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首页> 美国卫生研究院文献>Applied and Environmental Microbiology >Application of the Modular Approach to an In-House Validation Study of Real-Time PCR Methods for the Detection and Serogroup Determination of Verocytotoxigenic Escherichia coli
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Application of the Modular Approach to an In-House Validation Study of Real-Time PCR Methods for the Detection and Serogroup Determination of Verocytotoxigenic Escherichia coli

机译：模块化方法在实时PCR方法室内验证研究中的应用该方法用于检测和鉴定血清中的产毒毒素大肠埃希菌

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European Commission regulation 2073/2005 on the microbiological criteria for food requires that Escherichia coli is monitored as an indicator of hygienic conditions. Since verocytotoxigenic E. coli (VTEC) strains often cause food-borne infections by the consumption of raw food, the Biological Hazards (BIOHAZ) panel of the European Food Safety Authority (EFSA) recommended their monitoring in food as well. In particular, VTEC strains belonging to serogroups such as O26, O103, O111, O145, and O157 are known causative agents of several human outbreaks. Eight real-time PCR methods for the detection of E. coli toxin genes and their variants (stx1, stx2), the intimin gene (eae), and five serogroup-specific genes have been proposed by the European Reference Laboratory for VTEC (EURL-VTEC) as a technical specification to the European Normalization Committee (CEN TC275/WG6). Here we applied a “modular approach” to the in-house validation of these PCR methods. The modular approach subdivides an analytical process into separate parts called “modules,” which are independently validated based on method performance criteria for a limited set of critical parameters. For the VTEC real-time PCR module, the following parameters are being assessed: specificity, dynamic range, PCR efficiency, and limit of detection (LOD). This study describes the modular approach for the validation of PCR methods to be used in food microbiology, using single-target plasmids as positive controls and showing their applicability with food matrices.
机译：欧盟委员会关于食品微生物标准的法规2073/2005要求对大肠杆菌进行监测，以作为卫生状况的指标。由于病毒致毒素性大肠杆菌（VTEC）菌株通常通过食用生食而引起食源性感染，因此，欧洲食品安全局（EFSA）的生物危害（BIOHAZ）小组也建议对食品进行监测。特别地，属于血清群的VTEC菌株，例如O26，O103，O111，O145和O157是已知的几种人类暴发的病原体。欧洲参考实验室（VTEC）已提出了八种实时PCR方法，用于检测大肠杆菌毒素基因及其变体（stx1，stx2），内膜素基因（eae）和五个血清群特异性基因。 VTEC）作为欧洲标准化委员会（CEN TC275 / WG6）的技术规范。在这里，我们将“模块化方法”应用于这些PCR方法的内部验证。模块化方法将分析过程细分为称为“模块”的单独部分，这些部分根据方法性能标准针对一组有限的关键参数进行独立验证。对于VTEC实时PCR模块，正在评估以下参数：特异性，动态范围，PCR效率和检测限（LOD）。这项研究描述了用于验证食品微生物学中PCR方法的模块化方法，该方法使用单靶质粒作为阳性对照，并显示了它们在食品基质中的适用性。

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