Veterinary PCR Diagnostics


by

Chengming Wang, Bernhard Kaltenboeck, Mark D. Freeman

DOI: 10.2174/97816080534831120101
eISBN: 978-1-60805-348-3, 2012
ISBN: 978-1-60805-572-2



Recommend this eBook to your Library

Indexed in: Book Citation Index, Science (BKCI-S), Web of Science, Scopus

PCR (Polymerase Chain Reaction) technology has become an indispensable component of routine veterinary diagnostics. However, a number ...[view complete introduction]

Table of Contents

Foreword

- Pp. i- (1)

Konrad Sachse

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Preface

- Pp. ii

Chengming Wang, Bernhard Kaltenboeck and Mark D. Freeman

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List of Contributors

- Pp. iii-v (3)

Chengming Wang, Bernhard Kaltenboeck and Mark D. Freeman

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Principles of Real-Time PCR

- Pp. 3-17 (15)

Amanda D. Loftis and Will K. Reeves

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Design and Optimization of Real-Time PCR Assays

- Pp. 18-32 (15)

Raymaekers Marijke

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Antimicrobial Resistance in Bacterial Pathogens: Mechanisms and PCRBased Detection Technologies

- Pp. 33-58 (26)

Bashar W. Shaheen, Rajesh Nayak and Dawn M. Boothe

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PCR-Based Diagnosis of Veterinary Bacterial Pathogens

- Pp. 59-79 (21)

Walter Lilenbaum, Renata Fernandes Rabello and Rubens Clayton da Silva Dias

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PCR Detection of Viruses in Veterinary Medicine

- Pp. 80-97 (18)

Yihang Li, Sudhir K. Ahluwalia and Mark D. Freeman

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Quantitative PCR as a Diagnostic Technique in Veterinary Parasitology

- Pp. 98-105 (8)

Hongzhuan Wu, Kirsten Jaegersen, Boakai K. Robertson, Robert Villafane and Chengming Wang

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PCR and Veterinary Cancer Diagnostics

- Pp. 106-135 (30)

Fabio Gentilini, Maria Elena Turba and Claudia Calzolari

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Index

- Pp. 136-137 (2)

Chengming Wang, Bernhard Kaltenboeck and Mark D. Freeman

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Foreword

It was little more than a decade ago that PCR users were given the possibility to follow the amplification cycles in real time. The new machines were able to measure the accumulation of amplified product via a fluorescent signal generated by exonuclease digestion of a specifically annealed dual-labeled fluorogenic probe. This meant that users could take a closer look at the kinetics of their amplification reaction, so that, as a consequence, optimization was no longer a matter of gut feeling, but became tangible thanks to the quantitative data provided by the real-time thermocycler. The omission of post-amplification sample handling, such as gel electrophoresis, was another advantage because it significantly reduced the risk of contamination by product carry-over and enabled more rapid and high-throughput testing. Moreover, while conventional PCR would only confirm the presence or absence of a given pathogen in a sample, the real-time technology enabled the investigator to quantitate the amount of this agent.

The quantitation option has opened up new horizons to researchers by facilitating a rigorously quantitative approach to characterize cellular processes, the course of an infection, as well as the spread and dissemination of a pathogen. A whole area of research, i.e. transcriptomics, could not have reached the high level it has today without the use of quantitative real-time PCR as a gold standard.

The present book is filling a notable gap. As the amount of scientific publications is steadily increasing, so is the number of PCR-based methodologies and diagnostic assays. Harmonization and standardization of protocols, both in a national and worldwide context, remain important issues on the agenda of diagnostic laboratories. In this situation, there is a necessity to systematically digest the existing literature and give an overview to already specialized users. Furthermore, newcomers need guidance and technical advice before they are able to become productive in the field. This book will be a valuable orientation guide to the community of investigators involved in research and diagnosis of microbial infections, as well as cancer and other diseases that are amenable to nucleic acid-based analysis.

Konrad Sachse
Friedrich-Loeffler-Institute
(Federal Research Institute for Animal Health)
Institute of Molecular Pathogenesis
Germany


Preface

A speedy and accurate identification of pathogens is of vital importance for the effective control and management of veterinary infectious diseases. Infectious agents have been traditionally identified with the use of various phenotypic procedures, such as morphological, biochemical and serological assays. However, the phenotypic diagnoses are usually slow and lack proper specificity and sensitivity. The revolutionary invention of the nucleic acid amplification technologies such as PCR allows the detection of pathogens at nucleic acid level, and has played an increasing role in the laboratory diagnosis of infectious diseases. PCR-based technologies offer the ability to detect a single copy of nucleic acid template with supreme sensitivity, specificity, speed and precision for the detection of pathogens. Furthermore, the recent advances in probe chemistries, availability of multiple fluorescent channels in the PCR machines as well as instrumentation automation have facilitated the development of quantitative PCR that provides a convenient platform for high through-put quantitation and differentiation of pathogens in clinical specimens of veterinary medicine.

