该研究组建了基于高分辨熔解曲线分析的大肠杆菌O157:H7实时荧光定量PCR快速检测试剂盒，对试剂盒的灵敏度、特异性、抗干扰能力和检测人工污染样品的情况进行了评估，并在组建试剂盒的基础上，利用叠氮溴化丙锭（PMAxx）对活/死细胞的选择性，构建了大肠杆菌O157:H7活菌定量检测技术。结果显示，试剂盒特异性强，仅大肠杆菌O157:H7血清型有阳性扩增；最低检测限为84 CFU/mL；在四种常见食源性致病菌干扰下能准确检测出大肠杆菌O157:H7；在人工污染食品样品的实际检测中，可检测到初始污染量为7.97×100 CFU/mL的样品；通过单因素变化试验对PMAxx处理的各项参数进行优化，确立了曝光时间11 min、暗孵育时间20 min、PMAxx浓度30 μmol/L的最优反应体系，并与本研究组建的实时荧光定量PCR试剂盒相结合，建立了在3.4×107~3.4×102 CFU/mL浓度范围内Ct值与菌液浓度线性关系良好的大肠杆菌O157:H7活菌定量检测方法，为食源性致病菌快速定量检测提供技术支持。

A fluorescence-based quantitative real-time polymerase chain reaction (PCR) rapid detection kit for Escherichia coli O157: H7, developed based on high-resolution melt curve analysis, was assembled and evaluated for sensitivity, specificity, anti-interference ability, and detection of artificially contaminated samples. On the basis of kit assembly, we developed a quantitative detection technique for viable E. coli O157:H7 by utilizing the selectivity of propidium monoazide (PMAxx) for viable/dead cells. Our results indicated that the kit was highly specific, with positive amplification occurring only for the E. coli O157:H7 serotype, with a minimum detection limit of 84 CFU/mL. E. coli O157:H7 could be accurately detected under the interference of four common foodborne pathogens. While testing artificially contaminated food, samples with an initial contamination level of 7.97×100 CFU/mL could be detected. Subsequently, various parameters of the PMAxx treatment system were optimized by single-factor experiments and the following optimum reaction conditions were determined: exposure time: 11 min, dark incubation time: 20 min, PMAxx concentration: 30 μmol/L. By combining the optimized parameters with our assembled kit, we established a quantitative method for the detection of E. coli O157:H7 with a good linear relationship between Ct value and bacterial solution concentration within the concentration range of 3.4×107~3.4×102 CFU/mL. The results of this study provide a scientific basis for the rapid quantitative detection of foodborne pathogens.

国家重点研发计划重点专项（2018YFC1602500）；江苏省农业科技自主创新项目（CX(18)3053）