Food Safety Department, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland.
J Microbiol Methods. 2011 Jan;84(1):19-26. doi: 10.1016/j.mimet.2010.10.004. Epub 2010 Oct 14.
In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR technology in tandem with an adapted two-step enrichment protocol were assessed and validated against a reference culture method, ISO 6579, 2004. The DNA and RNA real-time PCR assays amplified a 270 bp region of the hilA gene of Salmonella enterica serovars, and incorporated an internal amplification control (IAC) which was co-amplified with the hilA gene to monitor potential PCR inhibitors and ensure successful amplification. The inclusivity and exclusivity of the hilA primer set was examined for both the DNA and RNA methods and detected the 30 S. enterica serovars but not the 30 non-salmonellae strains. The inoculation of meat carcass swabs with five different S. enterica serovars at five different inocula, indicated both PCR methods were able to detect between 1 and 10 CFU per carcass swab. The real-time DNA PCR assay performed as well as the traditional cultural method in detecting Salmonella sp. in artificially contaminated salad, chocolate, fish and cheese samples. The relative accuracy, relative sensitivity and relative specificity of the DNA PCR real-time method were determined to be 98.5, 98.1 and 100%, respectively. The DNA method was further validated in a collaborative inter-laboratory trial according to ISO 16140, 2003. The validated methods provide an accurate means for the rapid detection and tracking of S. enterica serovars giving equivalent results to the standard method within three days, thus providing an alternative testing method to the reference microbiological method. The real-time PCR methodology not only offers significant time-saving advantages compared to traditional methods, it can also be applied to a wide range of samples types.
在欧洲，如果替代方法经过验证符合 ISO 16140:2003 中概述的协议，则可以替代标准的 ISO/CEN 参考方案，用于检测食源性病原体。在这项研究中，评估并验证了使用实时 PCR 技术与改良两步富集方案串联检测沙门氏菌的两种新型方法的性能，该方法与参考培养方法 ISO 6579:2004 相对照。DNA 和 RNA 实时 PCR 检测扩增了沙门氏菌血清型 hilA 基因的 270 bp 区域，并纳入了内部扩增对照 (IAC)，与 hilA 基因一起共扩增，以监测潜在的 PCR 抑制剂并确保成功扩增。检查了 DNA 和 RNA 方法中 hilA 引物对的包容性和排他性，检测到 30 种血清型沙门氏菌，但未检测到 30 种非沙门氏菌菌株。将 5 种不同血清型沙门氏菌以 5 种不同接种量接种到肉胴体拭子上，表明两种 PCR 方法均能够检测到每头胴体拭子 1 至 10 CFU。实时 DNA PCR 检测法与传统培养法在检测人工污染沙拉、巧克力、鱼和奶酪样品中的沙门氏菌效果相当。DNA PCR 实时法的相对准确性、相对灵敏度和相对特异性分别确定为 98.5%、98.1%和 100%。根据 ISO 16140:2003，该方法在协作实验室间试验中进一步得到验证。验证后的方法为快速检测和跟踪沙门氏菌血清型提供了一种准确的方法，在三天内可获得与标准方法等效的结果，因此为参考微生物方法提供了一种替代检测方法。与传统方法相比，实时 PCR 方法不仅具有显著的节省时间的优势，而且还可以应用于广泛的样本类型。
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