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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。
Objective To establish a double real-time PCR method for rapid detection of enterohemorrhagic Escherichia coli O157 and O104 in legumes and their products. Methods Specific genes rfbE of Escherichia coli O157 and wzy of Escherichia coli O104 were selected as target genes, and corresponding specific primer probes were designed to establish double real-time PCR reaction system. The reaction system and primer concentration The reaction conditions are optimized. Results The final reaction system was confirmed as follows: DNA of Escherichia coli O157 and Escherichia coli O104 was mixed in a ratio of 1: 3 as a template, template DNA of 2 μL, Master Mix of 12.5 μL, and 1 μL of each of the upstream primers (10 μmol / L) / L), 0.5 μL each of probe (10 μmol / L) and 25 μL of sterile water. The simultaneous detection of two serotypes of enterohemorrhagic Escherichia coli in the same reaction system with the same treatment sample was achieved, with a lower limit of detection of 102 CFU / mL (g). Conclusion The method is of good specificity, no amplification of other common pathogens, high sensitivity, rapid and simple, and provides an ideal means for the detection of food-borne pathogens.