We are very fortunate and honored to have international specialists as chapter contributors in their respective specialty of veterinary PCR diagnostics. Mrs. Salma Sarfaraz at Bentham Science Publishers is a constant source of encouragement and discipline for the production of this book. This book provides a reliable, convenient and comprehensive reference on molecular detection and identification of pathogens of veterinary significance. Chapter 1 (Loftis et al.) outlines the principles of real-time PCR, and chapter 2 (Marijke) describes a practical guiding standard that can be used in different steps of the design and validation of in-house developed real-time PCR assays. Chapter 3 (Lilenbaum et al. ) focuses on molecular diagnosis of veterinary bacterial infections, exemplified with Brucella sp., Leptospira sp., Mycobacterium bovis, Staphylococcus aureus and Mycoplasma sp. Chapter 4 (Shaheen et al.) provides valuable knowledge on the molecular mechanisms of drug resistance in microbial pathogens and the potential advantages and disadvantages of PCR-based methods. Chapter 5 (Li et al.) highlights the diagnosis of viral disease in livestock and companion animals with PCR, and Chapter 6 (Wu et al.) focuses on the application of real-time PCR for detection and differentiation of significant parasites in veterinary medicine and public health, exemplified in protozoa, helminthes and arthropods. In chapter 7, Gentilini et al. focus their attention on PCR applications for the diagnosis and prognosis of cancer in pets which are already currently available, albeit not diffusely, at both academic and private laboratories around the world.

Chengming Wang
Yangzhou University
Jiangsu, China

Bernhard Kaltenboeck
Auburn University
AL, USA

Mark D. Freeman
Ross University School of Veterinary Medicine
West Indies

List of Contributors

Editor(s):
Chengming Wang
Yangzhou University
Jiangsu
China


Bernhard Kaltenboeck
Auburn University
AL
USA


Mark D. Freeman
Ross University School of Veterinary Medicine
West Indies




Contributor(s):
Sudhir K. Ahluwalia
Banfield Pet Hospital
10 Traders Way
Salem
MA, 01970
USA


Dawn M. Boothe
Department of Anatomy
Physiology & Pharmacology Auburn University
Auburn
AL
USA


Claudia Calzolari
Department of Veterinary Medical Sciences
University of Bologna
Italy


Mark D. Freeman
Ross University School of Veterinary Medicine
Basseterre St. Kitts
West Indies


Fabio Gentilini
Department of Veterinary Medical Sciences
University of Bologna
Italy


Kirsten Jaegersen
Ross University School of Veterinary Medicine
Basseterre St. Kitts
West Indies


Bernhard Kaltenboeck
Department of Pathobiology
Auburn University
AL
USA


Yihang Li
Department of Veterans Affairs Palo Alto Health Care System
Geriatrics Research, Education and Clinical Center
California
USA


Walter Lilenbaum
Biomedical Institute
Fluminense Federal University
RJ
Brazil


Amanda D. Loftis
Ross University School of Veterinary Medicine
Basseterre St. Kitts
West Indies


Raymaekers Marijke
Clinical Laboratory, Jessa Hospital
Site Virga Jesse
Belgium


Rajesh Nayak
Division of Microbiology
National Center for Toxicological Research
US Food and Drug Administration
Jefferson
AR
USA


Renata F. Rabello
Biomedical Institute
Fluminense Federal University
RJ
Brazil


Will K. Reeves
USAF School of Aerospace Medicine
Wright-Patterson Air Force Base
Ohio
USA


Boakai K. Robertson
Department of Biological Sciences
Alabama State University
Montgomery
AL
USA


Konrad Sachse
Friedrich-Loeffler-Institute
(Federal Research Institute for Animal Health)
Institute of Molecular Pathogenesis
Jena
Germany


Bashar W. Shaheen
Division of Microbiology
National Center for Toxicological Research
US Food and Drug Administration
Jefferson
AR
USA


Rubens C. da Silva Dias
Biomedical Institute
Federal University of State of Rio de Janeiro
Rio de Janeiro
RJ
Brazil


Maria E. Turba
Department of Veterinary Medical Sciences
University of Bologna
Italy


Robert Villafane
Department of Biological Sciences
Alabama State University
Montgomery
AL
USA


Chengming Wang
School of Veterinary Medicine
Yangzhou University
Jiangsu
USA


Hongzhuan Wu
Department of Biological Sciences
Alabama State University
Montgomery
AL
USA




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