Real-Time PCR in Microbiology: From Diagnosis to Characterization
Caister Academic Press
Ian M. Mackay
Sir Albert Sakzewski Virus Research Centre, Queensland, Australia
x + 454
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Real-time PCR has established itself as a sensitive and specific qualitative and quantitative technique that has become important to all areas of microbiology. This invaluable book describes and explains some of the more complex aspects of real-time PCR presenting a background for the novice, a theoretical reference for the experienced user, and useful discussions of future developments. Chapters address the basics of PCR history, oligonucleotide design, target preparation, standardisation, quantification, various applications, and future challenges. The final chapter is presented in the format of a roundtable discussion providing an insightful, topical and interesting discourse with contributions from over 30 authorities and experts on real-time PCR.
The editor and authors have produced an excellent book that will be extremely useful for all microbiologists. A recommended book for all microbiology laboratories.
"I am impressed with the amount of valuable information in this book - information that is difficult to find in a single source. I have purchased three additional copies for each laboratory and teaching area. It is clear that I will use this book many times ..." from Rebecca T. Horvat, University of Kansas Medical Center (from Doody.com)
"The book's greatest contribution is the piece recounting popular standards and controls ... indispensable for laboratory leadership considering real-time PCR and designing assays for the first time" from Curr. Issues Mol. Biol. (2007)
"This up-to-date book on real-time PCR should be available on the bench in all diagnostic microbiology and virology laboratories. I wish I had access to this book when I started doing real-time PCR some years ago. I really enjoyed reading it and can recommend it to anyone using the technique in a diagnostic laboratory." from Susan Engelbrecht, University of Stellenbosch, South Africa
"... enjoyable and easy to read ... well-referenced and provide an easy to follow explanation, especially the chapter on fluorogenic chemistry which also has clear illustrations. The table of design rules for oligonucleotide and probes provided a good summary in an easy-to-read format. ... an interesting insight into the thoughts of the current leaders in their fields. This book shows what a versatile and sensitive tool real-time PCR can be and the many uses it can be put too. In my opinion this book would be useful for the novice because of the background information it provides." from Microbiology Today 2008
Table of contents
1. Real-Time PCR: History and Fluorogenic Chemistries
Ian M. Mackay, John F. Mackay, Michael D. Nissen, and Theo P. Sloots
The use of real-time PCR in microbial diagnostics has increased to the point where it has evolved from a novelty into a mature and essential technology for the field. In doing so, real-time PCR has driven significant changes in the way we detect microbes. The predominantly phenotype-related methods of culture and antigen detection, while still of considerable value, are being supplanted by the detection, characterization and quantification of microbial nucleic acids. Real-time PCR has engendered wider acceptance of the PCR technique due to its improved rapidity, sensitivity, reproducibility and the considerably reduced risk of carry-over contamination. There are many fluorogenic chemistries that can detect PCR product as it accumulates but only a few are useful, popular or exciting enough to be the subject of publication in the field of microbiology. We review how real-time PCR has come to be; especially the essential role of fluorescence and we critically review the plethora of detection chemistries available to the end user.
2. Oligonucleotide Design for In-House Real-Time PCR Applications in Microbiology
Today, real-time PCR has entered nearly every field in the biosciences. Numerous applications of real-time PCR have been described for basic scientific purposes and for diagnosis of hereditary and infectious diseases. Ready-to-use assays for profitable targets are commercially available and can be easily implemented in the laboratory's methods repertoire. However, for special situations or just to reduce expenses, it is often desirable to design a real-time PCR assays by oneself. Although often considered a daunting task, a manageable number of design rules permit the generation of reproducible real-time PCR assays. The basic guidelines for successful real-time PCR assay design are presented here.
3. QPCR: Target Preparation
Tania Nolan, Reinhold Mueller and Stephen Bustin
Application of modern molecular biology techniques, for the detection of pathogens has resulted in a radical change in the methodology adopted by many microbiology laboratories for the diagnosis of pathogens. Use of real-time quantitative PCR technology has resulted in much more sensitive and specific detection. It is becoming apparent that the most vulnerable stages of the qPCR and RT-qPCR protocols are those concerned with target preparation. Each specimen, organism and nucleic acid combination requires a different set of considerations for collection, transport and nucleic acid purification and the most appropriate combination should be ascertained for each project and laboratory. In order to ensure that the data collected are reliable the relevant controls should also be included. In this way a set of standard operating procedures can be defined that ensure reliable, reproducible and robust data.
