实时荧光多重聚合酶链反应-高分辨率溶解曲线分析鉴别6种致腹泻性大肠埃希菌的研究

摘要

Objective To develop a real-time fluorescence-based multiplex PCR and high resolution melting-curve analyze method to detect diarrheagenic Escherichia coii (E.coii) strains.Methods Eight pairs of primers were designed to specifically amplify 8 different virulence genes of 6 strains of diarrheagenic E.coli.The real-time fluorescence-based multiplex PCR and high resolution melting-curve analyses were used to identify different pathogenic bacteria according to the peak value,altitude,width and area of the melting-curve.Results Eight pairs of primers were successfully designed to amplify 8 different virulence genes for 6 diarrheagenic E.coii,and the specific dissolution curves were obtained.After optimizing the reaction system,the dissolution curves for 8 specific genes were successfully obtained in the same reaction tube.Five kinds of diarrheal Escherichia coii could be identified clearly;however,EIEC and DAEC could not be identified because two genes were the same.Conclusion Real-time fluorescence-based multiplex PCR and high resolution melting-curve analysis (PCR-HRM) is a simple,specific and rapid method to identify diarrheagenic Escherichia coii,which may be used in the routine monitoring and outbreak survey of diarrhea and foodborne infectious disease.%目的 建立一种鉴别6种致腹泻性大肠埃希菌的实时荧光多重聚合酶链反应-高分辨率溶解曲线分析方法.方法 根据6种致腹泻大肠埃希菌的8种特异性毒力基因设计8对引物,采用实时荧光多重聚合酶链反应后溶解曲线分析,根据曲线的峰值、高度、宽度、面积不同鉴别不同的致病菌.结果 成功设计出8对引物,扩增出针对6种致腹泻大肠埃希菌的8种不同毒力基因,获得了特异性的溶解曲线.优化反应体系后在同一反应管中成功获得针对8种特异基因的溶解曲线,除肠侵袭性大肠杆菌、细胞致死膨胀性大肠杆菌具有相同的两种基因不能鉴别外,其中5种致腹泻性大肠埃希菌均可以明确鉴别.结论 多重聚合酶链反应-高分辨率溶解曲线分析鉴别致腹泻性大肠埃希菌是一种简单、特异、高效的方法,在腹泻病与食源性疾病的暴发调查和日常监测中具有广泛的应用价值.