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.

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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.

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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.

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"This volume provides a good overview of how newer techniques are being used to study environmental microbial populations, primarily in water. It is a very useful starting point for those who are looking for an introduction to some of the methods or need to come up to speed on developments over the last decade or so ... The chapters are very well referenced ... it provides quite a comprehensive and useful look at the applications of a range of methodologies to aquatic microbiology in recent years. " from Jean MacRae (University of Maine, Orono, USA) writing in The Quarterly Review of Biology (2011) 86: 354-355. read more ...

Environmental Microbiology: Current Technology and Water Applications Edited by: Keya Sen and Nicholas J. Ashbolt ISBN: 978-1-904455-70-7 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback "comprehensive and useful" (Quar. Rev. Biol.)

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).

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