【摘要】 目的建立一种快速检测豆类及其制品中肠出血性大肠杆菌O157、O104的双重real-time PCR方法。方法分别选取大肠杆菌O157的特异性基因rfbE和大肠杆菌O104的特异性基因wzy作为靶基因,设计相应的特异性引物探针,建立双重real-time PCR反应体系,并对反应体系及引物浓度等反应条件进行优化。结果最终确定反应体系为:以大肠杆菌O157、大肠杆菌O104菌株的DNA按照1∶3比例混合作为模板,模板DNA 2μL,Master Mix 12.5μL,上游引物(10μmol/L)各1μL,下游引物(10μmol/L)各1μL,探针(10μmol/L)各0.5μL,灭菌水补齐至25μL。实现了同一次处理样品在同一反应体系中同时检测肠出血性大肠杆菌的两种血清型,检测灵敏度的下限可达102CFU/m L(g)。结论该方法特异性好,对其他常见病原菌无扩增,灵敏度高、快速、简便,为食源性致病菌的检测提供了理想手段。更多还原

【Abstract】 Objective To establish a rapid assay to detect enterohemorrhagic Escherichia coli O157（ E. coli O157） and enterohemorrhagic Escherichia coli O104（ E. coli O104） in the legume plants and its products by using the duplex real-time PCR. Methods Based on part fragments of E. coli O157 specific gene rfb E and E. coli O104 specific gene wzy,primers and Taq-Man probes were designed. Results DNA（ O157） ∶ DNA（ O104） = 1 ∶ 3,DNA 2 μL,Master Mix 12. 5 μL,primers（ 10 μmol/L） 1 μL,Taq-Man probes（ 10 μmol/L） 0. 5 μL,dd H₂O 25 μL. The two pathogens could be detected in one reaction system,the lowest limit of detection sensitivity was 10² CFU/mL（ g）. Conclusion This method is rapid and convenient,with good specificity,high sensitivity. The duplex real-time PCR assay could provide a cost-effective and convenient method for detect foodborne pathogenic bacteria.更多还原