Food Safety Department, Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland.
Food Microbiol. 2011 May;28(3):447-56. doi: 10.1016/j.fm.2010.10.012. Epub 2010 Oct 27.
The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.
本研究的目的是开发用于检测食源性沙门氏菌的 DNA 和 RNA 实时 PCR 方法，作为传统文化方法（ISO 6579，2004）的快速替代方法，用于新鲜肉胴体和加工肉样品。这些 PCR 方法基于 hilA 序列，针对该基因靶标设计了引物和杂交探针。评估了引物和探针的效率和动态范围，随后使用 106 株沙门氏菌亚种 enterica 菌株和 30 株非沙门氏菌菌株测试了该方法的特异性。还开发了内部扩增对照（IAC）进行合并。确定 IAC 的最佳拷贝数为每个反应 500 个拷贝。适应现有的标准 ISO 6579:2004 开发了补充富集方案，包括在缓冲蛋白胨水（BPW）中 22 ± 2 小时的富集和在含有大豆的瑞普氏肉汤（RVS）中的第二次选择性富集 6 小时。DNA 和 RNA 实时 PCR 方案应用于接种了沙门氏菌亚种 enterica 菌株的肉样品，包括肉胴体拭子和经过热处理或冷冻的碎牛肉样品。所开发的方法有可能替代标准 ISO 6579:2004 方法，用于在屠宰后 3 天内或在食品生产过程中使用 hilA 作为靶标对胴体拭子和生肉中的沙门氏菌亚种 enterica 进行检测。DNA 检测是在屠宰场对肉样品进行筛选的有用工具，可在 3 天内或在食品生产过程中使用 hilA 作为靶标对肉样品进行筛选。RNA 基检测方法有可能检测到即食产品中的存活沙门氏菌亚种 enterica。
