title: 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.
authors: Christian Doppler Laboratory for Molecular Biological Food Analytics, Veterinärplatz 1, 1210 Vienna, Austria. peter.rossmanith@vu-wien.ac.at
authors: Peter
authors: P
authors: Rossmanith
authors: Martin
authors: M
authors: Wagner
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D008089:Listeria monocytogenes / Q000302:isolation & purification; D016012:Poisson Distribution; D016133:Polymerase Chain Reaction / Q000592:standards*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D004269:DNA, Bacterial