HTK Hygiene Technologie Kompetenzzentrum GmbH Bamberg, Bayern, Germany.
Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria.
Methods Mol Biol. 2021;2220:41-53. doi: 10.1007/978-1-0716-0982-8_4.
Quantitative real-time polymerase chain reaction (qPCR) is one of the most used molecular methods. There are numerous qPCR assays on the market, some of them for pathogen detection, and the development of new assays still continues. However, what methods are suitable for assay performance validation and which information do they provide? For conclusions based on qPCR data, it is essential to know which capacities and limitations an assay has. This chapter gives an overview of methods for qPCR assay performance validation and the respective insights and how to combine them. Most of those validation methods have been published in connection with the prfA assay, which specifically detects Listeria monocytogenes. Thereby, it could be demonstrated that this assay reliably quantifies even a single copy of the prfA gene and is thus suitable for detection of Listeria monocytogenes.
实时荧光定量聚合酶链反应（qPCR）是最常用的分子方法之一。市场上有许多 qPCR 检测方法，其中一些用于病原体检测，并且新的检测方法仍在不断开发中。然而，哪些方法适合检测性能验证，它们提供了哪些信息？对于基于 qPCR 数据的结论，了解检测的能力和局限性至关重要。本章概述了 qPCR 检测性能验证方法以及各自的见解，以及如何将它们结合起来。这些验证方法中的大多数都是与 prfA 检测方法相关联发表的，该方法专门检测单核细胞增生李斯特菌。由此可以证明，该检测方法能够可靠地定量检测到单个 prfA 基因拷贝，因此适用于单核细胞增生李斯特菌的检测。
