Christian Doppler Laboratory for Molecular Biological Food Analytics, Veterinärplatz 1, 1210 Vienna, Austria.
J Food Prot. 2011 Sep;74(9):1404-12. doi: 10.4315/0362-028X.JFP-10-458.
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.
定量实时 PCR 系统的验证，尤其是该方法检测限的证明，是食品病原体检测中经常讨论的话题。在适当的样本采集、分析设计、仔细的实验设计、实时 PCR 的执行和数据分析中，方法本身的验证对于确保可靠的定量数据至关重要。本研究的目的是评估一种新的实时 PCR 分析验证工具，该工具基于单个 DNA 靶标扩增的理论可能性。这项工作的理论基础是泊松分布，它描述了体积中低粒子数的模式。在这方面，我们首次关注实时 PCR 的定量方面。这使得可靠地扩增单个目标 DNA 分子成为可能，并证明了当使用低初始拷贝数时，整数分子数之间存在明显的区分。这项工作使用实时 PCR 扩增李斯特菌阳性调节蛋白 A 基因座的 274bp 片段。实验证明了从单个目标拷贝到 10ng 目标 DNA 的定量线性范围，并提出了这种新验证方法的重要性的证据。
