牛病毒性腹泻病毒SYBR Green荧光定量PCR方法的建立及其应用

Establishment and application of a SYBR Green real-time PCR assay for detection of bovine viral diarrhea virus

为了建立BVDV快速诊断方法,针对BVDV基因组5’UTR序列保守区域设计了荧光定量PCR引物,并建立了快速检测BVDV的荧光定量PCR方法。结果表明,以构建的重组质粒pMD-5’UTR为标准品建立的SYBR Green荧光定量PCR方法标准曲线具有良好的线性关系,线性相关系数达0.997 3;敏感性试验结果显示,该方法敏感性较好,其最低检测下限为10拷贝。重复性试验结果显示,该方法批内和批间的变异系数为均小于5%,表明该方法的重复性良好。特异性试验显示,该方法与牛副流感病毒3型、牛传染性鼻气管炎病毒不存在交叉反应,但其不可区别鉴定BVDV与猪瘟病毒。应用所建立的SYBR Green荧光定量PCR方法测定非致细胞病变型BVDV(BVDV-BJ-2016株)分离毒株的一步生长曲线,发现在感染后12～48 h为其对数生长期。该研究为研究非致细胞病变型生长特性提供了参考。

To develop a rapidly diagnostic method for bovine viral diarrhea virus(BVDV),a SYBR Green I RT-qPCR assay was established.The assay had a sensitivity of 10 copies.The assay was reproducibility with less than 5% of the coefficient of valuationwithin a batch and among batch - es.There was no cross-reaction with bovine parainfluenza virus type 3 and Infectious bovine rhi - notracheitis virus.But it was not able to distinguishably identified BVDV and classical swine fever virus.The one-step growth curve of noncytopathic type BVDV was detected using the estab - fished assay.The results indicated that the logarithmic growth phase of BVDV-BJ-2016 strain was during 12h to 48h post infection.This study provides a reference for studying the growth char - acteristics of noncytopathic type BVDV isolates.