplification of many targets in a single reaction by using over one pair of primers. Amplicon sizes should be different or be labeled

It is an assay that monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (realtime) as opposed to the endpoint detection

direction of the usual orientation. The template for the reverse primers is a restriction fragment

This technique uses primers whose nucleotide sequence is randomly chosen. Amplification occurs under low stringency conditions

This PCR transcribes RNA into DNA. It allows the synthesis of complementary DNA (cDNA)

This PCR variant runs at a single temperature (usually 63–67°C) and requires 6 primers, and it is amenable to visual detection. Amplification

multiple compartments so that only few molecules are present in each partition. Thus, each droplet will be an independent PCR

Inverse PCR In this PCR, primers are oriented in the reverse

that has been self-ligated

occurs in less than 30 min

Droplet PCR It includes a separation step of sample into

*Comparison of several PCR-based methods.*

**12. Variants of the PCR methods**

results, and epidemiologic data are essential. Because cluster investigations are an epidemiologic activity, the infectious disease programs should maintain the principal databases for spread analysis and control measurements [43]. The information in these databases can enable infectious disease programs to identify easily patients with matching genotypes and epidemiologic links [44]. Today, information available regarding bacterial genotyping at both traditional MLST and whole-genome sequencing (WGS) are available (**Table 3**) [10, 15, 45–47]. For more applications, public resources also store primer information for quantitative gene expression

Beyond PCR use in the lab, availability of an improved version of thermocyclers, dyes, primers, probes, and DNA polymerases have extended applications.

**Technique Principle Use Reference**

in a clinical sample

Increase specificity of an amplification reaction when targeting a gene

Screening for a set of genes at once in a DNA sample. Analysis of microsatellites and SNPs

DNA quantification in a sample. Level of gene expression. Copy number variation. Genotyping. Multi-species analysis

Cloning of sequences flanking a known sequence. Amplification and identification of flanking sequences

Evidence regarding the transcription of a gene. Complementary DNA

Point of care methods for pathogen screening

Rare species detection and mutations with low

synthesis

frequency

DNA fingerprinting [55–57]

[62]

[61]

[60]

[59]

[58]

[51]

[52]

[53]

*In house* PCR Conventional PCR targeting a single gene Detection of a pathogen

**12**

**Table 4.**

Many PCR variants allow quantify gene expression, improving diagnostic sensitivity and genotyping without further procedures as restriction analysis (**Table 4**) [51–64]. Advanced technique such as digital PCR (dPCR) has showed to improve sensitivity and reliability until single-cell applications and tolerance to PCR inhibitors such as chelating agents [64]. Finally, another common application of several PCR-based techniques (conventional PCR, RT-PCR, qPCR, and LAMP assays) is the detection of antimicrobial resistance against first-line drugs or even last-resort antibiotics [65].

#### **13. Educational tools for beginners**

Biotechnology students face challenges understanding molecular biology principles and techniques. E-learning resources simulate a real environment where students create a real experience when running protocols in the lab [66, 67]. The genetic science learning center provides a flash-simulation of PCR principles useful for beginners (https://learn.genetics.utah.edu/content/labs/pcr/). This virtual lab explains step-by-step concepts behind the technique principles and concept background. Another educational tool to train students not only in the method but also regarding interpretation results is the virtual application for bacterial identification (available on http://www.hhmi.org/biointeractive/vlabs/bacterial\_id/index.html). The experiment's purpose is to use molecular tools to identify different bacterial species based mainly on their 16S rRNA genes. This virtual lab also requires that students prepare a patient's sample, isolate the whole bacterial DNA, perform a DNA sequencing analysis, run the query sequence in the BLAST tool, and identify the pathogen. Finally, students can practice PCR-RFLP simulations through online exercises based on available bacterial genomes [32].

#### **14. Case study: identification at species and subspecies level**

PCR-based detection based on the conserved regions of the 16S rRNA sequence of bacterial pathogens is currently performed by several groups [4, 18, 21]. The rRNA at SSU contains segments that are conserved in species, genus, and kingdom level. In this case, *Klebsiella pneumoniae* is divided into three subspecies: *K. pneumoniae pneumoniae*, *K. pneumoniae rhinoscleromatis*, and *K. pneumoniae ozaenae*. All together are phenotypically closely related and difficult to differentiate based on conventional tests [68].

