Real-time PCR technology is an established powerful research tool used in many scientific disciplines and is also utilised for mainstream testing in the regulated markets such as food, veterinary and human in-vitro diagnostics.
This essential manual provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and provides an overview of the theory of this increasingly important technique. Renowned international authors present detailed technical insights into the underlying principles, methods and practice of real-time PCR. The initial chapters cover the important aspects of real-time PCR including choosing an instrument and probe system, set-up, nucleic acid synthesis, sample extraction controls, and validation and data analysis. Further chapters provide a comprehensive overview of important real-time PCR methodologies such as quantification, expression analysis and mutation detection. This is complemented by the final chapters, which address the application of real-time PCR to diagnosis of infectious diseases, biodefence, veterinary science, food authenticity and molecular haplotyping. This timely and authoritative volume serves both as a basic introduction to real-time PCR and as a source of current trends and applications for those already familiar with the technology. The editors also aim to stimulate readers of all levels to develop their own innovative approaches to real-time PCR.

An essential book for all laboratories using PCR.

Chapter 1 Homogenous Fluorescent Chemistries for Real-time PCR
Martin A. Lee, David J. Squirrell, Dario L. Leslie and Tom Brown
The development of fluorescent methods for the closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes. There are also a number of strand-specific probes that use the phenomenon of Fluorescent Energy Transfer. In this chapter we describe these methods in detail, outline the principles of each process, and describe published examples. This text has been written to provide an impartial overview of the utility of different assays and to show how they may be used on various commercially available thermal cyclers.

Chapter 2 Internal and Other Controls for Real-time PCR Validation
Martin A. Lee, David J. Squirrell and Dario L. Leslie
A range of factors can cause false negative results in real-time PCR through effects on one or more of the reaction components. Consequently applications requiring a high level of confidence need to be designed to control for the occurrence of false negatives. Whilst an external, or batch, control is often used, the ideal control is an internal one included in the reaction cocktail in a multiplex assay. Here we discuss the application and development of molecular mimics as controls in real-time PCR and explain concepts and experimental considerations to aid in the optimisation of controlled multiplexed assays.

Chapter 3 Analysis of mRNA Expression by Real-time PCR
Stephen A. Bustin and Tania Nolan
The last few years have witnessed the transformation of the real-time, fluorescence-based reverse transcription polymerase chain reaction (RT-qPCR) from an experimental technology into a mainstream scientific tool for the detection and quantification of RNA with an enormous range of uses in basic research, molecular medicine and biotechnology. The continuous improvement of reagents and instruments, combined with the trend towards high throughput and miniaturisation, is likely to reinforce that pre-eminence and continue to open up new application areas. Nonetheless, although in principle undoubtedly a straightforward technology, the reliability of RT-qPCR assays depends a series of sequential steps that include careful experimental design, optimisation and validation, which must be implemented pragmatically to obtain meaningful, biologically relevant data.

Chapter 4 Applications of Real-time PCR to Biothreat Analysis
Christina Egan and Cassandra D. Kelly-Cirino
With the public's reawakened concern regarding use of biological agents as weapons, the rapid detection, discrimination, and identification of pathogenic organisms and toxins has become a priority for state and federal government agencies. High confidence, cost effective, and near real-time diagnostic methods are essential to protecting national health security whether the target is public health, agriculture, commodities, or water supply infrastructures. While culture-based methods have been, and will likely remain, the gold standard for microbiological diagnostics, PCR-based tests offer significant advantages in sensitivity, specificity, speed, and data richness that make them invaluable to diagnostic laboratories. In this chapter, we will describe the application of real-time PCR methods in biodefense. We will discuss the use of real-time PCR in biodefense in terms of general workflow and processing considerations, clinical diagnostic applications, environmental diagnostic applications, and multiplex screening. Real-time PCR assays can be either quantitative (qPCR) or qualitative, depending on whether a standard curve is included with the analytical run. Most diagnostic and biodefense applications utilize the qualitative nature of real-time PCR as a detection platform; this chapter will focus on the benefits of these types of assays. Finally, we will consider the future uses and anticipated advances in real-time PCR applications as related to biodefense.

