Methods in
Molecular Biology 2967
Lucília Domingues Editor
PCR
Methods and Protocols
Second Edition
,METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:
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,For over 35 years, biological scientists have come to rely on the research protocols and
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, PCR
Methods and Protocols
Second Edition
Edited by
Lucília Domingues
CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal
,Editor
Lucı́lia Domingues
CEB - Centre of Biological Engineering
University of Minho
Braga, Portugal
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3357-1 ISBN 978-1-0716-3358-8 (eBook)
https://doi.org/10.1007/978-1-0716-3358-8
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,Preface
With 40 years since its official discovery and 51 years since its theoretical proposition, the
polymerase chain reaction (PCR) has revolutionized the fields of biotechnology, medicine,
food microbiology, environmental microbiology, industry, and science, in general. The
concept is so perfectly simple that the elemental scheme remains unchanged since its
foundation. There are very few inventions that can compete with the importance of PCR.
PCR is still today a fundamental tool in current scientific research and its importance has
been recently disclosed to the general public with its widespread use during the COVID-19
pandemic that began in 2019. Being such a relevant technique with wide-range applications,
significant literature exists on the basics of PCR. Still, the specificities for its application in
diverse areas of the biotechnology and bioengineering field are mostly dispersed and are
preferentially found in the health area. PCR is a powerful and flexible tool in modern
biotechnology and the continued development of this technology is still expanding its
wide range of applications.
This new edition of PCR Methods and Protocols maintains the focus of the first edition
on PCR application specificities to the biotechnology and bioengineering field with updated
content on recently developed cutting-edge methodologies and novel applications. While
the previous edition almost exclusively covered end-point PCR, this volume is balanced with
real-time PCR and with fresh applications in the biotechnology and bioengineering field, in
particular in the food sector, in which a growing trend for the use of this technology is
observed. Topics such as detection of foodborne microbial contaminants, toxins, and
allergens are included as well as food authentication. Two protocols involving high-
resolution melting assays are illustrated for the detection of foodborne pathogens and
fungi detection in plant matrices. Relevant applications in biotechnology are emphasized
with protocols for accurate absolute quantification of bacterial populations in mixed cultures
and for gene expression quantification from pathogenic bacterial biofilms. Applications in
synthetic biology for the assessment of recombination efficiency in minicircle production
and quantification of plasmid copy number are also included. More recently developed PCR
techniques like digital PCR protocols were incorporated in this new edition highlighting the
applications for SARS-Cov-2 detection and surveillance from sewage samples and food
herbal spices and products authentication. Emulsion PCR coupled with denaturing gradient
gel electrophoresis is described in the context of microbial diversity studies. New develop-
ments for end-point PCR like the use of disruptors for PCR improvement are included as
well as novel applications such as the use of mitochondrial DNA D-loop amplification and
sequencing for species differentiation in milk. Highly used end-point PCR applications from
the previous edition were kept and updated like long fragment PCR and megaprimer
applications in the synthesis of fusion genes, colony PCR, inverse PCR for site-directed
mutagenesis, and degenerate PCR.
It is amazing as such a straightforward methodology like PCR has evolved and expanded
its applications over 40 years. The ongoing development of PCR technology has enabled
PCR to continue to play an indispensable role in the biotechnology and bioengineering
field. The trend is the development of new PCR technologies and applications with digitali-
v
,vi Preface
zation, accessibility, and adaptability to different settings and contexts, contributing to
further streamlined analyses while maintaining and improving the sensitivity and specificity
required for PCR’s wide range of applications. This book aims to contribute to a current
update of the dynamic field of PCR-dependent methods and, thus, to be a valuable,
indispensable, and useful resource to wet-lab researchers, particularly within the biotech-
nology and bioengineering field.
Braga, Portugal Lucı́lia Domingues
,Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Digital PCR: A Partitioning-Based Application for Detection
and Surveillance of SARS-CoV-2 from Sewage Samples. . . . . . . . . . . . . . . . . . . . . . 1
Bhumika Prajapati, Dalipsingh Rathore, Chaitanya Joshi,
and Madhvi Joshi
2 Digital PCR: A Tool to Authenticate Herbal Products and Spices . . . . . . . . . . . . . 17
Abhi P. Shah, Tasnim Travadi, Sonal Sharma, Ramesh Pandit,
Chaitanya Joshi, and Madhvi Joshi
3 Emulsion Polymerase Chain Reaction Coupled with Denaturing
Gradient Gel Electrophoresis for Microbial Diversity Studies . . . . . . . . . . . . . . . . . 31
Maria-Eleni Dimitrakopoulou, Dimosthenis Tzimotoudis,
and Apostolos Vantarakis
4 Real-Time PCR High-Resolution Melting Assays for the Detection
of Foodborne Pathogens. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Prashant Singh and Frank J. Velez
5 High-Throughput Real-Time qPCR and High-Resolution Melting
