from Martin A. Lee, David J. Squirrell, Dario L. Leslie and Tom Brown writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

from Martin A. Lee, David J. Squirrell and Dario L. Leslie writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

from Jacob Moran-Gilad and Nick Saunders writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

from Jim Huggett, Tania Nolan and Stephen A. Bustin writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

from Scott D. Rose, Richard Owczarzy, Joseph R. Dobosy and Mark A. Behlke writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

from Nick A. Saunders writing in Real-Time PCR: Advanced Technologies and Applications:

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.

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

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 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 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 Patricia Elízaquível, Gloria Sánchez and Rosa Aznar writing in Real-Time PCR in Food Science: Current Technology and Applications:

Foodborne diarrheagenic Escherichia coli strains belong to a minor number of O:H serotypes. Of them the shiga toxin-producing E. coli O157:H7 is the most frequently reported. Besides, non-O157 strains are increasingly being isolated from a variety of food products. E. coli infections are typically associated with transmission through animal products. However, in the last years, contaminated fresh produce is increasingly being implicated in E. coli O157:H7 outbreaks. Currently real-time PCR (qPCR) is considered as an alternative to standard culture methods for E. coli detection in food due to its high speed, specificity, sensitivity, reproducibility and minimization of cross-contamination. Moreover, quantification is possible when an enrichment step is omitted. Although qPCR is a very promising technique for pathogen detection in food, food laboratories and industries are still reluctant to extensively apply it. Real-time qPCR presents some challenges when applying in food, e.g. presence of inhibitors, low levels of cells, detection of dead cells. Besides, the selection of appropriate target regions is another challenge in E. coli detection because of their high genetic heterogeneity. In this review the different approaches proposed to circumvent the difficulties to detect pathogenic E. coli in food and the most frequently targeted genes are presented.

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

from Kathie Grant and Corinne Amar writing in Real-Time PCR in Food Science: Current Technology and Applications:

The principle Clostridal foodborne pathogens, Clostridium botulinum and Clostridium perfringens are responsible, respectively, for two different toxin mediated foodborne diseases, namely botulism and C. perfringens food poisoning. Foodborne botulism is a severe, life-threatening disease which can affect a large number of people and although incidence is rare, it is considered a public health emergency. Whilst C. perfringens type A food poisoning is far less severe, it is one of the most common causes of bacterial food poisoning in both the UK and US. It is important to have rapid, accurate methods to detect these two clostridial pathogens and their toxins in order to confirm the cause of illness and identify the food source so that appropriate control and preventative interventions can be implemented. However, conventional laboratory methods to detect C. botulinum and C. perfringens in foods and clinical samples are lengthy, complex, may involve the use of animals and are not always very informative. Real-time PCR assays have been developed to rapidly detect the toxin genes of both pathogens and have been used, in conjunction with culture techniques, to: improve the diagnostic procedure; enhance incident and outbreak investigations and provide information on the pathogenicity of isolates. Real-time PCR detection assays for clostridial foodborne pathogens are also highly valuable to food producers providing faster methods for monitoring growth in food enabling the safety of food products to be assessed more rapidly and effectively. The reliability of real-time PCR detection assays depends upon a range of factors from the bacterial pathogen being detected and the sample matrix to the effective use of controls to ensure the efficiency of the nucleic acid extraction and accuracy of the amplification procedure. This review focuses on the practical application of real-time PCR detection assays for these two clostridial foodborne pathogens.

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

from George D. Di Giovanni, Gregory D. Sturbaum, and Huw V. Smith writing in Real-Time PCR in Food Science: Current Technology and Applications:

Many parasites are capable of infecting humans, with zoonotic and environmental transmission pathways having significant roles. Of particular significance are pathways involving contaminated food and water. Despite increasing risks posed by food and waterborne parasites due to global sourcing of food, cosmopolitan eating habits, and increased international travel; detection and epidemiologic methods for many of these parasites remains underdeveloped. Microscopy-based detection and diagnostic techniques are still revered as the gold standard for the detection of many food and waterborne parasites. However, the advent and employment of molecular methodologies has proven to surpass microscopy in three major aspects: sensitivity, specificity and the ability to speciate. While molecular methodologies have clear advantages over many traditional detection techniques, standardized PCR methods for the detection of food and waterborne parasites are lacking. This is largely due to multiple obstacles, such as: the diversity of test matrices (e.g. fruits, vegetables, meat products, shellfish, and water); different approaches needed for recovery, concentration, and DNA extraction for different parasites; intrinsically low levels of parasites present in samples; and a lack of multi-laboratory validation of promising methods. Although the PCR detection of food and waterborne parasites may be complex and challenging, recent advances in sample processing techniques and the development of real-time PCR assays are bringing the goal of standardized methodology within view. This review covers an overview of some important food and waterborne parasites, a description of conventional detection methodology, and advances in sample processing and real-time PCR assays. Research needs are discussed along with the benefits of real-time PCR detection and typing.

Further reading: Real-Time PCR in Food Science: Current Technology 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 paper 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 review 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 review 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

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 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

"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 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   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 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 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 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 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 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