花生制品中金黄色葡萄球菌SYBR Green qPCR快检方法的评价研究

The Evaluation Study of SYBR Green qPCR Rapid Detection Method for Staphylococcus aureus in Peanut Products

金黄色葡萄球菌是花生及其制品中常见的一种食源性致病菌,针对金黄色葡萄球菌的快速检测方法对实现花生及其制品中的食品安全防控有重要意义.根据金黄色葡萄球菌的耐热核酸酶基因(nuc)设计了3对特异性引物,同时优化了退火温度,成功建立了一种特异性引物的SYBR Green实时荧光定量PCR(qPCR)快速检测方法,进一步对该方法的特异性、重现性及灵敏度进行了验证.以8种模拟污染花生加工产品为实验对象,对该方法与荧光定量PCR试剂盒-探针法及商品化Baird-Parker干粉培养基法进行了评价研究.结果表明,建立的SYBR Green qPCR快速检测方法特异性和重现性良好,最低检测限为8.99 copies/μL,其灵敏度比荧光定量PCR试剂盒-探针法高2个数量级.SYBR Green qPCR快速检测方法具有成本低、检测快速、特异性好、灵敏度高的优点,可用于花生及其制品中金黄色葡萄球菌的快速检测,也可为防控其他食品中的金黄色葡萄球菌污染提供参考.

Staphylococcus aureus is a common causative pathogen in peanuts and peanut products.Rapid detection of S.aureus is of great significance for the prevention and control of microbial contamination of peanut products.In this study,we design three pairs of specific primers according to the nuc gene of S.aureus,and a SYBR Green qPCR rapid detection method is established by optimizing the annealing temperature.The specificity,repeatability and sensitivity of the method are further evaluated.Finally,eight simulated contaminated peanut products are used as experimental objects to evaluate SYBR Green qPCR method,fluorescence quantitative PCR kit-probe method and commercial Baird-Parker dry powder medium method.The results show that the established method can specificity and repeatability detect S.aureus,the minimum detection limit is 8.99 copies/μL,and its sensitivity is two orders of magnitude higher than that of probe PCR kit method.The SYBR Green qPCR method has the advantage of economy,rapidity,specificity and high sensitivity,is suitable for the rapid detection of S.aureus in peanuts and peanut products,and can also provide reference for the prevention and control of S.aureus contamination in other foods.