(HRM) Assay for Fungal Detection in Plant Matrices . . . . . . . . . . . . . . . . . . . . . . . 53
Filipe Azevedo-Nogueira, Sara Barrias, and Paula Martins-Lopes
6 Multiplex Real-Time PCR for the Detection of Shiga Toxin-Producing
Escherichia coli in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Ana Costa-Ribeiro, Sarah Azinheiro, Foteini Roumani,
Marta Prado, Alexandre Lamas, and Alejandro Garrido-Maestu
7 DNA Isolation from Cocoa-Derived Products and Cocoa Authentication
by TaqMan Real-Time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Ana Caroline De Oliveira, Yordan Muhovski, Herve Rogez,
and Frédéric Debode
8 Quantitative Real-Time PCR for the Detection of Allergenic Species
in Foods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Joana Costa, Caterina Villa, and Isabel Mafra
9 Accurate Absolute Quantification of Bacterial Populations in Mixed
Cultures by qPCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Ângela Lima, Lúcia G. V. Sousa, and Nuno Cerca
10 Real-Time PCR Method for Assessment of ParA-Mediated
Recombination Efficiency in Minicircle Production . . . . . . . . . . . . . . . . . . . . . . . . . 117
Cláudia P. A. Alves, Duarte Miguel F. Prazeres,
and Gabriel A. Monteiro
vii
,viii Contents
11 Gene Expression Quantification from Pathogenic Bacterial Biofilms
by Quantitative PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Angela França and Nuno Cerca
12 A Real-Time Quantitative PCR Protocol for the Quantification
of Plasmid Copy Number in Lactococcus lactis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Sofia O. D. Duarte and Gabriel A. Monteiro
13 Improved PCR by the Use of Disruptors, a New Class of Oligonucleotide
Reagents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
Yong Ma and Minxue Zheng
14 Mitochondrial DNA D-Loop Amplification and Sequencing for Species
Differentiation in Milk . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Marlene Baptista and Lucı́lia Domingues
15 Long-Range Polymerase Chain Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Ping Siu Kee, Harsheni Karunanathie, Simran D. S. Maggo,
Martin A. Kennedy, and Eng Wee Chua
16 Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning . . . . . . . . . . . . . 193
Tatiana Q. Aguiar, Carla Oliveira, and Lucı́lia Domingues
17 Bacteria and Yeast Colony PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Humberto Pereira, Paulo César Silva, and Björn Johansson
18 Inverse PCR for Site-Directed Mutagenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Diogo Silva, Gustavo Santos, Mário Barroca, Diogo Costa,
and Tony Collins
19 Optimized Design of Degenerate Primers for PCR Based on DNA
or Protein Sequence Comparisons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Maria Jorge Campos, Alejandro Gallardo, and Alberto Quesada
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
, Contributors
TATIANA Q. AGUIAR • CEB - Centre of Biological Engineering, University of Minho, Braga,
Portugal; LABBELS - Associate Laboratory, Braga/Guimarães, Portugal
CLÁUDIA P. A. ALVES • iBB- Institute for Bioengineering and Biosciences, Department of
Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Lisbon, Portugal;
Associate Laboratory i4HB – Institute for Health and Bioeconomy at Instituto Superior Té
cnico, Universidade de Lisboa, Lisbon, Portugal
FILIPE AZEVEDO-NOGUEIRA • DNA & RNA Sensing Lab, University of Trás-os-Montes e Alto
Douro, Department of Genetics and Biotechnology, School of Life Science and Environment,
Vila Real, Portugal; BioISI – Biosystems & Integrative Sciences Institute, University of
Lisboa, Faculty of Sciences, Lisbon, Portugal
SARAH AZINHEIRO • International Iberian Nanotechnology Laboratory, Food Quality and
Safety Research Group, Braga, Portugal; Department of Analytical Chemistry, Nutrition
and Food Science, Faculty of Veterinary Science, University of Santiago de Compostela,
Lugo, Spain
MARLENE BAPTISTA • CEB-Centre of Biological Engineering, University of Minho, Braga,
Portugal
SARA BARRIAS • DNA & RNA Sensing Lab, University of Trás-os-Montes e Alto Douro,
Department of Genetics and Biotechnology, School of Life Science and Environment, Vila
Real, Portugal; BioISI – Biosystems & Integrative Sciences Institute, University of Lisboa,
Faculty of Sciences, Lisbon, Portugal
MÁRIO BARROCA • CBMA - Centre of Molecular and Environmental Biology, Department of
Biology, University of Minho, Braga, Portugal
MARIA JORGE CAMPOS • MARE-Marine and Environmental Sciences Centre & ARNET—
Aquatic Research Network Associated Laboratory, ESTM, Polytechnic of Leiria, Peniche,
Portugal
NUNO CERCA • Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), CEB – Centre
of Biological Engineering, University of Minho, Braga, Portugal; LABBELS –Associate
Laboratory, Braga, Guimarães, Portugal
ENG WEE CHUA • Centre for Drug and Herbal Development, Faculty of Pharmacy,
Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
TONY COLLINS • CBMA - Centre of Molecular and Environmental Biology, Department of
Biology, University of Minho, Braga, Portugal
DIOGO COSTA • CBMA - Centre of Molecular and Environmental Biology, Department of
Biology, University of Minho, Braga, Portugal
JOANA COSTA • REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Porto,
Portugal
ANA COSTA-RIBEIRO • International Iberian Nanotechnology Laboratory, Food Quality and
Safety Research Group, Braga, Portugal; Department of Biochemistry, Genetics and
Immunology, University of Vigo, Vigo, Spain
ANA CAROLINE DE OLIVEIRA • Department of Life Sciences, Unit Bioengineering, Walloon
Agricultural Research Centre (CRA-W), Gembloux, Belgium
FRÉDÉRIC DEBODE • Department of Life Sciences, Unit Bioengineering, Walloon
Agricultural Research Centre (CRA-W), Gembloux, Belgium
ix
