from Chaminda Salgado and Waqar Hussain writing in Real-Time PCR: Advanced Technologies and Applications:

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.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Alan McNally writing in Real-Time PCR: Advanced Technologies and Applications:

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.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Melvyn Smith writing in Real-Time PCR: Advanced Technologies and Applications:

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.

Further reading: Real-Time PCR: Advanced Technologies and Applications

from Doris Helen D'Souza, Marta Hernández, Nigel Cook and David Rodríguez-Lázaro writing in Real-Time PCR in Food Science: Current Technology and Applications:

Analysis of foodstuffs for virus contamination requires profoundly sensitive and accurate methods, due to the potentially low number of viruses and the complexity of the sample matrix. In view of these criteria, the polymerase chain reaction is the assay type of choice, with its rapidity being another useful factor. Real-time PCR (qPCR) is superceding conventional PCR in several areas of molecular diagnostics, and a large variety of published qPCR-based methods for foodborne pathogen detection is available in the scientific literature. In common with other molecular-based methods, qPCR-based analysis of foodstuffs for viruses requires effective controls to ensure that issues associated with low virus numbers and the complexity of the matrix do not result in false negative or positive interpretations of results. These controls are essential for implementation of qPCR-based methods for foodborne virus detection, but in most cases are not included in those which have been published hitherto. Alternative molecular techniques, such as nucleic acid sequence-based amplification (NASBA) and loop-mediated amplification (LAMP) are also suitable for utilization in detection methods for viruses in foods, the same requirements regarding controls pertaining. All molecular-based methods for foodborne virus detection must of necessity contain sample treatment procedures to extract the virus or its nucleic acid out of the food matrix, and these procedures can be elaborate due to matrix complexity. Nonetheless efficient sample treatment methods have been devised, and in combination with molecular assays effective methods for virus analysis are now available for foods. Implementation of these methods in routine diagnostics will support food safety management programs and assist in outbreak investigation, and help to ensure a safe food supply.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Arne Holst-Jensen writing in Real-Time PCR in Food Science: Current Technology and Applications:

Genetic modification (GM) alters the phenotype of the GM organism (GMO). This is achieved through application of gene technology and modification of genetic information stored in nucleic acids. The logical choice of methodology to detect and characterise GM is therefore analytical methods targeting nucleic acids. The polymerase chain reaction (PCR) methodology has been the preferred methodology of this type for two decades, and the following chapter will review its applications and derivatives in relation to detection and characterisation of GM organisms (GMOs). The need for detection, identification, characterization and quantitation of GMOs depends on issues such as the legal status of the GMOs in question (authorized or not), labeling or contractual requirements, authentication, traceability and co-existence, environmental monitoring and risk assessments. The fitness for purpose of a specific analytical method is often limited to certain applications. Guidelines to establishment of analytical strategy and method selection can be very useful to those who order as well as to those who provide GMO analyses. A fundamental distinction can be made between screening and identification methods, respectively. The former may be used to group and separate putatively GMO-free samples from samples containing GMO. Both classes of methods may provide qualitative and quantitative information, but only the identification methods can provide accurate quantitation. GMO quantification is achieved almost exclusively with real-time PCR methods, but other alternatives are also available. PCR is also commonly used in combination with other techniques such as Southern blot analyses and DNA sequencing to characterize the genetic constitution of GMOs. Over the last decade extensive resources have been put into validation and critical assessment of performance characteristics and requirements for real-time PCR based GMO detection methods. GMO analyses can be particularly challenging because quantitation is required at very low concentrations, in products of highly variable nature, and where the introduced novel sequences of different GMOs belonging to the same or different species may result in misinterpretation and analytical interference. Consequently, there is a lot to learn from this field of science also for others working with real-time PCR methods. This chapter will provide several examples.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Carmen Diaz-Amigo and Bert Popping writing in Real-Time PCR in Food Science: Current Technology and Applications:

Food allergens, responsible for IgE-mediated allergic responses and listed in European, North American and Japanese regulations, are exclusively proteins and are ideally detected by analytical methods targeting either peptides or proteins. However, in some cases where no suitable methods for proteins exist or as an alternative method to substantiate results from protein-based methods, DNA-targeting methods can be used as indicators of the presence of potentially allergenic proteins. The advantage of DNA-targeting methods like PCR, real-time PCR is presently the lower cost and availability of free literature on several detection systems, including a certain degree of multiplexing. Clear disadvantages include the poor sensitivity for egg, milk and samples containing inhibitors (like polyphenols in chocolate) as well as its limited applicability in some industrial protein concentrates. In addition, if quantitative results need to be obtained, the DNA-based system needs to be calibrated for each matrix tested as protein-to-DNA composition is typically matrix specific. However, PCR based methods are well established in many laboratories and still regularly used. This chapter discusses suitable systems for detection of DNA of ingredients and foods containing allergenic proteins, potential pitfalls and multiplex capabilities of such systems.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from David Rodríguez-Lázaro and Marta Hernández writing in Real-Time PCR in Food Science: Current Technology and Applications:

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.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Nigel Cook, Gabriel A de Ridder, Martin D'Agostino and Maureen B Taylor writing in Real-Time PCR in Food Science: Current Technology and Applications:

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.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from Dietrich Mäde writing in Real-Time PCR in Food Science: Current Technology and Applications:

Yersinia enterocolitica ranks as the third bacterial food pathogen in Europe. Because cultural assays are labour and time consuming, the routine analyses of food samples need to be improved. The domestic pig is considered as the moost important carrier of the zoontic strains but the data set for food samples is limited due to the limitations of the labour intensive cultural method. Duplex real-time PCR systems targeting the chromosomally encoded ail-gene allow a sensitive and specific detection. A heterologous internal amplification control based on the plasmid pUC18 or pUC19 is applied to monitor for PCR inhibitions. The duplex real-time PCR including the heterologous IAC is a robust method for screening food samples. The combination with the cultural standard method allows the detection and cultural confirmation of a high percentage of PCR positive samples. The molecular system can be successfully applied to the test of suspect colonies.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from L. Jesús Garcia-Gil writing in Real-Time PCR in Food Science: Current Technology and Applications:

Campylobacter is a microaerophilic, spiral shaped, Gram-negative bacterium comprising 16 species. Although many of these species are thermotolerant, i.e. able to grow at 42 degrees C, C. jejuni, C. coli, C lari, and C. upsaliensis are the most prevalent foodborne pathogens. The need for a fast detection of these bacteria in foodstuff has fostered the development of rapid method, most of them based on PCR techniques. Nevertheless, the use of the appropriate targets has limited the development of reliable methods. This difficulty arises, in part, from the fact that target genes used commonly, either virulence genes or ribosomal, contain high variability, even among strains. This has serious implications, for instance, as false negative results. As a consequence, the number of available PCR protocols to detect thermotolerant Campylobacters is very limited. The use of strongly conserved, housekeeping genes as PCR targets has resulted in a good approach to the ideal real-time PCR based method. The difficulty in such a task is actually reflected in the scarce officially certified tools commercially available.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

from David Rodríguez-Lázaro, Nigel Cook and Marta Hernández writing in Real-Time PCR in Food Science: Current Technology and Applications:

A principal consumer demand is a guarantee of the safety and quality of food. The presence of foodborne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labeling in foods suitable for vegetarians are among the subjects where society demands total transparency. The application of controls within the quality assessment programs of the food industry is a way to satisfy these demands, and is necessary to ensure efficient analytical methodologies are possessed and correctly applied by the Food Sector. The use of real-time PCR has become a promising alternative approach in food diagnostics. It possesses a number of advantages over conventional culturing approaches, including rapidity, excellent analytical sensitivity and selectivity, and potential for quantification. However, the use of expensive equipment and reagents, the need for qualified personnel, and the lack of standardized protocols are impairing its practical implementation for food monitoring and control.

Further reading: Real-Time PCR in Food Science: Current Technology and Applications

"reviews and illustrates the use of quantitative real-time PCR for a number of different purposes. It covers the basic process as well as the technology that has improved its performance, while also exploring the various scientific fields that use this technique routinely. It provides a complete description of what scientists need to design and perform a quantitative PCR ... useful to scientists in many different types of laboratories, including public health, environmental, clinical diagnostic, and food industry. It also can be useful to students and young investigators as well as experienced scientists. The authors clearly are familiar with the development and application of quantitative PCR and share their experience here ... This useful book is filled with valuable information for any laboratory using PCR to detect microbial agents and will serve as a resource for many years to come. " from Rebecca T. Horvat (University of Kansas, USA) writing in Doodys read more ...