The 16S rRNA gene sequences from *K. pneumoniae* subspecies were retrieved in FASTA format and aligned by using MACAW program (download link http://en.biosoft.net/format/MACAW.html). Subsequently, sequences were virtually cleaved according to restriction enzyme database obtained from Rebase (GCG format) implemented in GeneDoc program (available in https://www.softpedia.com/get/ Science-CAD/GeneDoc.shtml). Restriction patterns were predicted by testing each enzyme and considering specific patterns. Primers were custom-designed using Gene Runner program targeting 16S rRNA gene (**Table 2**). Experimental validation was performed by harvesting the reference strains and extracting their genomic DNA. After PCR, amplification products and restriction enzyme digests were resolved by using agarose gel electrophoresis. Restriction enzyme combinations generated fragments that allowed easy identification of subspecies after separation of digested DNA. Primer design entailed a challenge since 16S rRNA genes are highly conserved in all bacterial genomes and may be present in multiple copies [69, 70]. PCR-RFLP protocol was designed specifically to detect single nucleotide

polymorphisms in the 16S rRNA genes from *K. pneumoniae* subspecies. In conclusion, our method combined the detection of specific nucleotide polymorphisms with the easy identification and categorization of the three bacterial subspecies from *K. pneumoniae* [68].

In another case, it was the implementation of *in-house* PCR method for tuberculosis diagnosis [62]. This molecular method might be an important tool in high-incidence areas due to its speed, sensitivity, and discriminatory power overcoming conventional methods (acid-fast stain and culture). The method was designed based on the *IS6110* gene specific for *Mycobacterium tuberculosis* complex and was successfully tested in sputum, bronchoalveolar lavage fluid, blood, gastric fluid aspirate, urine, cerebrospinal fluid, ascitic fluid, and abscess secretions. The method improved diagnostic accuracy and confirmed to be fast, low cost, and feasible and can be implemented in a middle-income resource setting [62].

#### **15. Conclusions**

Some bacterial pathogens may be undetectable by traditional culture methods due to their nutrient requirements, growth conditions or the bacterial inoculum per sample. Therefore, PCR emerged as the effective method which overcomes the detection limit of certain pathogens in clinical samples. The success in the PCR experiment implies planning and results prediction with available tools and resources. Web-based tools and programs are useful for primer design, calculating accurate thermodynamic and physicochemical parameters, changing the thermal cycling protocol, and performing a good experimental design. Success in the PCR-based protocol depends on performing an accurate *in silico* simulation which would allow the optimal selection of reagents and test conditions and to avoid troubleshooting on inefficient reactions. Recommendations in this chapter might enable the researcher to customize and troubleshoot a wide variety of PCR-based methods. Hence, PCR remains as a versatile technique in molecular biology that allows changes in adjustable standard protocol to any gene target choosing the most suitable option for pathogen identification.

#### **Acknowledgements**

We would like to acknowledge the Office for Research Funding from Universidad de Cundinamarca.

**15**

**Author details**

Colombia

Nelson Enrique Arenas1

provided the original work is properly cited.

Ciudad Universitaria, Bogotá, Colombia

\*Address all correspondence to: nearenass@unal.edu.co

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 Faculty of Agricultural Sciences, Universidad de Cundinamarca, Cundinamarca,

2 Chemistry Department, Faculty of Sciences, Universidad Nacional de Colombia,

\* and Luz Mary Salazar2

#### **Conflict of interest**

The authors declare not to have any conflict of interests.

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

*Biotechnology and Bioengineering*

*pneumoniae* [68].

setting [62].

**15. Conclusions**

pathogen identification.

**Acknowledgements**

**Conflict of interest**

Universidad de Cundinamarca.

polymorphisms in the 16S rRNA genes from *K. pneumoniae* subspecies. In conclusion, our method combined the detection of specific nucleotide polymorphisms with the easy identification and categorization of the three bacterial subspecies from *K.* 

In another case, it was the implementation of *in-house* PCR method for tuberculosis diagnosis [62]. This molecular method might be an important tool in high-incidence areas due to its speed, sensitivity, and discriminatory power overcoming conventional methods (acid-fast stain and culture). The method was designed based on the *IS6110* gene specific for *Mycobacterium tuberculosis* complex and was successfully tested in sputum, bronchoalveolar lavage fluid, blood, gastric fluid aspirate, urine, cerebrospinal fluid, ascitic fluid, and abscess secretions. The method improved diagnostic accuracy and confirmed to be fast, low cost, and feasible and can be implemented in a middle-income resource

Some bacterial pathogens may be undetectable by traditional culture methods due to their nutrient requirements, growth conditions or the bacterial inoculum per sample. Therefore, PCR emerged as the effective method which overcomes the detection limit of certain pathogens in clinical samples. The success in the PCR experiment implies planning and results prediction with available tools and resources. Web-based tools and programs are useful for primer design, calculating accurate thermodynamic and physicochemical parameters, changing the thermal cycling protocol, and performing a good experimental design. Success in the PCR-based protocol depends on performing an accurate *in silico* simulation which would allow the optimal selection of reagents and test conditions and to avoid troubleshooting on inefficient reactions. Recommendations in this chapter might enable the researcher to customize and troubleshoot a wide variety of PCR-based methods. Hence, PCR remains as a versatile technique in molecular biology that allows changes in adjustable standard protocol to any gene target choosing the most suitable option for

We would like to acknowledge the Office for Research Funding from

The authors declare not to have any conflict of interests.

**14**

### **Author details**

Nelson Enrique Arenas1 \* and Luz Mary Salazar2

1 Faculty of Agricultural Sciences, Universidad de Cundinamarca, Cundinamarca, Colombia

2 Chemistry Department, Faculty of Sciences, Universidad Nacional de Colombia, Ciudad Universitaria, Bogotá, Colombia

\*Address all correspondence to: nearenass@unal.edu.co

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **References**

[1] Donelli G, Vuotto C, Mastromarino P. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Microbial Ecology in Health and Disease. 2013;**24**(1):20105

[2] Yamamoto Y. PCR in diagnosis of infection: Detection of bacteria in cerebrospinal fluids. Clinical and Diagnostic Laboratory Immunology. 2002;**9**(3):508-514

[3] Ludwig W. Nucleic acid techniques in bacterial systematics and identification. International Journal of Food Microbiology. 2007;**120**(3):225-236

[4] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology. 1991;**173**(2):697-703

[5] Reller LB, Weinstein MP, Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clinical Infectious Diseases. 2007;**44**(8):1108-1114

[6] Savelkoul P, Aarts H, De Haas J, Dijkshoorn L, Duim B, Otsen M, et al. Amplified-fragment length polymorphism analysis: The state of an art. Journal of Clinical Microbiology. 1999;**37**(10):3083-3091

[7] Fredlund H, Falk L, Jurstrand M, Unemo M. Molecular genetic methods for diagnosis and characterization of *Chlamydia trachomatis* and *Neisseria gonorrhoeae*: Impact on epidemiological surveillance and interventions. APMIS. 2004;**112**(11-12):771-784

[8] Olive DM, Bean P. Principles and applications of methods for DNAbased typing of microbial organisms. Journal of Clinical Microbiology. 1999;**37**(6):1661-1669

[9] Tyler K, Wang G, Tyler S, Johnson W. Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. Journal of Clinical Microbiology. 1997;**35**(2):339

[10] Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Research. 2018;**3**:124

[11] Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, et al. *Mycobacterium tuberculosis* complex genetic diversity: Mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiology. 2006;**6**(1):23

[12] Sola C, Filliol I, Gutierrez MC, Mokrousov I, Vincent V, Rastogi N. Spoligotype database of *Mycobacterium tuberculosis*: Biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerging Infectious Diseases. 2001;**7**(3):390

[13] Demay C, Liens B, Burguière T, Hill V, Couvin D, Millet J, et al. SITVITWEB—A publicly available international multimarker database for studying *Mycobacterium tuberculosis* genetic diversity and molecular epidemiology. Infection, Genetics and Evolution. 2012;**12**(4):755-766

[14] Pearce ME, Alikhan N-F, Dallman TJ, Zhou Z, Grant K, Maiden MCJ. Comparative analysis of core genome MLST and SNP typing within a European *Salmonella* serovar Enteritidis outbreak. International Journal of Food Microbiology. 2018;**274**:1-11

**17**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

> [23] Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal database project: Data and tools for high throughput rRNA analysis. Nucleic Acids Research. 2014;**42**(Database

[24] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology. 2007;**73**(16):5261-5267

[25] Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Research. 2012;**41**(D1):D590-D5D6

[26] Nakao A, Yoshihama M, Kenmochi N. RPG: The ribosomal protein gene database. Nucleic Acids Research. 2004;**32**(suppl\_1):D168-DD70

[27] Bellizzi D, Sgaramella V. Bacteria identification by PCR-based techniques. Microbial Genome Methods. 1996:167

[28] Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications. Oval road, London, UK: Academic Press; 2012

[29] White BA. PCR Protocols: Current Methods and Applications. Totowa, New Jersey, USA: Springer Science &

[30] Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols. Totowa, New Jersey, USA: Springer; 2000. pp. 365-386

[31] Giegerich R, Meyer F, Schleiermacher C, editors. GeneFisher-software support for the detection of postulated genes. In: Proc Int Conf Intell Syst Mol Biol. (ISMB). Vol. 4. 1996. pp. 68-77

Ballesteros I, Rementeria A, Garaizar J,

[32] San Millán RM, Martínez-

Business Media; 1993

Issue):D633-D642

[16] Bromham L, Penny D. The modern molecular clock. Nature Reviews.

[17] Rajendhran J, Gunasekaran P. Microbial phylogeny and diversity: Small subunit ribosomal RNA sequence analysis and beyond. Microbiological

Research. 2011;**166**(2):99-110

[19] Gironde S, Manceau C. Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within *Pseudomonas syringae* pv. *maculicola* and *Pseudomonas syringae* pv. tomato that correlate with host specificity. Applied and Environmental Microbiology.