4. Standards and Controls: Concepts for Preparation and Use in Real-Time PCR Applications
Amy Muska, Edith Peck and Stuart Palmer
The use of real-time PCR for molecular diagnostic applications requires a high degree of assurance that the analytic result reflects accurately the true concentration of the target nucleic acid and is not affected by inhibitors of the reaction. A variety of strategies have been developed to ensure confidence in the resulting assay information: Well-characterized reference standards, established by extensive collaborative studies using a variety of amplification methodologies can provide a basis for accurate calibration and a means for preparation of secondary and tertiary standards while co-amplification of internal controls provides assurance the PCR has functioned without inhibition. When considering the increasing complexity of reagent composition for multiplexed real-time PCR and the associated hardware for kinetic fluorescent signal acquisition, such measures would appear not only desirable but necessary. The preparation methods for reference standards, calibration standards and internal controls often present unique challenges. Consistent, stable formulation of low copy number targets requires stringent control of the laboratory environment to prevent contamination by exogenous RNAse or DNAse and a reliable means of measuring the target at such low concentrations. Depending upon the intended application for the real-time PCR, linkage of the assay calibration to a reference standard may also be desirable. The quantity of information yielded by a single multiplex real-time PCR can now readily discriminate the signal output from four or more individual target probes. This output of enhanced information content from a single reaction will continue to rapidly increase with advances in detection chemistries and hardware capabilities. This has particularly beneficial implications for molecular diagnostics. However, further challenges will be posed for the development of standards and controls that monitor and assure the accuracy of large numbers of potentially related target sequences. In the present chapter, we discuss concepts for the application of standards to assay calibration and discuss the development, preparation and use of reference standards and controls in real-time PCR assays.
5. Quantification of Micro-Organisms: Not Human, Not Simple, Not Quick
Ian M. Mackay, Stephen A. Bustin, José Manuel Andrade, Mikael Kubista and Theo P Sloots
The majority of real-time PCR applications in microbiology are for qualitative (resulting in a yes or no answer) detection of a virus, bacterium, fungus or parasite. In terms of disease relevance, the importance of quantitative PCR (qPCR) to microbiology has been proven, however it is less clear just how punctilious the clinical microbiology laboratory must be to produce relevant quantitative results. Despite a decade of qPCR experience, commercial development of applications is limited and many of our approaches remain entrenched among the PCR techniques used to monitor human mRNA levels rather than addressing adequately the diverse needs of the microbiology field. Real-time PCR has permeated every aspect of microbiology, but its applications have particular value in the clinical microbiology laboratory where the speed, sensitivity, reproducibility and accuracy of this tool help to produce robust data in a clinically relevant timeframe. Other areas within microbiology have also gained from the use of real-time PCR; gene therapy has found benefit from qPCR applications that monitor the production, replication and administration of viral vectors used to transport therapeutic genes into host cells or tissues. Studies of the host's response to microbial replication suggest a vision of the future wherein patient specimens may be used to provide an indication not only of the type of micro-organism present and its replicative status, but the stage of disease and the type of immune response underway. To make such vision reality, we must first discuss and reach consensus on the best, microbiology-specific qPCR approaches to permit the production of comparable microbial load data. This process must include the development of clear definitions associating microbial load with clinical outcome, the production of more reference materials, the development of more quality assessment schemes and of commercial kits. It may be that we find the perfect estimate of micro-organism numbers is not as important as reproducible and clinically relevant data. The increased identification of newly emergent or previously unknown endemic pathogens demands that we must strive harder than ever to expand our understanding of infectious diseases, and for that we need reliable results from reliable tools.
6. Multiplex rtPCR in Microbiology
Nick M. Cirino, Norma P. Tavakoli, Susan Madison-Antenucci and Christina Egan
Real-time PCR, or rtPCR, with its ability to detect and identify microorganisms is contributing to improvements in public health, and is facilitating more rapid attribution of disease-causing agents, whether they are old foes or newly emerging pathogens. Capitalizing on the strengths of rtPCR, we can further expand the capabilities of the methodology, so as to detect multiple target nucleic acid sequences in a single reaction. We refer to this type of an assay as a multiplex diagnostic assay, capable of detecting two (i.e., duplex) or more target signatures simultaneously. While multiplexing of rtPCR assays is achieved on a limited basis, typically two to four target sequences, there are examples of highly multiplexed assays such as DNA arrays or gene chips that can interrogate >10,000 oligonucleotide sequences in a single sample. This chapter will focus on the multiplex ability of rtPCR assays. Through integration of multiple assays into a single reaction, information about assay quality (e.g., internal positive or inhibition controls) can be simultaneously generated, and additional target pathogen sequences queried. Multiplexing therefore reduces analytical costs, improves turnaround time, expands testing capability and capacity, and adds data richness to analyses. In this chapter, we will describe the current applications of multiplex rtPCR to clinical diagnostics and public health, and we will review current applications of rtPCR to various classes of pathogenic microorganisms including viruses, bacteria, fungi, and parasites. Given a basic understanding of multiplexing concepts and critical parameters, it is fairly simple to convert conventional or rtPCR assays to multiplex formats.
7. The Role of Real-time PCR in Routine Microbial Diagnostics
Eric C.J. Claas, Willem J.G. Melchers and Adriaan J.C. van den Brule
Routine microbial diagnostics have changed significantly over the last two decades. Initially, implementation of the polymerase chain reaction (PCR) resulted in sensitive detection of microbes that were hard or impossible to diagnose by conventional diagnostic procedures such as culture and serology. A further improved version is now rapidly replacing end-point detection PCR in many diagnostic laboratories. In real-time PCR, amplified products are detected by fluorescence at the moment that they are generated and directly related to the input target quantity, so that quantitation is possible. For research purposes this has been shown to be a valuable tool in for example quantifying expression levels of genes, but also routine clinical laboratories have implemented numerous applications of this powerful method. This chapter provides an overview of different routine microbial applications in clinical diagnostics and shows the increasing importance of real-time PCR in the different fields of microbiology.