Chapter 5 Veterinary Applications of Real-time PCR for Detection and Diagnosis of Infectious Agents
Alan McNally
The detection and diagnosis of veterinary infectious diseases is an area in which the potential of Real-time PCR has been best demonstrated. In particular Real-time PCR has been successfully applied as a front line tool in the diagnostic algorithm for notifiable veterinary viral pathogens such as Avian Influenza, foot-and-mouth disease, bluetongue virus, as well as rabies and Newcastle disease virus. The rapidly transmissible nature of these agents necessitates near real-time detection and diagnosis in suspected infected animals to allow implementation of control procedures. This chapter will highlight the importance of Real-time PCR in facilitating this rapid diagnosis, and the effect such rapid detection has had on containing and controlling veterinary infectious disease outbreaks.

Chapter 6 Applications in Clinical Microbiology
Andrew D. Sails
The introduction of real-time PCR technology to diagnostic clinical microbiology laboratories has led to significant improvements in the diagnosis of infectious disease. It has been particularly useful to detect slow growing or difficult to grow infectious agents therefore much of its initial impact was in diagnostic virology. However, in more recent years real-time PCR-based methods have been introduced in diagnostic bacteriology, mycology and parasitology and there are few areas of clinical microbiology which remain unaffected by real-time PCR methodologies. One area where it has had great impact is its use for quantitation of viral pathogens. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. In addition, the closed-tube format removes the need for post-amplification manipulation of the PCR products also reducing the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. The inherent sensitivity of real-time PCR means that contamination between samples and from previously amplified product can lead to false positive results. Therefore diagnostic labs utilising real-time PCR methods have to strictly adhere to good laboratory practice to reduce the likelihood of cross contamination. In addition individual laboratories must ensure quality of diagnostic testing by participating in external quality assurance schemes.

Chapter 7 The Extraction and Purification of Nucleic Acids for Analysis by PCR
Chaminda Salgado and Waqar Hussain
Myriad methods for the extraction and purification of nucleic acids prior to PCR are currently used throughout the community. While these methods have many unique and bespoke aspects, they broadly follow a sequence of lysis, isolation, washing and elution to get from a complex biological sample to purified nucleic acid that can be used in a PCR reaction. Various common methods available for each stage are described and potential sequences for particular sample types can be discerned. The potential for these methods to be automated are discussed and the process options summarized with respect to the speed of the methods, technical skill required and the resultant purity and yield that can be expected.

Chapter 8 Oligonucleotide Primers and Probes: Use of Chemical Modifications to Increase or Decrease the Specificity of qPCR
Scott D. Rose, Richard Owczarzy, Joseph R. Dobosy and Mark A. Behlke
Although the vast majority of primers and probes employed in qPCR applications today are synthesized using unmodified DNA bases, selective use of chemically-modified bases and non-base modifying groups can prevent primer-dimer artifacts, improve specificity, and allow for selective amplification of sequences that differ by as little as a single base. A wide variety of chemical modifications have been characterized for use in qPCR. As a general class, the modifications that are in greatest use today increase the binding affinity of the oligonucleotides (i.e., increase the melting temperature, Tm). Tm-enhancing modifications allows both primers and probes to be shorter, improving the differential Tm (DTm=Tm match-Tm mismatch) between perfect match and mismatch hybridization. These modifications have widespread application in allele-specific PCR and in the detection of single nucleotide polymorphisms (SNPs). Conversely, a second class of base modifications are in common use that decrease specificity and improve duplex formation in the presence of base mismatches. Although these modifications lower Tm, they have less of an impact on primer stability than do actual mismatched bases. Universal bases permit use of primers and probes in polymorphic loci when it is desirable to detect all sequence variants and minimize mismatch discrimination.

Chapter 9 Real-time PCR Arrays
Nick A. Saunders
Real-time PCR arrays are tools that allow convenient testing of samples in many assays concurrently, parallel testing of many samples or testing of multiple samples and targets simultaneously. It is desirable to standardise and automate primer and probe selection due to the large number of assays that must be designed. Furthermore, it is useful to use probe selection techniques that increase the robustness of the individual assays since this will increase the level of compatibility between the assays and decrease the complexity of interpretation of the outputs. A simple approach to creating real-time PCR arrays is to use microtitre plates which currently have capacities of 96, 384 or 1536 features. Such arrays can be populated with user designed assays or with tests selected form a menu of over one million that are commercially available. A primary application of such arrays has been to verify gene expression data obtained using hybridisation. Cramming additional features into a device of manageable scale has led to the introduction of nanolitre volume arrays that diverge from the microtitre plate pattern. Several thousand different reactions can now be included in a single real-time PCR array. The reduction in scale also has advantages in terms of the volumes of materials required. As real-time arrays are miniaturised the number of pipetting steps required increases and it is often necessary to pre-configure them commercially leading to relative inflexibility. This limitation has prompted the development of arrays that include microfluidic channels and valves. These 'chips' can be loaded via relatively few liquid handling steps to create custom applications.

