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