A Novel Poisson Distribution–Based Approach for Testing Boundaries of Real-Time PCR Assays for Food Pathogen Quantification
Abstract:The validation of quantitative real-time PCR systems and above all, proof of the detection limit of this method, is a frequently and intensively discussed topic in food pathogen detection. Among proper sample collection, assay design, careful experimental design, execution of real-time PCR, and data analysis, the validation of the method per se ensuring reliable quantification data is of prime importance. The purpose of this study was to evaluate a novel validation tool for real-time PCR assays, based on the theoretical possibility of the amplification of a single DNA target. The underlying mathematical basis for the work is Poisson distribution, which describes patterns of low particle numbers in a volume. In this context, we focused on the quantitative aspect of real-time PCR for the first time. This allowed for demonstration of the reliable amplification of a lone target DNA molecule and the demonstration of the distinct discrimination between integer molecular numbers when using low initial copy numbers. A real-time PCR assay amplifying a 274-bp fragment of the positive regulatory protein A locus of Listeria monocytogenes was used for this work. Evidence for a linear range of quantification from a single target copy to 10 ng of target DNA was experimentally demonstrated, and evidence for the significance of this novel validation approach is presented here.
Document Type: Research Article
Affiliations: 1: Christian Doppler Laboratory for Molecular Biological Food Analytics, Veterinärplatz 1, 1210 Vienna, Austria 2: Institute of Milk Hygiene, Milk Technology, and Food Science, Department of Veterinary Public Health and Food Science, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria
Publication date: September 1, 2011
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