8. Challenges Facing Real-Time PCR Characteriztaion of Acute Respiratory Tract Infections
Ian M. Mackay, Katherine E. Arden, Michael D. Nissen and Theo P Sloots
The age of reliance upon in vitro cell culture for routine laboratory diagnosis of respiratory virus infections has well and truly passed. We are much more comfortable with the application of molecular methods to detect and characterise the most common and frequent infectious agents of humans. The increased acceptance of molecular tools is not at the complete expense of other biological or serological methods; they will always have a place in microbiology, however the era of the high-throughput laboratory has driven the use of faster, more sensitive and more specific methods to diagnose of viral pathogens. Unfortunately these methods have some inherent limitations and the use of molecular techniques pose a number of serious problems to overcome, especially apparent in the area of respiratory virus detection and characterization. The scope and diversity of respiratory viruses mean that scientists in this field have to design and evaluate their own assays "in-house" because commercial options are extremely limited. The question of our ability to reliably detect so many viruses and so much subtle nucleotide variation is at the forefront of assay design and implementation problems, and we must address this aggressively. We are also faced with the extreme difficulty of quantifying respiratory viruses from essentially acellular fluids, secreted from within the host onto its surface (albeit a humid, highly convoluted invagination housed within our body) which we sample using a variety of collection methods. Quantification is further complicated by issues of specimen quality, handling and storage. Recently, the appearance of newly identified viruses or NIVs have both challenged and stymied respiratory virus real-time PCR assay designers and will undoubtedly continue to do so into the foreseeable future. We also see that real-time PCR cannot be used in isolation; for maximum success it must be accompanied by nucleotide sequencing, phylogenetic analyses and constant vigilance over the relevant literature. There is also a driving need to delve into the increased co-detection of multiple viral sequences and the ill-defined impact of quasispecies variation on oligonucleotide design. Real-time PCR is a mature technology and an extremely useful one for the study of acute respiratory tract infections yet it is not the perfect tool. In the meantime, we have much work to do in order to make best use of what we have to work with today.
9. Rapid Detection of Bioterror Agents
The detection of infectious agents is one of the most important tasks in the modern diagnostic laboratory. Many PCR-based methods for clinically relevant infectious agents have been implemented in routine diagnostics during recent years. However, due to the discussions about biowarfare and possible bioterrorist attacks, an arsenal of additional agents now has to be identified in a rapid and reliable manner by the diagnostic laboratory. In recent years, natural infections with pathogens such as Yersinia pestis or Bacillus anthracis have occurred only sporadically in humans and their diagnosis was restricted to selected expert laboratories. Since the anthrax letter attacks in 2001 the further abuse of these pathogens for bioterror is feared and their identification has become a routine task in expert laboratories. Similarly, variola virus, which was eradicated by a global vaccination program at the end of the last century, has not been considered as a realistic infectious threat for the last 25 years. But today, variola virus is the most dangerous viral candidate for bioterrorism and diagnosis and the treatment and prevention of poxvirus infections are of heightened public interest. These re-emerging pathogens have posed no global danger to the human population until recently therefore methods to verify their presence have not been developed during recent decades. Real-time PCR based methods are promising to fill this gap rapidly. This chapter gives a brief summary of the present applications of real-time PCR for the diagnosis of infectious agents of bioterrorism.
10. Experts Roundtable: Real-Time PCR and Microbiology
M.G.H.M. Beld, C. Birch, P.A. Cane, W. Carman, E.C.J. Claas, J.P. Clewley, E. Domingo, J. Druce, C. Escarmis, R.A.M. Fouchier, V. Foulongne, M.G. Ison, L.C. Jennings, B. Kaltenboeck, I.D. Kay, M. Kubista, O. Landt, I.M. Mackay, J. Mackay, H.G.M. Niesters, M.D. Nissen, S. Palladino, N.G. Papadopoulos, A. Petrich, M, W. Pfaffl, W. Rawlinson, U. Reischl, N.A. Saunders, C. Savolainen-Kopra, O. Schildgen, G.M. Scott, M. Segondy, R. Seibl, T.P. Sloots, Y-W. Tang, R. Tellier and P.C.Y. Woo
This chapter attempts to document informal discussions about topics of interest to those of us who develop, employ or make decisions based upon the use of real-time PCR in microbiology. It is also intended to complement the preceding chapters. The layout for this chapter was loosely based on the format of published conference discussions and popular magazine interviews. The authors were most generous with their time and very responsive to badgering. The result is a wander through the minds of those whose papers we read, whose work we admire and whose methods we often employ. Please enjoy reading the opinions provided by a diverse selection of the foremost scientists and clinicians actively working in and around the field of microbiology.
How to buy this book
(EAN: 9781904455189 9781913652357 Subjects: [microbiology] [medical microbiology] [molecular microbiology] [pcr] [molecular biology] )