2012;**78**(9):3266-3279

2008;**105**(7):2504-2509

[20] Koeppel A, Perry EB, Sikorski J, Krizanc D, Warner A, Ward DM, et al. Identifying the fundamental units of bacterial diversity: A paradigm shift to incorporate ecology into bacterial systematics. Proceedings of the National Academy of Sciences.

[21] Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. Journal of Clinical Microbiology. 2007;**45**(9):2761-2764

[22] Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews.

2004;**17**(4):840-862

[18] Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S. Use of 16S rRNA and *rpoB* genes as molecular markers for microbial ecology studies. Applied and Environmental Microbiology. 2007;**73**(1):278-288

and source tracking bacterial pathogens. Nucleic Acids Research.

2016;**44**(D1):D682-D6D7

Genetics. 2003;**4**(3):216

[15] Ruan Z, Feng Y. BacWGSTdb, a database for genotyping

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

and source tracking bacterial pathogens. Nucleic Acids Research. 2016;**44**(D1):D682-D6D7

[16] Bromham L, Penny D. The modern molecular clock. Nature Reviews. Genetics. 2003;**4**(3):216

[17] Rajendhran J, Gunasekaran P. Microbial phylogeny and diversity: Small subunit ribosomal RNA sequence analysis and beyond. Microbiological Research. 2011;**166**(2):99-110

[18] Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S. Use of 16S rRNA and *rpoB* genes as molecular markers for microbial ecology studies. Applied and Environmental Microbiology. 2007;**73**(1):278-288

[19] Gironde S, Manceau C. Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within *Pseudomonas syringae* pv. *maculicola* and *Pseudomonas syringae* pv. tomato that correlate with host specificity. Applied and Environmental Microbiology. 2012;**78**(9):3266-3279

[20] Koeppel A, Perry EB, Sikorski J, Krizanc D, Warner A, Ward DM, et al. Identifying the fundamental units of bacterial diversity: A paradigm shift to incorporate ecology into bacterial systematics. Proceedings of the National Academy of Sciences. 2008;**105**(7):2504-2509

[21] Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: Pluses, perils, and pitfalls. Journal of Clinical Microbiology. 2007;**45**(9):2761-2764

[22] Clarridge JE. Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews. 2004;**17**(4):840-862

[23] Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal database project: Data and tools for high throughput rRNA analysis. Nucleic Acids Research. 2014;**42**(Database Issue):D633-D642

[24] Wang Q, Garrity GM, Tiedje JM, Cole JR. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. Applied and Environmental Microbiology. 2007;**73**(16):5261-5267

[25] Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Research. 2012;**41**(D1):D590-D5D6

[26] Nakao A, Yoshihama M, Kenmochi N. RPG: The ribosomal protein gene database. Nucleic Acids Research. 2004;**32**(suppl\_1):D168-DD70

[27] Bellizzi D, Sgaramella V. Bacteria identification by PCR-based techniques. Microbial Genome Methods. 1996:167

[28] Innis MA, Gelfand DH, Sninsky JJ, White TJ. PCR Protocols: A Guide to Methods and Applications. Oval road, London, UK: Academic Press; 2012

[29] White BA. PCR Protocols: Current Methods and Applications. Totowa, New Jersey, USA: Springer Science & Business Media; 1993

[30] Rozen S, Skaletsky H. Primer3 on the WWW for general users and for biologist programmers. Bioinformatics Methods and Protocols. Totowa, New Jersey, USA: Springer; 2000. pp. 365-386

[31] Giegerich R, Meyer F, Schleiermacher C, editors. GeneFisher-software support for the detection of postulated genes. In: Proc Int Conf Intell Syst Mol Biol. (ISMB). Vol. 4. 1996. pp. 68-77

[32] San Millán RM, Martínez-Ballesteros I, Rementeria A, Garaizar J,

**16**

*Biotechnology and Bioengineering*

[1] Donelli G, Vuotto C, Mastromarino P. Phenotyping and genotyping are both essential to identify and classify a probiotic microorganism. Microbial Ecology in Health and Disease.

[9] Tyler K, Wang G, Tyler S, Johnson W. Factors affecting reliability and reproducibility of amplification-based DNA fingerprinting of representative bacterial pathogens. Journal of Clinical

[10] Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their

applications. Wellcome Open Research.

