基于qPCR检测金黄色葡萄球菌3种肠毒素基因的方法研究

Detection of three enterotoxin genes of Staphylococcus aureus based on qPCR

目的建立同时检测3种可引起食物中毒金黄色葡萄球菌肠毒素sea、seb、sec基因的方法。方法对GenBank中公布的sea、seb、sec基因序列进行分析,针对3种肠毒素基因分别设计3对特异性引物和探针,通过条件优化建立检测肠毒素sea、seb、sec基因的三重qPCR方法。结果该方法可以特异性检测到sea、seb、sec基因,而与其他病原菌或肠毒素基因无交叉反应;对肠毒素sea、seb、sec基因的最低检测量分别为10.2、1.63、9.4 copies/μL;组内和组间的变异系数均小于5%。结论该方法快速灵敏、准确可靠,为金黄色葡萄球菌肠毒素的快速检测及多重qPCR试剂盒的开发奠定基础。

Objective To establish a method for simultaneous detection of sea,seb and sec genes of three common Staphylococcus aureus enterotoxins causing food poisoning.Methods By analyzing the sea,seb and sec gene sequences published in GenBank,three pairs of specific primers and probes were designed for the three enterotoxin genes respectively,and a triple qPCR method for detecting the sea,seb and sec genes of enterotoxin was established by optimizing the conditions.Results This method could specifically detect sea,seb and sec genes without cross-reaction with other pathogens or enterotoxin genes.The minimum detection values of enterotoxin sea,seb and sec genes were 10.2,1.63,9.4 copies/μL,respectively.The coefficient of variation within and between groups was less than 5%.Conclusion This method is fast,sensitive,accurate and reliable,which lays a foundation for the rapid detection of Staphylococcus aureus enterotoxin and the development of multiple qPCR kits.