牛病毒性腹泻病毒巢式RT-PCR方法的建立及初步应用

Establishment and preliminary application of nested RT-PCR method for Bovine viral diarrhea virus

为了建立牛病毒性腹泻病毒(Bovine viral diarrhea virus, BVDV)巢式RT-PCR方法,试验通过对我国近5年流行的BVDV毒株的全基因序列进行比对分析,选择BVDV的保守5′端非编码区(5′UTR)片段作为靶点,设计巢式RT-PCR引物,对内外套引物进行筛选,优化反应条件,并分析建立的巢式RT-PCR方法的敏感性和特异性,同时利用该方法对172份临床样品进行检测并与实时荧光定量PCR方法进行比较。结果表明:巢式RT-PCR方法的敏感性为1.8×10^(1)copies/μL,敏感性是仅使用一对引物进行一轮PCR扩增的100倍;该方法对BVDV具有较好的特异性,与水疱性口炎病毒(VSV)、猪瘟病毒(CSFV)、口蹄疫病毒(FMDV)、小反刍兽疫病毒(PPRV)及蓝舌病病毒(BTV)均无交叉反应。从172份临床样品中共检出27份BVDV阳性样品,阳性率为15.70%,与实时荧光定量RT-PCR方法比较,符合率为81.82%。说明试验建立的BVDV巢式RT-PCR方法特异性、敏感性较好,可作为基层检测机构中诊断BVD的辅助检测方法。

In order to establish the nested RT-PCR method of Bovine viral diarrhea virus(BVDV),in this experiment,the whole gene sequences of the BVDV strains circulating in China in the past 5 years was compared and analyzed.Conservative 5′UTR fragments of BVDV were selected as targets and nested RT-PCR primers were designed.Inner and outer primers were screened;reaction conditions were optimized,and sensitivity and specificity of the method were analyzed.At the same time,172 clinical samples were detected by this method and compared with real-time RT-PCR method.The results showed that the sensitivity of the nested RT-PCR method was 1.80×10^(1) copies/μL.The sensitivity was the use of one pair of primers for one-step PCR,which was 100-fold increased.This method had good specificity for BVDV,and there was no cross-reaction with Vesicular stomatitis virus(VSV),Classical swine fever virus(CSFV),Foot-and-mouth disease virus(FMDV),Peste des petits ruminants virus(PPRV)and Bluetongue virus(BTV).A total of 27 positive BVDV samples were detected from 172 clinical samples,with a positive rate of 15.70%;compared with the real-time RT-PCR method,the compliance rate was 81.82%.The results showed that the BVDV nested RT-PCR method established by the test had good specificity and sensitivity,and could be used as an auxiliary detection method for the diagnosis of BVD in primary testing institutions.