[11] Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, Al-Hajoj SA, et al. *Mycobacterium tuberculosis* complex genetic diversity: Mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiology. 2006;**6**(1):23

[12] Sola C, Filliol I, Gutierrez MC, Mokrousov I, Vincent V, Rastogi N. Spoligotype database of *Mycobacterium tuberculosis*: Biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives. Emerging Infectious Diseases. 2001;**7**(3):390

[13] Demay C, Liens B, Burguière T, Hill V, Couvin D, Millet J, et al. SITVITWEB—A publicly available international multimarker database for studying *Mycobacterium tuberculosis* genetic diversity and molecular epidemiology. Infection, Genetics and

Evolution. 2012;**12**(4):755-766

Microbiology. 2018;**274**:1-11

a database for genotyping

[15] Ruan Z, Feng Y. BacWGSTdb,

[14] Pearce ME, Alikhan N-F, Dallman TJ, Zhou Z, Grant K, Maiden MCJ. Comparative analysis of core genome MLST and SNP typing within a

European *Salmonella* serovar Enteritidis outbreak. International Journal of Food

Microbiology. 1997;**35**(2):339

2018;**3**:124

[2] Yamamoto Y. PCR in diagnosis of infection: Detection of bacteria in cerebrospinal fluids. Clinical and Diagnostic Laboratory Immunology.

International Journal of Food Microbiology. 2007;**120**(3):225-236

[3] Ludwig W. Nucleic acid techniques in bacterial systematics and identification.

[4] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology.

[5] Reller LB, Weinstein MP, Petti CA. Detection and identification of microorganisms by gene amplification and sequencing. Clinical Infectious Diseases. 2007;**44**(8):1108-1114

[6] Savelkoul P, Aarts H, De Haas J, Dijkshoorn L, Duim B, Otsen M, et al. Amplified-fragment length polymorphism analysis: The state of an art. Journal of Clinical Microbiology.

[7] Fredlund H, Falk L, Jurstrand M, Unemo M. Molecular genetic methods for diagnosis and characterization of *Chlamydia trachomatis* and *Neisseria gonorrhoeae*: Impact on epidemiological surveillance and interventions. APMIS. 2004;**112**(11-12):771-784

[8] Olive DM, Bean P. Principles and applications of methods for DNAbased typing of microbial organisms. Journal of Clinical Microbiology.

1999;**37**(10):3083-3091

1999;**37**(6):1661-1669

**References**

2013;**24**(1):20105

2002;**9**(3):508-514

1991;**173**(2):697-703

Bikandi J. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Research Notes. 2013;**6**(1):513

[33] Bikandi J, Millán RS, Rementeria A, Garaizar J. *In silico* analysis of complete bacterial genomes: PCR, AFLP–PCR and endonuclease restriction. Bioinformatics. 2004;**20**(5):798-799

[34] Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile *Thermus aquaticus*. Journal of Bacteriology. 1976;**127**(3):1550-1557

[35] Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, et al. High-level expression, purification, and enzymatic characterization of full-length *Thermus aquaticus* DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. PCR Methods and Applications. 1993;**2**(4):275-287

[36] Kainz P. The PCR plateau phase– towards an understanding of its limitations. Biochimica et Biophysica Acta. 2000;**1494**(1-2):23-27

[37] D'Aquila RT, Bechtel LJ, Videler JA, Eron JJ, Gorczyca P, Kaplan JC. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Research. 1991;**19**(13):3749

[38] Korbie DJ, Mattick JS. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature Protocols. 2008;**3**(9):1452

[39] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research. 1997;**25**(17):3389-3402

[40] Yang S, Lin S, Kelen GD, Quinn TC, Dick JD, Gaydos CA, et al. Quantitative

multiprobe PCR assay for simultaneous detection and identification to species level of bacterial pathogens. Journal of Clinical Microbiology. 2002;**40**(9):3449-3454

[41] Hulton C, Higgins C, Sharp P. ERIC sequences: A novel family of repetitive elements in the genomes of *Escherichia coli*, *Salmonella typhimurium* and other enterobacteria. Molecular Microbiology. 1991;**5**(4):825-834

[42] Sachdeva P, Virdi JS. Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of *Yersinia enterocolitica* biotype 1A reveal existence of limited number of clonal groups. FEMS Microbiology Letters. 2004;**240**(2):193-201

[43] Urwin R, Maiden MC. Multi-locus sequence typing: A tool for global epidemiology. Trends in Microbiology. 2003;**11**(10):479-487

[44] Sabat A, Budimir A, Nashev D, Sá-Leão R, Van Dijl J, Laurent F, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Eurosurveillance. 2013;**18**(4):20380

[45] Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al. Multilocus sequence typing of total-genome-sequenced bacteria. Journal of Clinical Microbiolology. 2012;**50**(4):1355-1361

[46] Thomsen MCF, Hasman H, Westh H, Kaya H, Lund O. RUCS: Rapid identification of PCR primers for unique core sequences. Bioinformatics. 2017;**33**(24):3917-3921

[47] Brinkac LM, Beck E, Inman J, Venepally P, Fouts DE, Sutton G. LOCUST: A custom sequence locus typer for classifying microbial isolates. Bioinformatics. 2017;**33**(11):1725-1726

**19**

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

> with arbitrary primers. Nucleic Acids Research. 1990;**18**(24):7213-7218

[57] McClelland M, Welsh J. DNA fingerprinting by arbitrarily primed PCR. PCR Methods and Applications.

