大肠杆菌O157H7实时荧光PCR快速检测方法建立.docVIP

大肠杆菌O157H7实时荧光PCR快速检测方法建立 　　（浙江省湖州市疾病预防控制中心， 浙江 湖州313000） 　　 　　摘要 [目的] 利用特异性荧光探针为特点的TaqMan荧光定量PCR技术，建立大肠杆菌O157:H7污染的快速敏感特异的检测方法。[方法] 以大肠杆菌O157H7的rfbE基因作为靶序列，设计一对引物和探针，以大肠杆菌O157H7菌株提取核酸DNA作为模板，优化引物和探针的浓度比和Mg2+浓度，以大肠杆菌O157:H7和10种相7关细菌考核检测体系的灵敏性、稳定性和特异性。[结果] 本研究建立的反应体系在引物和探针的浓度为0.6μmoL/L、0.8μmoL/L；Mg2+浓度为4 mmoL/L时，具有良好的特异性和敏感性。在10株相关菌株的检测中，除大肠杆菌O157:H7出现很好的阳性外，其余菌株均为阴性。在纯菌条件下，定量检测低限17cfu/mL。稳定性分析表明：同一样品重复检测3次Ct值的变异系数均小于5％。检测样品结果显示Real-time PCR方法较传统方法敏感、快捷、简便。[结论] 该方法特异性强，稳定性高，操作简便快捷，适应食品微生物检验发展需要，具有较大的推广及应用价值。 　　 [关键词] 荧光定量PCR；TaqMan探针；大肠杆菌O157:H7 　　 　　Construction and primary application of a TAQMAN PCR detection method for detection of escherichia coli O157H7 　　DE Shun-XuYUE Hua-Shen CHENG Ping-Qing (Huzhou Municipal Center for Disease Control and Prevention，Huzhou 313000, China) 　　Abstract[Objective] To establish a rapid sensitive and specific detection method of Escherichia coli O157:H7 with TaqMan PCR. [Methods] To design a pair of primers and probe depending on rfbE gene by way of target sequence of Escherichia coli O157:H7, and apply Escherichia coli O157:H7 of standard bacterium strain for template to appraise Escherichia coli O157:H7. The best Mg2+ concentration. primer and probe ratio were optimized. Specificity，sensitivity and stability analysis test were performed by Escherichia coli O157:H7 and 10 other associated bacteria strains. [Results] The best Mg2+ concentration was 4mmoL/L. Concentretion of primer s and probe was 0.6μmoL/L, 0.8μmoL/L.Test showed that the probe were highly conservative and specific. The results of all 10 bacteria strains were negative except of strains of Escherichia coli O157:H7.The quantitative detection Limit of the method was 17cfu/mL in pure cultured broth . Stability test show that Co-efficient Variables were all less than 5% in 4 different concentrations. The results show that real-time PCR method is more sensitive, more easier and more faster than conventional culture method for detection of Escherichia coli O157:H7