Quantitative Real-time PCR in Applied Microbiology Edited by: Martin Filion ISBN: 978-1-908230-01-0 Publisher: Caister Academic Press Publication Date: May 2012 Cover: hardback "useful book ... filled with valuable information" (Doodys)

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

The application of the RT-qPCR technology has contributed immensely to our understanding of gene expression in various biological systems; however in certain areas of research, for example applied microbiology, application of this technique has not progressed as much as one would have liked. This application gap could at best be attributed to the extreme difficulties in extracting nucleic acids from environmental samples and the high sensitivity of the RT-qPCR system towards chemical components inherently co-extracted from environmental samples. Development of a more robust RT-qPCR platform is one possible solution to overcome this problem. A cross-adaption of some new developments in amplification and enzymatic technology would alleviate some of the drawbacks inherent to the RT-qPCR technology so that itÕs potentially is fully realized in areas like applied microbiology.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

The first step towards analysing microbial gene expression requires a quantitative extraction of RNA. This step has proven to be highly problematic for environmental matrices, due to compounded inefficiencies in individual steps which include, but not limited to, incomplete cell lysis, RNA degradation by ubiquitous RNases, co-extraction of inhibitors and their interaction with the enzymes used. One straightforward approach to quantify such losses and apply the necessary correction is to include an internal amplification control (IAC), so as to make the final gene expression meaningful and reproducible. An IAC is essentially a non-target DNA/RNA sequence co-amplified, preferably in the same reaction tube, under the same reaction conditions. While attempts to develop IACÕs have met with some success for experimental systems which are highly controlled, developing such controls have proven to be highly problematic for certain experimental set-ups, for example complex environmental matrices. The main difficulty in these cases has been in our inability to identify an inert IAC which is able to (a) withstand the harsh nucleic acid extraction procedures usually employed for environmental matrices, and if such a sequence is indeed developed/ identified (b) designing a primer/probe combination which would not cross-react with other non-target (nucleic acids) components of the matrices. While few potential IAC based solutions have been proposed, for example the Biotrove OpenArray platform, high costs and proprietary issues of some IAC sequences have served as a deterrent for researchers who are seriously interested in rigorously implementing this external normalization strategy.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

The helicase-dependent (HDA) amplification system is one such novel Ônon-PCRÕ system for amplifying target DNA (Vincent et al., 2004) and RNA, under isothermal conditions. This revolutionary amplification system makes use of a novel enzymatic cocktail which does not require the DNA to be cycled between different temperatures, like that done for reactions based on Taq DNA polymerase amplification or any of its variants. In lieu of a standard denaturation step, the HDA system uses the helicase enzyme to unwind the double-stranded DNA and with the aid of other polymerizing enzymes, an exponential amplification is achieved. All these steps are performed at a fixed, user defined temperature. Though extremely novel when first introduced, the HDA system suffers from one major limitation- its inability to amplify DNA targets greater than 200 bp. As a result, in its present state, it is seriously unable to challenge and act as a viable alternative to the highly versatile PCR, or any amplification system based on it. Adapting such Ônon-PCRÕ amplification technologies could in the near future lead to detection platforms which are more robust and would not suffer from the inherent drawbacks, for example spurious amplification, cycling parameter standardization, typically associated with the classical three-stage PCR system.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

The current RT-qPCR technology is based on the classical three-step thermal cycling process which is, template denaturation, followed by primer/ probe annealing and finally, extension/detection of the fluorescence signal, to amplify and detect the target transcripts all under real-time conditions. A very commonly observed phenomenon in this multistep thermocycling amplification system is the generation of spurious fluorescence signal due to mispriming of primer/probes. To overcome such limitations, detection platforms have been proposed which amplify the target exponentially like PCR, but under isothermal conditions, i.e. at a fixed, user-defined temperature.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Jorge Santo Domingo, Mary Schoen, Nicholas Ashbolt and Hodon Ryu writing in Quantitative Real-time PCR in Applied Microbiology:

Microbial risk assessment (MRA) has traditionally utilized microbiological data that was obtained by culture-based techniques that are expensive and time consuming. With the advent of PCR methods there is a realistic opportunity to conduct MRA studies economically, in less time, and simultaneously targeting multiple pathogens and their sources. More importantly, recently developed qPCR assays provide the opportunity to estimate the densities of the reference pathogens and their sources, which is critical to quantitative MRA (QMRA) analyses. In this chapter we discuss the use of qPCR-based methods to identify risks associated with exposure to water, namely, drinking and recreational waters. We discuss the advantages associated with the current qPCR approaches used in microbial water quality studies and critically evaluate some of the limitations as they relate to the use of QMRA in the assessment of microbial water quality and public health protection.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these "substrate defined" limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this chapter, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Dan Tulpan, Michelle Davey and Mark Laflamme writing in Quantitative Real-time PCR in Applied Microbiology:

The ability of DNA microarray technology to identify and quantify microbial entities and genes of interest in various environments, such as soil, water, air, compost, and blood, propelled biological, environmental and clinical research into the post-genomic era. Nevertheless, as it is valid for any new technology, errors may occur at different stages along the experimental process. Three sources of errors associated with DNA microarray utilization have been identified by Taniguchi et al. (2001), namely: (i) the microarray fabrication, (ii) the microarray experiment, and (iii) the interpretation of results (data analysis). Validation strategies are typically required to alleviate and eventually repair the undesired errors that may arise in a microarray experiment. One of the validation techniques widely accepted and used worldwide is the quantitative Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR). This chapter will provide succinct introductions to microarray technologies applied to microbial research and fundamental notions regarding RT-qPCR and its use to validate microarray results. A discussion including advantages and disadvantages of microbial microarray validation using RT-qPCR will be presented and current and future trends and research directions will be summarized towards the end of the chapter.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Michael W. Pfaffl writing in Quantitative Real-time PCR in Applied Microbiology:

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.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Lia C.R.S. Teixeira and Etienne Yergeau writing in Quantitative Real-time PCR in Applied Microbiology:

Quantitative polymerase chain reaction (qPCR) represents an effective method to quantify genes or transcripts within environmental samples. For that reason, qPCR has been widely used to characterize the functional patterns of complex microbial communities. In this chapter we summarize some recent applications of different qPCR approaches targeting functional genes encoding key enzymes in the N-, C- and S-cycles and also functional genes related to antibiotic resistance. We also point out some limitation of qPCR approaches. The ongoing development of new molecular techniques, like metagenomics, will have positive impacts on the specificity and the coverage of qPCR assays, since the availability of more sequence data will help to improve the design of primers targeting functional genes.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Claudia Goyer and Catherine E. Dandie writing in Quantitative Real-time PCR in Applied Microbiology:

Development of quantitative PCR (qPCR) has facilitated major advances in assessment of microbial community abundances in complex environmental samples including water, soil, sediments, compost and manure and in our understanding of factors influencing community sizes in situ. qPCR has increasingly been used in environmental studies due to its sensitivity, ease of use, and the capacity to run large numbers of samples. However, qPCR has some limitations, which are specifically caused by the nature of environmental samples, including the variability in microorganism distribution, the efficiency of DNA recovery and purification, and the amount and type of PCR inhibitors co-extracted with the target nucleic acids. The heterogeneity of the templates amplified by qPCR can generate PCR biases and artifacts. Accuracy of the quantification of broad groups of microorganisms is influenced by the number of gene copies per genome of the selected marker. In this review, we will discuss the main experimental considerations for using qPCR in environmental studies, including the factors affecting key steps in the process of performing quantification of microorganisms in environmental samples. Although quantification of microorganisms is challenging, it is possible to reliably quantify microorganisms in complex environmental samples using qPCR. We will also briefly review the findings of studies which have used qPCR to quantify microorganisms from complex matrices.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these "substrate defined" limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this chapter, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Luca Cocolin and Kalliopi Rantsiou writing in Quantitative Real-time PCR in Applied Microbiology:

Since its first application in food microbiology in the late '90s, quantitative PCR (qPCR) has attracted the interest of researchers, working mainly in the field of food safety, but lately also of microbiologists studying spoilage and fermentation processes. In addition to the advantages that conventional PCR offers in microbiological testing, i.e. specificity, reduced time of analysis and detection of viable but not culturable cells, qPCR allows quantification of target populations. This aspect is particularly relevant for foodborne pathogens, for which specific microbiological criteria exist, but also for spoilage and technological important microorganisms, in order to follow their population kinetics in foods. Although advancements in food microbiology have been made from its application, qPCR has not yet been utilized to its full potential: the quantification step is only rarely carried out and qPCR is often used as an alternative of conventional PCR. In this chapter we will critically describe the application of qPCR in food microbiology based on the available literature, taking into account the specific problems and suggesting some possible solutions.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Mathilde H. Josefsen, Charlotta Löfström, Trine Hansen, Eyjólfur Reynisson and Jeffrey Hoorfar writing in Quantitative Real-time PCR in Applied Microbiology:

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.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Mikael Kubista, Vendula Rusnakova, David Svec, Björn Sjögreen and Ales Tichopad writing in Quantitative Real-time PCR in Applied Microbiology:

As the qPCR field advances, the design of experiments and the analysis of data is becoming more important and more challenging. Calculation of relative expression of a reporter gene to a reference gene in pairs of samples using the ΔΔCq method is no longer sufficient. Studies are now designed using multiple markers, nested levels, exploring or confirming the effect of multiple factors, occasionally in paired designs, etc. Proper handling of such data requires software that support the planning and design of experiments, and data analysis. Several software with these capacities are emerging. This chapter describes some of the features of one of the most powerful of those: GenEx from MultiD Analysis.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this chapter, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Stephen A Bustin, Sara Zaccara and Tania Nolan writing in Quantitative Real-time PCR in Applied Microbiology:

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).

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide:

The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:

Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. This chapter discusses the strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. It also highlights the significance of standard curves for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It also provides guidance on how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. In this chapter the optimization of the whole RT-PCR workflow will be discussed and recommendations to reduce experimental variance and produce more reproducible and reliable results are put forward.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide:

A paper from 2008 mentions that quantitative PCR is 25 years old (VanGuilder et al., 2008) but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems (Gibson et al., 1996; Heid et al., 1996). Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instrument available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). In this chapter we highlight some key features that differentiate Real-Time PCR instruments, with the goal of simplifying the criteria needed to select the instrument that best fit a specific scientist's research needs.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide:

Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. This chapter will cover all data analysis aspects from the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Gavin Meredith, Miro Dudas, Mark Landers, Vasiliki Anest, Jonathan Wang, Caifu Chen, Peter Jozsi and Christopher Adams writing in PCR Troubleshooting and Optimization: The Essential Guide:

The field of epigenetics transcends traditional genetics, genomics, molecular biology, and is poised to revolutionize the field of medical research and healthcare. It is a diverse field that encompasses the study of nuclear components such as chromatin structure, including histone modifications, protein/DNA interactions, protein/RNA interactions, and how these factors influence gene function. It also includes the study of DNA methylation and the role that non-coding RNAs play in influencing DNA methylation patterns, chromatin structure and ultimately regulating gene expression. Just as the field of epigenetics is broad and complex, so is the molecular technology of polymerase chain reaction (PCR). For every question one would like to address in any of these areas of epigenetics, there is a PCR application and instrumentation suitable to address it. For example there are numerous PCR-based approaches to look at DNA methylation patterns, densities, and even the methylation status of individual cytosine residues by PCR. Additionally, there are PCR methods to survey ncRNA expression and identify regions of the genome where proteins and RNA interact or where certain functional histone marks are located. This chapter provides an overview of these methodologies with a focus on the advantages and disadvantages of each approach.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. This chapter will address all of these factors to aid the investigator in designing high quality PCR assays.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from N. Reginald Beer and John H. Leamon writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR has traditionally been performed in microliter-scale reactions because larger scale volumes are prohibitively expensive and wasteful while the smaller scales (nanoliter and below) are impractical with available sample handling tools and detection systems. At the microliter scale, samples can contain mutually competitive and distinct targets, introducing amplification bias and competitive inhibition that degrade assay performance. Microfluidic Emulsion PCR has emerged as a technique to resolve these challenges by a combination of two enabling technologies. Emulsion PCR provides the advantages of fluid partitioning, namely elimination of sample bias and the ability to run millions of reactions in discrete volumes, while microfluidics simultaneously reduces the sample volume, introduces a level of control over emulsion parameters, and provides optical observability of the partitioned microreactors. Furthermore, since microfluidic emulsions can be made monodisperse in size, they allow the assumption of an average dilution per reactor to permit the exploitation of Poisson statistics for very accurate titer estimation. Microfluidic emulsions can also be employed to perform solid-phase amplification with bead-based assays, combining yet another useful technique with the sample partitioning benefits of droplets. We expect the advantages of both emulsion PCR and microfluidics will encourage new applications and the integration of these enabling technologies will improve PCR performance.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:

The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. The origins of PCR and its early evolution are described, including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. Early real-time instruments are compared. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide:

Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA)_ is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation. This chapter covers some of these developments and provides a guide to those wishing to establish this technique, as well as troubleshooting advice for those already underway.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:

One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay. To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, this chapter briefly explores some of the current strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. It also discusses strategies to amplify difficult templates and optimize reverse transcription reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide