三重荧光PCR检测沙门氏菌、金黄色葡萄球菌和大肠埃希氏菌O157:H7

Detection of Salmonella,Staphylococcus aureus and Escherichia coli O157:H7 by triple fluorescent PCR

目的建立一种检验沙门氏菌、金黄色葡萄球菌、大肠埃希氏菌O157:H7的TaqMan探针三重荧光PCR方法。方法针对沙门氏菌属特异性invA基因、金黄色葡萄球菌rpoB基因、大肠埃希氏菌O157:H7的rfbE基因设计引物与探针,建立三重荧光PCR体系,对引物与探针浓度及退火温度优化,并进行特异性和敏感性研究。结果引物与探针的最优添加量为:invA 0.2μL、ropB 0.4μL、rfbE 0.5μL;最优退火温度为60℃。特异性试验显示16株非目标菌株扩增结果为阴性;目标菌株均出现显著扩增。敏感性试验显示,3种微生物对应的最低菌体数量检出量依次为:230、38、670 CFU。结论该方法特异性好、灵敏度高,能够快速检测沙门氏菌、金黄色葡萄球菌、大肠埃希氏菌O157:H7。

Objective To establish a method for detecting Salmonella,Staphylococcus aureus and Escherichia coli O157:H7 by TaqMan probe triple fluorescent PCR.Methods Primers and probes were designed for Salmonella spp.specific invA gene,Staphylococcus aureus rpoB gene and Escherichia coli O157:H7 rfbE gene,and a triple fluorescent PCR system was established.The concentration of primers and probes and annealing temperature were optimized,and the specificity and sensitivity were studied.Results The optimal amount of primer and probe were invA 0.2μL,ropB 0.4μL and rfbE 0.5μL,and the optimal annealing temperature was 60℃.The specificity test showed that 16 non-target strains were negative and target strains were significantly amplified.The sensitivity test showed that the minimum number of bacteria detected for the 3 microorganisms were 230,38 and 670 CFU,respectively.Conclusion This method is specific,sensitive,and can quickly detect Salmonella,Staphylococcus aureus and Escherichia coli O157:H7.