BVDV和IBRV双重实时荧光定量PCR检测方法建立  

The Development of the Duplex Real-time PCR for BVDV and IBRV

作　　者：刘梦瑶 张秋楠[1] 吴文学[1] 李金祥[2] LIU Meng-yao;ZHANG Qiu-nan;WU Wen-xue;LI Jin-xiang(Lab of Rapid Diagnostic Technology of Animal Diseases,College of Veterinary Medicine,China Agricultural University,Beijing 100193,China;Chinese Acaderny of Agricultural Sciences,Beijing 100083,China)

机构地区：[1]中国农业大学动物医学院,北京海淀100193 [2]中国农业科学院,北京海淀100083

出　　处：《中国兽医杂志》2019年第11期28-31,36,I0003,共6页Chinese Journal of Veterinary Medicine

基　　金：现代农业(奶牛)产业技术体系建设专项(CARS-36).

摘　　要：根据牛病毒性腹泻病毒(BVDV)5′端非编码区和牛传染性鼻气管炎病毒(IBRV)gB基因保守区基因序列,参考文献分别合成了2对特异性引物和探针。利用构建的包含BVDV和IBRV目标基因片段的阳性质粒,对退火温度、引物和探针的浓度以及反应条件进行了优化,构建了实时荧光定量PCR(qPCR)标准曲线,并对双重qPCR方法的特异性、敏感性和重复性进行了评价。试验结果显示,qPCR最适退火温度为54.0℃,BVDV引物浓度为0.2μmol/L,IBRV引物浓度为0.3μmol/L,BVDV探针浓度0.2μmol/L,IBRV探针浓度为0.1μmol/L。BVDV的RNA最低检测限为100拷贝/μL,IBRV的DNA最低检测限为1拷贝/μL,并具有良好的特异性和重复性,为BVDV和IBRV的同时、快速、定量检测提供了有效的方法。Two pairs of primers and probes were synthesized targeting 5'-UTR of BVDV and gB Gene conserved region of IBRV,and the concentrations of the primers and probes,the reaction parameters and conditions of the real-time PCR reaction system were optimized with two positive plasmids containing the target fragments of BVDV and IBRV respectively constructed by our lab.The standard curves were drawn to assess the specificity,sensitivity and reproducibility of the Real-time PCR reaction system.The results showed that the optimum concentrations of the primers were 0.2μmol/L and 0.3μmol/L,and the optimum concentrations of the probes were 0.2μmol/L and 0.1μmol/L for BVDV and IBRV respectively,and the annealing temperature was 54.0℃.The minimum detectable limits of BVDV and IBRV were 100copies/μL and 1 copies/μL respectively,and the Duplex Real-time PCR had good specificity and repeatability.In conclusion,we develop a rapid,quantitative and simultaneous detection method for BVDV and IBRV in clinical samples.

关 键 词：BVDV IBRV 实时荧光定量PCR(qPCR):探针 

分 类 号：S858.23[农业科学—临床兽医学]