应用实时荧光定量TaqManRT-PCR检测口蹄疫病毒  

Detection of Foot-and-mouth Disease Virus by Real-time,Fluorogenic TaqMan Reverse Transcription Polymerase Chain Reaction Assay

作　　者：花群义[1] 金宁一[2] 周晓黎[1] 董俊[1] 杨云庆[1] 徐自忠[1] 

机构地区：[1]云南出入境检验检疫局技术中心,云南昆明650228 [2]解放军军需大学军事兽医研究所,吉林长春130062

出　　处：《中国兽医学报》2005年第2期116-119,共4页Chinese Journal of Veterinary Science

基　　金：国家"十五"重大科技攻关项目(2001BA804A22);云南省计委科技攻关重点项目((98)636

摘　　要：按照口蹄疫病毒 (FMDV )聚合酶 3D基因序列 ,设计合成了引物和探针 ,经各反应条件的优化 ,建立了实时荧光定量 RT- PCR技术 ,对细胞培养物、水泡液、水泡皮及分泌物、血液中的 FMDV进行了特异性检测和敏感性试验。结果 ,用 30 0 nmol/ L 的引物浓度和 2 0 0 nmol/ L 探针浓度 ,获得的 CT值较小 ,而 ΔRn最大 ;可检测到相当于 0 .1 TCID50 的病毒 RNA;与 VSV和其他水泡性病毒不发生交叉反应 ;制作的标准曲线中各浓度范围内有极好的线性关系 ,且线性范围宽 ,相关系数为 0 .984 ;组内和组间试验重复性的变异系数 (CV)分别为 5 .4 %和 6 .7% ;与常规 RT- PCR相比较 ,该方法具有快速、特异、敏感、可定量 。Foot-and-mouth disease virus(FMDV) causes a highly contagious and economically important vesicular disease of wild and domestic cloven-hoofed animals.Fast and reliable diagnosis of the disease is essential for preventing the spread of the disease and tracing the source of an outbreak.Method:A fluorogenic TaqMan RT-PCR assay using a primer/probe set designed from the 3D region of the virus genome was developed for the specific detection of foot-and-mouth disease virus in epithelial suspensions and cell culture virus preparations.Result:The reverse transcription polymerase chain reaction(RT-PCR) specifically detected FMD virus in samples with greater sensitivity than conventional RT-PCR procedure.The fluorogenic RT-PCR provides relatively fast results,enables a quantitative assessment to be made of virus amounts and can handle more samples and/or replicates of samples in a single assay than the conventional RT-PCR procedure.Conclusion:The results suggest that it is a valuable procedure for FMD virus diagnosis.

关 键 词：口蹄疫病毒 荧光定量RT-PCR 实时检测 口蹄疫 

分 类 号：S855.3[农业科学—临床兽医学] S852.659.6[农业科学—兽医学]