牛病毒性腹泻病毒RT-PCR方法的建立及应用

Establishment and application of a RT-PCR detection method for bovine viral diarrhea virus

目的建立用于牛源样本中牛病毒性腹泻病毒(BVDV)双重RT-PCR检测方法。方法选择已发表的BVDV 1型和BVDV 2型包含5’UTR区高保守区域基因作为靶基因,分别设计合成引物,建立双重BVDV RT-PCR方法,并对方法的特异性、敏感性、稳定性等进行方法学评价。同时用建立的RT-PCR方法检测了小牛血清、小牛血清去蛋白提取液、脾多肽注射液等41批次牛源性样本和64份牛血浆样本。牛源样本和64份牛临床样本。结果建立的BVDV RT-PCR检测方法与牛副流感病毒III型(BPIV3)、猪瘟病毒(CSFV)、乙型脑炎病毒(JEV)均无交叉反应;检测BVDV 1型和BVDV 2型DNA模板最低浓度分别为每微升8.87×10~2拷贝和6.31×10~2拷贝,BVDV 1型和2型c DNA在-30℃冰箱放置12个月仍可检测出目的条带。应用建立的BVDV RT-PCR方法检测41批次牛源样本和64份牛血浆样本,核酸阳性率分别为14.6%和29.7%。结论建立的BVDV RT-PCR检测方法具有快速、特异、敏感及稳定的特点,可用于牛源样本携带BVDV核酸的检测。

ObjectiveTo establish a dual RT PCR detection method for bovine viral diarrhea virus(BVDV)in bovine derived samples.MethodsThe primers were designed and synthesized according to the published BVDV1and BVDV2genes containing highly conservative sequences in the5’untranslated regions(5’UTR)to establish the dual RT PCR method.The specificity,sensitivity,stability of this method were evaluated.Then41bovine derived samples and64bovine plasma samples including bovine calf serum,deproteinized calf serum extract and one lienal polypeptide injection were detected with this method.ResultsThere was no cross reaction with bovine parainfluenza virus type3(BPIV3),classical swine fever virus(CSFV)and Japanese encephalitis virus(JEV)when samples were detected with the established dual RT PCR method.The lowest concentration of template DNA for detection of BVDV1and BVDV2was887×102copies and631×102copies per microliter,respectively.Electrophoresis bands of about151bp and303bp were still amplified and detected after the BVDV1and BVDV2cDNA was stored at-30℃for12months.The BVDV positive rate of41bovine derived samples and64bovine plasma samples detected with this dual RT PCR method was146%and297%,respectively.ConclusionsThe established dual RT PCR method has the advantages of high efficiency,specificity,sensitivity and stability,and can be used for the detection of BVDV in bovine derived samples.