狂犬病病毒荧光定量rt-pcr检测方法的建立及应用.pdf

摘要本实验参照SRV9株全基因序列,选取N基因组保守区序列设计一对引物和一条Taqman探针。通过与狂犬病病毒7个基因型代表毒株N基因保守区序列进行比较,可以得出与基因I型的代表毒株具有高度的同源性,在优化反应条件的基础上, 本实验成功地建立了可以特异的针对RV基因I型两步法荧光定量(Real-Trine Quantitative)-21细胞毒提取总RNA,转录合成cDNA,10倍稀释后建立了标准品,该标准品可以用于准确定量临床样品中狂犬病病毒滴度,以标准品验证建立的荧光定量RT--I:'CR方法对134份I临床样品进行检测并同时进行免疫荧光和普通RT-PCR。结果表明,荧光定量RT-PCR方法与免疫荧光的符合率为100%。而且比普通RT-PCR敏感性高,证明建立的荧光定量PCR方法具有特异、敏感、快速、。关键词;狂犬病病毒;荧光定量PCR:总RNA Development and Application ofReal-Time RT-PCR Assay fordetection ofRabies Virus Abstract Inthis武udy,他fered plete sequence ofrabiesvi吣Urmn of relative conservation ofsubcloned rabies virusstrainSRV9 Ngene WaS selected鹊target regions forplacement oftwoprimers and aTaqMan with7genotypes representative strainsar娜of relativeconservation ofrabies virusN to have height homology with representative strains ofgenotype I ofrabies systematic optimization,we have essfully developed aspecificquantitative method for genogype I ofrabiesvirus(R的detection,using Va,o-mbe Real-time RT-PCR assay. Furthermore,the l'吖erse缸'anscfipfion但:D oftotal鼢弧product WaS made from rabies virus strainSRV9 infected BHK-21 celllinewith known titerWaS used togenerate 10 times serialviralcDNA standards in theReal-time RT- standards may have accurated toquantitative rabiesvirus暇”titer ofthe method iS abletodetect as lessamount 8, oftheviruswiththe standard preparation flLrther of134 clinicaltissue$pecimeiis byReal-time RT-PCR parison withi咖urIofIuon} showed thattherasalt wascoincidence. Moreover,Real-time is sensitiver than routine RT-PCR Has been found to Real-time RT-PCR besensitive with theadvantages of atestthatis rapid,and amenable tohigh-thronghput itWaS able tOappliea:e indetection of rabies virusthathasbecame apowerful instrument inRV monitoring inthehfitialstage