Christian Doppler Laboratory for Monitoring of Microbial Contaminants, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria.
HTK Hygiene Technologie Kompetenzzentrum GmbH Bamberg, Bayern, Germany.
Methods Mol Biol. 2021;2220:31-40. doi: 10.1007/978-1-0716-0982-8_3.
Quantitative PCR, if performed properly, is a highly sensitive and robust tool. Nevertheless, its application to the particular case of pathogen detection from foodstuffs necessitates special requirements for reliable results. Firstly, a robust analytical chain, involving sample preparation and DNA isolation with purification, is necessary to ensure optimal performance. Secondly, for reliable quantification of Listeria monocytogenes from food, reproducible controls for all steps of the analytical chain are needed, which can give quantitative information about the performance of each individual step of the detection chain. Ideally, each individual sample should include a so-called internal sample process control (ISPC) which passes through all steps of the analytical chain and is phenotypically similar to the target organism (in this case L. monocytogenes).This chapter describes the modular and rapid (3 h) sample preparation method "matrix lysis" for the quantification of L. monocytogenes from food and gives detailed information regarding the application of an ISPC based on the example of the L. monocytogenes Δ-prfA/+IAC strain.
如果操作得当，定量 PCR 是一种高度敏感和稳健的工具。然而，将其应用于从食品中检测病原体的特殊情况，需要可靠结果的特殊要求。首先，需要一个稳健的分析链，包括样品制备和带有纯化的 DNA 分离，以确保最佳性能。其次，为了可靠地从食品中定量检测李斯特菌单核细胞增生李斯特菌，需要对分析链的所有步骤进行可重复的控制，这些控制可以提供有关检测链每个单独步骤性能的定量信息。理想情况下，每个单独的样本都应包含一个所谓的内部样本过程控制 (ISPC)，该控制应通过分析链的所有步骤，并且表型上与目标生物体（在这种情况下为单核细胞增生李斯特菌）相似。本章描述了用于从食品中定量检测单核细胞增生李斯特菌的模块化和快速（3 小时）样品制备方法“基质裂解”，并详细介绍了基于单核细胞增生李斯特菌 Δ-prfA/+IAC 菌株的 ISPC 应用的信息。
