1. Introduction to the Real-time Polymerase Chain Reaction

David Rodríguez-Lázaro and Marta Hernández

Food safety and quality control programs are increasingly applied throughout the production food chain in order to guarantee added value products as well as to minimize the risk of infection for the consumer. The development of real-time PCR has represented one of the most significant advances in food diagnostics as it provides rapid, reliable and quantitative results. These aspects become increasingly important for the agricultural and food industry. Different strategies for real-time PCR diagnostic have been developed including unspecific detection independent of the target sequence using fluorescent dyes such as SYBR Green, or by sequence-specific fluorescent oligonucleotide probes such as TaqMan probes or molecular beacons.

2. Principles of the Real-time Polymerase Chain Reaction

Stephen A Bustin, Sara Zaccara and Tania Nolan

The real-time fluorescence-based quantitative polymerase chain reaction (qPCR) has become the benchmark technology for the detection of nucleic acids in every area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative end-point assay, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The cost of instruments and reagents is well within reach of individual laboratories, assays are easy to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to achieve accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR assay, starting with sample selection, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the significant problems associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE).

3. 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.

4. Instrumentation and Fluorescent Chemistries Used in Quantitative Polymerase Chain Reaction

Mathilde H. Josefsen, Charlotta Löfström, Trine Hansen, Eyjólfur Reynisson and Jeffrey Hoorfar

The polymerase chain reaction has revolutionized the world of scientific research and its broad application has caused a tremendous development of versatile PCR instruments and chemistries to fit its purpose. This chapter provides the reader with a general introduction to the basics of real-time PCR instrumentation, including the thermal and optical systems and the software. Performance parameters such as temperature uniformity, accuracy and ramp speed as well as reaction format, optical systems, calibration of dyes, software and comparison between different real-time PCR platforms will be discussed from a user perspective leading to an instrument selection guide. Differences between fluorescent DNA binding dyes and target-specific fluorescently labeled primers or probes for detection of amplicon accumulation will be discussed, along with the properties and applications of the most frequently applied chemistries. The fluorophores and quenchers used for primer and probe labeling and their compatibility will be presented, and finally the future challenges and trends within the field of qPCR instrumentation will be discussed.

5. Quantification Strategies in Real-time Polymerase Chain Reaction

Michael W. Pfaffl

The present chapter describes the quantification strategies used in real-time RT-PCR (RT-qPCR), focusing on the main elements that are essential to fulfil the MIQE guidelines. The necessity of initial proper data adjustment and background correction is discussed to allow reliable quantification. The advantages and disadvantages of the absolute and relative quantification approaches are also described. In conjunction with relative quantification, the importance of an amplification efficiency correction is shown, and software tools that are available to calculate relative expression changes are presented.

6. 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.

7. Sample Preparation for Real-time PCR in Food Science

Tomáš Kuchta

Sample preparation including DNA isolation is described as the first procedural step preceding the analysis of food by real-time PCR. Principles of, applications of and prerequisites for direct DNA isolation from food matrix are presented, providing information on chaotropic solid phase extraction, solubilization with cetyltrimethylammonium bromide followed by liquid-liquid extraction, and on immunomagnetic separation. Importance of and procedures for testing the amplifiability of the isolated DNA, determination of the recovery and recovery rate of DNA from food as well as procedures improving the efficiency of DNA isolation from "difficult" food matrices are given. Various techniques for DNA quantitation, including UV spectrometry, fluorimetry and determination of amplifiable DNA by PCR, are described and their use and usefulness are discussed. The effectivity of individual approaches at the analysis of various food matrices is discussed. For the application field of rapid detection of pathogenic bacteria in food, information on cultivation enrichment, immunoseparation, centrifugation and microfiltration is provided. The applicability of different sample preparation methods at the detection of various pathogenic bacteria in several food product types is discussed. Attention is paid also to DNA isolation from enriched samples, including description of rapid techniques for partial DNA separation, bacterial cell lysis and removal of PCR inhibitors.

8. 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.

9. Oligonucleotide Primers and Probes: Use of Chemical Modifications to Increase or Decrease the Specificity of qPCR   Free download

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 (ΔTm=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.

10. Internal Amplification Controls in Real-time Polymerase Chain Reaction-Based Methods for Pathogen Detection

Nigel Cook, Gabriel A de Ridder, Martin D'Agostino and Maureen B Taylor

Assays based on nucleic acid amplification are highly efficient, but they can be affected by the presence of matrix-derived substances which can interfere or prevent the reaction from performing correctly. Careful sample treatment must be applied/used to remove these inhibitory substances. However no sample treatment can be relied on completely, thus an amplification control should be employed to be able to verify that the assay has performed correctly. An internal amplification control (IAC) is a non-target DNA sequence present in the very same reaction as the sample or target nucleic acid extract. If it is successfully amplified to produce a signal, any non-production of a target signal in the reaction is considered to signify that the sample did not contain the target pathogen or organism. If however the reaction produces neither a signal from the target nor the IAC, it signifies that the reaction has failed.

11. Standardization of Real-time PCR Methods in Food Microbiology

Kornelia Berghof-Jäger

International law requires that only food suitable for consumption may reach the market. To meet this demand, thorough microbiological testing must be performed on raw materials, the manufacturing process and finished products. Real-time PCR methods are particularly well-suited for this testing as they are fast, precise and very specific. Multiple methods, including real-time PCR, exist for testing the same analyte. These are favored according to regional preferences and regulatory requirements. However, global trade could be simplified if there was an international consensus on a set of analytical standards. The International Organization for Standardization (ISO) and the European Organization for Standardization (CEN) are platforms for generating standards through open, balanced and consensus-driven processes. To avoid duplication of work and structures, an agreement on technical co-operation between ISO and CEN (Vienna Agreement) was approved in 1991. This allows for focused expertise to be used efficiently to benefit international standardization. Currently, a few general Standards exist which describe the basic requirements of PCR methods. General standardization documents focusing on performance characteristics as well as basic requirements and definitions of real-time PCR are in development. Standards for specific detection of the food-borne pathogens Clostridium botulinum, Yersinia, STEC, Vibrio and viruses are also in progress. In parallel, standardization of real-time PCR-based methods for the detection of genetically modified organisms (GMO) and allergens in food are ongoing.

12. 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.

13. 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.

14. 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.