恒温隔绝式聚合酶链式反应检测食品中金黄色葡萄球菌

An Insulated Isothermal Polymerase Chain Reaction Method for Detection of Staphylococcus aureus in Foods

建立一种恒温隔绝式聚合酶链式反应(insulated isothermal polymerase chain reaction,iiPCR)检测食品中金黄色葡萄球菌的方法。根据nuc基因设计特异性引物、探针,并探索针对食品样品快速提取金黄色葡萄球菌模板DNA的方法;通过优化引物、探针及模板的用量,对iiPCR的特异性、灵敏度、稳定性进行评价,并利用该方法和传统PCR方法对人工污染样品和实际采集食品样品中的金黄色葡萄球菌进行检测和比较。结果表明:水浴法因操作简便、耗时短、仪器要求不高,适合快速提取模板DNA;建立的iiPCR具有特异性强、灵敏度高、稳定性好的特点,且与其他菌无交叉反应;对人工污染猪肉样品和牛奶样品中的金黄色葡萄球菌,iiPCR方法可在培养至第6小时完成检测,检出限为10^(3) CFU/mL和10^(2) CFU/mL,传统PCR方法8 h后完成检测,检出限为10^(5) CFU/mL;针对实际采集的食物样品,验证实验表明iiPCR方法在培养至第6小时的检出结果与传统PCR在培养至第12小时以及传统培养方法培养至第16小时的检出结果一致。证明建立的iiPCR方法能够更快速准确检测食品中金黄色葡萄球菌。

A method for detecting Staphylococcus aureus in foods was established by insulated isothermal polymerase chain reaction(iiPCR).In this study,specific primers and probes were designed based on the nuc gene,and the extraction of template DNA from S.aureus in food samples was explored.After optimizing the amount of primers,probes and templates,the specificity,sensitivity and stability of iiPCR were evaluated,and the application of iiPCR and traditional PCR was compared to detect S.aureus in artificially contaminated samples and actual food samples.The results showed that the water bath method was suitable for rapid extraction of template DNA due to its simple operation,short time consumption,and low instrument requirements.The iiPCR method had strong specificity,high sensitivity and good stability without cross-reaction with other bacteria.It took six hours to detect S.aureus in artificially contaminated pork samples and milk samples by the iiPCR method with a detection limit of 10^(3) and 10^(2) CFU/mL,respectively,while the traditional PCR method took eight hours with a detection limit of 10^(5) CFU/mL.For actual food samples,the verification experiments showed that the results of the iiPCR method at 6 h were consistent with those of traditional PCR at 12 h and the traditional culture method at 16 h.It was proved that the iiPCR method could detect S.aureus in foods more quickly and accurately.