致病性钩端螺旋体TaqManReal－timePCR检测-中华流行病学杂志.PDF

致病性钩端螺旋体TaqManReal-time PCR检测技术的建立及其应用 张翠彩 李秀文 聂一新 杨会棉 蒋秀高 [摘要] 目的 建立致病性钩端螺旋体(钩体)TaqManReal-timePCR检测技术。方法 以钩 体16SrRNA基因的部分片段rrs基因作为靶基因，设计引物、TaqMan探针，PCR产物克隆到 pMD19-T载体，制作标准曲线，建立定量分析质控标准。利用中国15群15型致病性钩体参考菌株、 16群21型非致病性钩体参考菌株、50株不同血清群致病性分离株及伯氏疏螺旋体、嗜肺军团菌、肺 炎链球菌、脑膜炎奈瑟菌等27株其他常见致病菌检验引物、探针的灵敏性、特异性。将Real-time PCR、普通PCR同时应用于倍比稀释致病性钩体染色体DNA及25份现场鼠肾标本的检测。结果 建立、优化致病性钩体Real-timePCR技术，致病性钩体扩增荧光信号阳性，非致病性钩体及其他非 钩体菌均无扩增。对于倍比稀释的质粒标准品，Real-timePCR和普通PCR的最低检测下限分别是 10copy/μl和104copy/μl。对于倍比稀释的钩体染色体DNA，两者的最低检测下限分别为：100f/μl 和1ng/μl。25份现场鼠肾标本检测显示，两种方法的检测结果一致。结论 以rrs为靶基因建立的 Real-timePCR技术，具有较高的灵敏度和特异度，可用于致病性钩体的病原学检测。 钩端螺旋体；TaqMan；Real-timePCR EstablishmentandapplicationofTaqManReal-timePCRforthedetectionofpathogenic Leptospiraspecies ZHANGCui-caiLIXiu-wenNIEYi-xin YANGHui-mianJIANGXiu-gao NationalInstituteforCommunicableDiseaseControlandPrevention,ChineseCenterforDiseaseControl andPrevention,Beijing102206,China ThisworkwassupportedbygrantsfromtheNationalScienceandTechnologyMega-ProjectsofChina (No.2008ZXi0004-002,2008ZX10004-008). [Abstract] Objective TodevelopandevaluateaTaqManReal-timePCRmethodforthe detectionofpathogenicLeptospiraspecies.Methods rrsgeneofpartfragmenton16SrRNAwas usedtodesignprimersandTaqManprobe.ThetargetgenewasclonedintovectorpMD19-Tinorder tomakethestandardcurveandbeusedforqualitycontrol.Todeterminethespecificityand specificity,DNAfromChineseLeptospirastrainsbelongingto15pathogenicreferencestrains,21 non-pathogenicreferencestrains,and50differentserotypesofpathogenicisolatesaswellas27other micro-organismswereincludedinthisstudy.EightserialDNAdilutionsfrompathogenicLeptospira andDNAfrom25kidneytissuesweredetectedbyReal-timePCRandconventionalPCR simultaneously.Results AReal-timePCRmethodologywasdevelopedandoptimised.Allthe pathogenicLeptospiragaveapositiveamplification.Non-pathogenicLeptospiraandalltheother micro-organismswerenotamplified.TheplasmidsensitivityofReal-timePCRandconventional PCRwere10copy/μland104copy