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

摘要

To establish a qualitative and quantitative detection method for Staphylococcus aureus in milk, a real-time PCR assay based on SYBR Green I 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 Green 1 was specific, highly sensitive, and could be further used in detection of S. Aureus in milk.%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.