Chapter 10 The Validation of Real-time PCR Assays for Infectious Diseases
Melvyn Smith
The real-time polymerase chain reaction is now established as one of the core technologies for diagnosing infectious diseases. The early stages of the technique's development were followed by a dramatic increase in the number of diagnostic assays being published, together with the introduction of commercially produced tests. Each of the numerous publications showed a number of differences in the approach to validating the newly-produced assays and in the quality and quantity of the data supporting their validation. As a result, many workers have, at times, found it difficult to reproduce the published results from other laboratories. These difficulties can arise from e.g. a lack of information in the publication, differences in equipment between laboratories, the use of different extraction methods and sequence variations in the pathogen being detected. Over the years a number of authors have voiced their concerns over the subject of what constitutes a properly validated assay, highlighting the issues of basic scientific good practice and the responsibilities of journals in publishing full validation data. This chapter summarises the recent work covering validation and verification methodology in order to provide a practical guide to help inform and standardise the process.

Chapter 11 MIQE: Guidelines for the Design and Publication of a Reliable Real-time PCR Assay
Jim Huggett, Tania Nolan and Stephen A. Bustin
The capacity to amplify and detect trace amounts of nucleic acids has made the polymerase chain reaction (PCR) the most formidable molecular technology in use today. Its versatility and scope was further broadened first with the development of reverse transcription (RT)-PCR, which opened up the entire RNA field to thorough exploration and then, most conspicuously, with its evolution into real-time quantitative PCR (qPCR). Speed, simplicity, specificity, wide linear dynamic range, multiplexing and high throughput potential, reduced contamination risk, simplified detection and data analysis procedures as well as availability of increasingly affordable instrumentation and reduced reagent cost have made qPCR the molecular method of choice when quantifying nucleic acids. Detection of pathogens, SNP analyses and quantification of RNA, even real-time analysis of gene expression in vivo have become routine applications and constant enhancements of chemistries, enzymes, mastermixes and instruments continue to extend the scope of qPCR technology by promising added benefits such as extremely short assay times measured in minutes, low reagent usage and exceptionally rapid heating/cooling rates. The whole process is driven by the insatiable demand for ever-more specific, sensitive, convenient and cost-effective protocols. However, it has also become clear that variable pre-assay conditions, poor assay design and incorrect data analysis have resulted in the regular publication of data that are often inconsistent, inaccurate and often simply wrong. The problem is exacerbated by a lack of transparency of reporting, with the details of technical information wholly inadequate for the purpose of assessing the validity of reported qPCR data. This has serious consequences for basic research, reducing the potential for translating findings into valuable applications and potentially devastating implications for clinical practice. In response, guidelines proposing a minimum standard for the provision of information for qPCR experiments ("MIQE") have been launched. These aim to establish a standard for accurate and reliable qPCR experimental design as well as recommendations to ensure comprehensive reporting of technical detail, indispensable conditions for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.

Chapter 12 Management Aspects of Real-time PCR based Assay Development, Validation, Verification and Implementation
Jacob Moran-Gilad and Nick Saunders
There are significant risks associated with the introduction of new diagnostic assays based on real-time PCR. A consistent approach to the management of the development, validation, verification and implementation of such assays is essential to meet good practice. Adoption of a strategic framework which is followed rigorously by the team is important to ensure that the project is successful. The project core team must have clearly defined roles and produce adequate documentation that can be assessed by a separate review team. Project planning should include aspects such as setting clear objectives, identification of materials and the resources required to complete the project. Of particular importance for real-time PCR diagnostics is the inclusion of adequate positive controls. Tools for the selection of appropriate positive controls are presented and discussed here as a key aspect of diagnostic PCR project management. Following the introduction of assays to practice it is vital to maintain the standard operating procedure to ensure that it is followed consistently and so that any necessary changes are documented and adequately validated. The users of diagnostic assays must be aware of the contents of the project dossiers or have other means to verify their provenance and performance. Project documentation should be maintained to ensure that the quality of the validation data is strengthened over time