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

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

read more ...

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

Theodoros N. Rampias, Emmanuel G. Fragoulis and Diamantis C. Sideris

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3 ́ RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3 ́UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3 ́ RACE procedures.
Recommended reading

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