实时荧光环介导等温扩增快速分型致泻大肠埃希氏菌方法的建立

Development of a real-time fluorescence loop-mediated isothermal amplification method for rapid subtyping of diarrheagenic Escherichia coli

致泻大肠埃希氏菌(diarrheagenic Escherichia coli,DEC)是一种重要的食源性致病菌,该研究立足DEC目前流行率高、基层分型困难的现状,建立了快速分型5种DEC的实时荧光环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法。针对DEC的8段特征性毒力基因pic、aggR、lt、st、stx 1、stx2、escV、invE及大肠埃希氏菌属特异性基因uidA设计LAMP引物,优化反应体系,并对方法的分析性能及临床菌株分型效果进行了评价。结果显示,所建立的方法对pic和escV的检出限是1 ng/μL,对aggR、stx 1、invE和uidA的检出限是100 pg/μL,对lt、st和stx 2的检出限是10 pg/μL,该方法灵敏度与特异度均为100%,DEC型别间无交叉反应,对临床菌株的分型与实际结果完全一致。方法快速简便、设备要求低,借助恒温荧光读取设备可在40 min内迅速判定待检菌株是否为DEC及其致病类型,可满足各机构现场快速检测的需求。

Diarrheagenic Escherichia coli(DEC)is a significant foodborne pathogen.Aiming at the current situations of high prevalence and subtyping difficulty of the DEC,a real-time fluorescence loop-mediated isothermal amplification(LAMP)method was developed for rapid subtyping of DEC.LAMP primers were designed for pic,aggR,lt,st,stx 1,stx2,escV,invE and the genus specific gene uidA of Escherichia coli.The key parameters was optimized,as well as the analytical performance and subtyping results for clinical DEC strains were evaluated.The detection limit of the proposed method were 1 ng/μL for pic and escV,100 pg/μL for aggR,stx 1,invE and uidA,and 10 pg/μL for lt,st and stx 2 respectively.The sensitivity and specificity of the method were both 100%.No cross-reaction was observed under different DEC types,and the subtyping results of clinical strains were consistent with actual results.The method was rapid and straightforward without demanding equipment requirements.By utilizing a constant temperature fluorescence reading device,it can rapidly identify whether the strain being tested is DEC and its pathogenic type within 40 min.Thus,the proposed method can satisfy the needs of on-site detection for most institutions.