Nagaraj S, Ramlal S, Sripathy M H, Batra H V
Microbiology Division, Defence Food Research Laboratory, Mysore, India.
J Appl Microbiol. 2014 Feb;116(2):435-46. doi: 10.1111/jam.12364. Epub 2013 Nov 20.
To develop a multiplex PCR assay coupled with selective enrichment step to detect major virulence-associated genes of enterotoxigenic Staphylococcus aureus and evaluate the same directly on contaminated food samples.
The most important virulence-associated genes of Staph. aureus, which are commonly related to food safety issues, are targeted in this study. They include five major enterotoxigenic genes-sea, seb, sec, seg and sei, tst-which encodes TSST-1, mecA-which confer methicillin resistance and coa-for the enzyme coagulase along with an internal amplification control (IAC) to rule out false-negative result. A modified mannitol salt broth (MSB) supplemented with sodium pyruvate was used for selective enrichment of Staph. aureus from food samples prior to PCR. Evaluation of efficiency of different media revealed that enrichment of samples in modified MSB followed by PCR resulted in specific, sensitive and effective amplification of the targeted genes in comparison with other enrichment media. Incorporation of bovine serum albumin (BSA) as PCR enhancer improved the intensity of amplicons. The standardized multiplex PCR (mPCR) format was able to detect all the target genes at a bacterial load of 10(6) CFU ml(-1) in any sample. The PCR results were unequivocally correlated with the conventional methods when the mPCR format was assessed on a total of 91 Staph. aureus isolates. The entire assay was found to be effectual when evaluated on naturally contaminated food samples.
The combinatorial approach involving selective enrichment followed by mPCR developed in this study was found to be effective for the detection of toxigenic Staph. aureus directly from various food sources.
The developed format would find a promising application in early detection of food contaminations as well as in the diagnosis of food poisoning due to Staph. aureus.
开发一种结合选择性富集步骤的多重PCR检测方法，以检测产肠毒素金黄色葡萄球菌的主要毒力相关基因，并直接在受污染的食品样本上进行评估。
本研究针对金黄色葡萄球菌中与食品安全问题通常相关的最重要的毒力相关基因。它们包括五个主要的产肠毒素基因——sea、seb、sec、seg和sei，编码TSST-1的tst，赋予耐甲氧西林特性的mecA，以及用于酶凝固酶的coa，同时还有一个内部扩增对照（IAC）以排除假阴性结果。在PCR之前，使用添加了丙酮酸钠的改良甘露醇盐肉汤（MSB）对食品样本中的金黄色葡萄球菌进行选择性富集。对不同培养基效率的评估表明，与其他富集培养基相比，在改良MSB中富集样本后进行PCR，能实现对目标基因的特异性、灵敏且有效的扩增。加入牛血清白蛋白（BSA）作为PCR增强剂可提高扩增子的强度。标准化的多重PCR（mPCR）方法能够在任何样本中细菌载量为10(6) CFU ml(-1)时检测到所有目标基因。当对总共91株金黄色葡萄球菌分离株评估mPCR方法时，PCR结果与传统方法明确相关。在对天然污染的食品样本进行评估时，发现整个检测方法是有效的。
本研究开发的先进行选择性富集然后进行mPCR的组合方法，被发现对于直接从各种食物来源中检测产毒金黄色葡萄球菌有效。
所开发的方法在食品污染的早期检测以及由金黄色葡萄球菌引起的食物中毒诊断中具有广阔的应用前景。
