荧光定量PCR检测牛乳中金黄色葡萄球菌肠毒素A基因方法的建立

为建立检测牛乳中金黄色葡萄球菌(S.aureus)肠毒素A基因(SEA)定性定量的检测方法,本研究针对S.aureus SEA基因片段设计1对引物,将构建的重组质粒作为阳性对照,建立了S.aureus SEA DNA的SYBR Green I real-time PCR检测方法.结果显示,特异性产物Tm值为78.2℃～78.5℃,最低可检测到49.5 fg/μL(16.5拷贝)的阳性质粒.标准曲线的相关系数为0.99.与其他常见的产SEB的S.aureus、产SEC的S.aureus、无乳链球菌、大肠杆菌、嗜热链球菌、伤寒沙门氏菌、大肠杆菌DH5 α及JM109均无交叉反应.该检测方法具有较好的特异性和敏感性,为牛乳中S.aureus的快速检测提供了新的技术手段.

Real-time PCR detection of Staphylococcus aureus enterotoxins A in milk

To establish a qualitative and quantitative detection method for Staphylococcus aureus in milk,a real-time PCR assay based on SYBR GreenⅠ was developed with a pair of primers designed according to the conserved sequence of heat-resistanting S.aureus enterotoxin A in Genbank and a recombinant plasmid containing the target gene was constructed as a standard control.Tm value of the amplified product was confirmed to be 78.2 ℃ to 78.5 ℃.This technique was highly sensitive with a detection limit of 49.5 fg/μL(16.5 copise/mL) of positive recombinant plasmid without any cross-reaction with S.aureus with SEB gene,S.aureus with SEC gene,Streptococcus agalactiae,Escherichia coli,Streptococcus thermophilus,Salmonella typhimurium,E.coli(DH5α and JM109).The correlation coefficient of the standard curve was 0.99.The developed real-time PCR using SYBR GreenⅠ was specific,highly sensitive,and could be further used in detection of S.aureus in milk.