[58] Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids

1994;**4**(1):S59-S65

Research. 1995;**23**(6):1087

2004;**10**(3):190-212

[59] Mackay IM. Real-time PCR in the microbiology laboratory. Clinical Microbiology and Infection.

[60] Alvarez J, Sota M, Vivanco AB, Perales I, Cisterna R, Rementeria A, et al. Development of a multiplex PCR technique for detection and epidemiological typing of *Salmonella* in human clinical samples. Journal of Clinical Microbiology. 2004;**42**(4):1734-1738

[61] Juck D, Ingram J, Prévost M, Coallier J, Greer C. Nested PCR protocol for the rapid detection of *Escherichia coli* in potable water. Canadian Journal of Microbiology. 1996;**42**(8):862-866

[62] Coronado-Ríos S, Arenas-Suárez NE, Gómez-Marín JE. Diagnosis of pulmonary and extrapulmonary tuberculosis using an *in-house* PCR method in clinical samples from a middle-income resource setting. Infection. 2011;**15**:177-183

[63] Ginzinger DG. Gene quantification using real-time quantitative PCR: An emerging technology hits the

mainstream. Experimental Hematology.

[64] Chang C-H, Mau-Hsu D, Chen K-C, Wei C-W, Chiu C-Y, Young T-H. Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis. Scientific Reports.

2002;**30**(6):503-512

2018;**8**(1):3432

[48] Lefever S, Vandesompele J, Speleman F, Pattyn F. RTPrimerDB: The portal for real-time PCR primers and probes. Nucleic Acids Research. 2008;**37**(suppl\_1):D942-D9D5

[49] Pattyn F, Robbrecht P, De Paepe A, Speleman F, Vandesompele J. RTPrimerDB: The real-time PCR primer and probe database, major update 2006. Nucleic Acids Research. 2006;**34**(suppl\_1):D684-D6D8

[50] Wang X, Spandidos A, Wang H, Seed B. PrimerBank: A PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Research. 2012;**40**(D1):D1144-D11D9

[51] Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription– polymerase chain reaction to study mRNA decay: Comparison of endpoint and real-time methods. Analytical Biochemistry. 2000;**285**(2):194-204

[52] Notomi T, Mori Y, Tomita N, Kanda H. Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. Journal

of Microbiology. 2015;**53**(1):1-5

[54] Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI. FastPCR: An *in silico* tool for fast primer and probe design and advanced sequence analysis. Genomics.

[55] Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by

arbitrary primers are useful as genetic markers. Nucleic Acids Research.

Fingerprinting genomes using PCR

2010;**50**(4):277-281

2017;**109**(3):312-319

1990;**18**(22):6531-6535

[56] Welsh J, McClelland M.

[53] Markey AL, Mohr S, Day PJ. Highthroughput droplet PCR. Methods.

*Steps and Tools for PCR-Based Technique Design DOI: http://dx.doi.org/10.5772/intechopen.83671*

*Biotechnology and Bioengineering*

2013;**6**(1):513

Bikandi J. Online exercise for the design and simulation of PCR and PCR-RFLP experiments. BMC Research Notes.

multiprobe PCR assay for simultaneous

[41] Hulton C, Higgins C, Sharp P. ERIC sequences: A novel family of repetitive elements in the genomes of *Escherichia coli*, *Salmonella typhimurium* and other enterobacteria. Molecular Microbiology.

[42] Sachdeva P, Virdi JS. Repetitive elements sequence (REP/ERIC)-PCR based genotyping of clinical and environmental strains of *Yersinia enterocolitica* biotype 1A reveal existence of limited number of clonal groups. FEMS Microbiology Letters.

[43] Urwin R, Maiden MC. Multi-locus sequence typing: A tool for global epidemiology. Trends in Microbiology.

[45] Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H, Marvig RL, et al. Multilocus sequence typing of total-genome-sequenced bacteria. Journal of Clinical Microbiolology.

[46] Thomsen MCF, Hasman H, Westh H, Kaya H, Lund O. RUCS: Rapid identification of PCR primers for unique core sequences. Bioinformatics.

[47] Brinkac LM, Beck E, Inman J, Venepally P, Fouts DE, Sutton G. LOCUST: A custom sequence locus typer for classifying

microbial isolates. Bioinformatics.

[44] Sabat A, Budimir A, Nashev D, Sá-Leão R, Van Dijl J, Laurent F, et al. Overview of molecular typing methods for outbreak detection and epidemiological surveillance. Eurosurveillance. 2013;**18**(4):20380

detection and identification to species level of bacterial pathogens. Journal of Clinical Microbiology.

