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A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything

Real time PCR (quantitative PCR, qPCR) is now a well-established method for the detection, quantification, and typing of different microbial agents in the areas of clinical and veterinary diagnostics and food safety. Although the concept of PCR is relatively simple, there are specific issues in qPCR that developers and users of this technology must bear in mind. These include the use of correct terminology and definitions, understanding of the principle of PCR, difficulties with interpretation and presentation of data, the limitations of qPCR in different areas of microbial diagnostics and parameters important for the description of qPCR performance. It is not our intention in this review to describe every single aspect of qPCR design, optimization, and validation; however, it is our hope that this basic guide will help to orient beginners and users of qPCR in the use of this powerful technique.

In this “A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything” , Petr Kralik & Matteo Ricchi is telling you what is qPCR. The phrase “Polymerase chain reaction” (PCR) was first used more than 30 years ago in a paper describing a novel enzymatic amplification of DNA (Saiki et al., 1985). The first applications of PCR were rather unpractical due to the usage of thermolabile Klenow fragment for amplification, which needed to be added to the reaction after each denaturation step.

In the conclusion part, the authors mentioned that qPCR technology represents a powerful tool in microbial diagnostics. In viral and parasitical detection, quantification and typing, the suitability of this technique is beyond doubt; in the area of bacterial diagnostics it can replace culture techniques, especially when rapid and sensitive diagnostic assays are required. The spread of qPCR to different areas of routine microbial diagnostics together with the lack of standard procedures for the determination of basic functional parameters of qPCR has led to a scenario in which standardization of methods is performed according to different rules by different laboratories. This issue was partially solved by the publication of MIQE guidelines (Bustin et al., 2009); however, there are differences in attitude to validation and standardization of qPCR assays across clinical, veterinary and food safety areas. Any contribution to the unification of standardization and validation procedures will improve the quality of qPCR assays in microbial detection, quantification and typing.

This articile is originally from “A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything” , by Petr Kralik & Matteo Ricchi , published in 2017.

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