同步增菌结合实时荧光聚合酶链式反应快速检测食品中5种致病菌

Rapid detection of 5 kinds of foodborne pathogens by real-time polymerase chain reaction with simultaneous enrichment

中文关键词:  同步增菌  食源性致病菌  实时荧光聚合酶链式反应  熔解曲线

英文关键词:simultaneous enrichment  foodborne pathogens  real-time polymerase chain reaction  melting curve

中文摘要:

      目的 实现食品中5种致病菌的同步增菌, 并结合实时荧光聚合酶链式反应(real-time polymerase chain reaction, RT-PCR)技术, 建立快速检测的方法体系。方法 比较金黄色葡萄球菌、单核增生李斯特氏菌、大肠埃希氏菌O157:H7、副溶血性弧菌和鼠伤寒沙门氏菌5种目标菌分别在营养肉汤和缓冲蛋白胨水中的生长曲线; 优化DNA模板提取的方法; 采用实时荧光PCR技术及熔解曲线分析, 建立5种致病菌的快速检测方法。结果 缓冲蛋白胨水可以作为5种目标菌的同步增菌液, 增菌3 h后5种目标菌能被检出, 最低检出的DNA浓度范围为10?3~10?4 ng/μL。经熔解曲线分析, 5种目标菌的平均特征熔解温度(Tm)值范围为79.51~87.39℃。结论 本研究建立的5种致病菌快速检测方法, 检测总时间大约为6 h, 且方法灵敏度和特异性均能满足检测要求。

英文摘要:

      Objective To realize the simultaneous enrichment of 5 kinds of pathogenic bacteria in food, and establish a rapid detection method system by combining real-time polymerase chain reaction (RT-PCR) technology. Methods The growth curves of 5 kinds of target bacteria including Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Vibrio parahaemolyticus and Salmonella typhimurium in nutrient broth and buffer peptone water were compared; the method of DNA template extraction was optimized; real-time fluorescent PCR and melting curve was used to analyze, a rapid detection method for 5 kinds of pathogenic bacteria was established. Results Buffer peptone water could be used as the simultaneous enrichment solution for 5 kinds of target bacteria. After enrichment for 3 hours, 5 kinds of target bacteria can be detected. The lowest detected DNA concentration range was 10?3?10?4 ng/μL. According to the melting curve analysis, the average characteristic melting temperature (Tm) of 5 kinds of target bacteria ranged from 79.51 to 87.39℃. Conclusion The rapid detection method for 5 kinds of pathogenic bacteria is established in this study, the total detection time is about 6 hours, and the sensitivity and specificity of the methods can meet the detection requirements.