2002;**40**(9):3449-3454

1991;**5**(4):825-834

2004;**240**(2):193-201

2003;**11**(10):479-487

2012;**50**(4):1355-1361

2017;**33**(24):3917-3921

2017;**33**(11):1725-1726

[33] Bikandi J, Millán RS, Rementeria A, Garaizar J. *In silico* analysis of complete bacterial genomes: PCR, AFLP–PCR and endonuclease restriction. Bioinformatics. 2004;**20**(5):798-799

[34] Chien A, Edgar DB, Trela JM. Deoxyribonucleic acid polymerase from the extreme thermophile *Thermus aquaticus*. Journal of Bacteriology.

[35] Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, et al. High-level expression, purification, and enzymatic

Applications. 1993;**2**(4):275-287

Acta. 2000;**1494**(1-2):23-27

1991;**19**(13):3749

Protocols. 2008;**3**(9):1452

1997;**25**(17):3389-3402

[36] Kainz P. The PCR plateau phase– towards an understanding of its limitations. Biochimica et Biophysica

[37] D'Aquila RT, Bechtel LJ, Videler JA, Eron JJ, Gorczyca P, Kaplan JC. Maximizing sensitivity and

specificity of PCR by pre-amplification heating. Nucleic Acids Research.

[38] Korbie DJ, Mattick JS. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nature

[39] Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research.

[40] Yang S, Lin S, Kelen GD, Quinn TC, Dick JD, Gaydos CA, et al. Quantitative

characterization of full-length *Thermus aquaticus* DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. PCR Methods and

1976;**127**(3):1550-1557

**18**

[48] Lefever S, Vandesompele J, Speleman F, Pattyn F. RTPrimerDB: The portal for real-time PCR primers and probes. Nucleic Acids Research. 2008;**37**(suppl\_1):D942-D9D5

[49] Pattyn F, Robbrecht P, De Paepe A, Speleman F, Vandesompele J. RTPrimerDB: The real-time PCR primer and probe database, major update 2006. Nucleic Acids Research. 2006;**34**(suppl\_1):D684-D6D8

[50] Wang X, Spandidos A, Wang H, Seed B. PrimerBank: A PCR primer database for quantitative gene expression analysis, 2012 update. Nucleic Acids Research. 2012;**40**(D1):D1144-D11D9

[51] Schmittgen TD, Zakrajsek BA, Mills AG, Gorn V, Singer MJ, Reed MW. Quantitative reverse transcription– polymerase chain reaction to study mRNA decay: Comparison of endpoint and real-time methods. Analytical Biochemistry. 2000;**285**(2):194-204

[52] Notomi T, Mori Y, Tomita N, Kanda H. Loop-mediated isothermal amplification (LAMP): Principle, features, and future prospects. Journal of Microbiology. 2015;**53**(1):1-5

[53] Markey AL, Mohr S, Day PJ. Highthroughput droplet PCR. Methods. 2010;**50**(4):277-281

[54] Kalendar R, Khassenov B, Ramankulov Y, Samuilova O, Ivanov KI. FastPCR: An *in silico* tool for fast primer and probe design and advanced sequence analysis. Genomics. 2017;**109**(3):312-319

[55] Williams JG, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research. 1990;**18**(22):6531-6535

[56] Welsh J, McClelland M. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Research. 1990;**18**(24):7213-7218

[57] McClelland M, Welsh J. DNA fingerprinting by arbitrarily primed PCR. PCR Methods and Applications. 1994;**4**(1):S59-S65

[58] Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Research. 1995;**23**(6):1087

[59] Mackay IM. Real-time PCR in the microbiology laboratory. Clinical Microbiology and Infection. 2004;**10**(3):190-212

[60] Alvarez J, Sota M, Vivanco AB, Perales I, Cisterna R, Rementeria A, et al. Development of a multiplex PCR technique for detection and epidemiological typing of *Salmonella* in human clinical samples. Journal of Clinical Microbiology. 2004;**42**(4):1734-1738

[61] Juck D, Ingram J, Prévost M, Coallier J, Greer C. Nested PCR protocol for the rapid detection of *Escherichia coli* in potable water. Canadian Journal of Microbiology. 1996;**42**(8):862-866

[62] Coronado-Ríos S, Arenas-Suárez NE, Gómez-Marín JE. Diagnosis of pulmonary and extrapulmonary tuberculosis using an *in-house* PCR method in clinical samples from a middle-income resource setting. Infection. 2011;**15**:177-183

[63] Ginzinger DG. Gene quantification using real-time quantitative PCR: An emerging technology hits the mainstream. Experimental Hematology. 2002;**30**(6):503-512

[64] Chang C-H, Mau-Hsu D, Chen K-C, Wei C-W, Chiu C-Y, Young T-H. Evaluation of digital real-time PCR assay as a molecular diagnostic tool for single-cell analysis. Scientific Reports. 2018;**8**(1):3432

[65] Anjum MF, Zankari E, Hasman H. Molecular methods for detection of antimicrobial resistance. Microbiology. 2017;**5**:6

[66] Cobb S, Heaney R, Corcoran O, Henderson-Begg S. The learning gains and student perceptions of a second life virtual lab. Bioscience Education. 2009;**13**(1):1-9

[67] Maldarelli GA, Hartmann EM, Cummings PJ, Horner RD, Obom KM, Shingles R, et al. Virtual lab demonstrations improve students' mastery of basic biology laboratory techniques. Journal of Microbiology & Biology Education: JMBE. 2009;**10**(1):51

[68] Arenas NE, Polanco JC, Coronado SM, Durango CJ, Gómez A. Design of a molecular method for subspecies specific identification of *Klebsiella pneumoniae* by using the 16S ribosomal subunit gene. Colombia Médica. 2009;**40**:194-201

[69] Klappenbach JA, Saxman PR, Cole JR, Schmidt TM. Rrndb: The ribosomal RNA operon copy number database. Nucleic Acids Research. 2001;**29**(1):181-184

[70] Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Applied and Environmental Microbiology. 2000;**66**(4):1328-1333

**21**

**Chapter 3**

**Abstract**

*Dermatophilosis*

**1. Introduction**

Developments and Perspectives in

The work described here covers an examination of new bioproducts based on sub-Saharan bryophytes. The work includes in vitro testing of extracts from moss and liverworts against plant pathogenic microbes causing food decay and field crop losses. Additionally, we have shown specific antimicrobial activities of *Marchantia debilis* and **moss** against *Erwinia spp* and *Pseudomonas spp*. The extracts were also tested against aflatoxin-producing fungi isolated from food crops such as maize and peanuts. The efficacy of the extracts on clinical dermatological fungal isolates like *Dermatophilus congolensis* has not been reported. This led to the production of an antifungal solution of bryophyte extracts, which was tested in vivo on animals with skin diseases caused by *Dermatophilosis*. Around 99.5% of the animals were treated. The antifungal solution for treatments has been labeled **Bryosol**, while the disinfectants solution is labeled Bryo-disinfectants and the crop-fungicide is labeled Bryo-fungicides. A mini field pilot trial with Bryo-fungicide showed that crops infected with pathogenic fungi were treated. The results provide the first attempt to demonstrate the use of bioproducts for organic treatment of agricultural crops and

**Keywords:** moss, Marchantia, Bryosol, disinfectants, antifungal, bioproducts, foods,

Bryophytes are non-vascular plants, which are constituted of mosses, liverworts, and hornworts [1]. Although not usually seen to have any importance, bryophytes have recently been used as bioindicators of pollution and are often used for decorations. However, the medicinal value of bryophytes is huge with a panoply of bioactive compounds isolated from bryophytes, especially liverworts [1, 2]. Bioactive compounds have been isolated from liverworts from Asia, Europe, and South Africa. For example, Allison in 1975 identified a number of bioactive compounds from liverworts in New Zealand. Volatile constituents have been identified in liverworts like *Tritomaria polita*, *Marsupella emarginata*, *M. aquatic*, and *M. alpine* [3]. It has been seen that bryophytes are rich in diverse phytochemicals such as sesquiterpenoids, norsesquiterpenoids, anthocyanidins, riccionidins etc. with interesting biological activities, such as antimicrobial, antifungal, insect-repellent, molluscicidal, cardiotonic activity, and fragrance compounds among others [1]. Bryophytes

Bryophyte Biotechnology in

*Kenneth Yongabi Anchang and Henrik Toft Simonsen*

Sub-Saharan Africa

diseases in animals based on sub-Saharan bryophytes.

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[65] Anjum MF, Zankari E, Hasman H. Molecular methods for detection of antimicrobial resistance. Microbiology.

[66] Cobb S, Heaney R, Corcoran O, Henderson-Begg S. The learning gains and student perceptions of a second life virtual lab. Bioscience Education.

[67] Maldarelli GA, Hartmann EM, Cummings PJ, Horner RD, Obom KM, Shingles R, et al. Virtual lab demonstrations improve students' mastery of basic biology laboratory techniques. Journal of Microbiology & Biology Education: JMBE. 2009;**10**(1):51

[68] Arenas NE, Polanco JC, Coronado SM, Durango CJ, Gómez A. Design of a molecular method for subspecies specific identification of *Klebsiella pneumoniae* by using the 16S ribosomal subunit gene. Colombia Médica.

[69] Klappenbach JA, Saxman PR, Cole JR, Schmidt TM. Rrndb: The ribosomal RNA operon copy number database. Nucleic Acids Research.

[70] Klappenbach JA, Dunbar JM, Schmidt TM. rRNA operon copy number reflects ecological strategies of bacteria. Applied and Environmental Microbiology. 2000;**66**(4):1328-1333

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