水貂肠炎病毒疫苗株和野毒株PCR-RFLP鉴别方法的建立

为了建立水貂肠炎病毒(MEV)疫苗株与野毒株的鉴别检测方法,根据GenBank中的水貂肠炎病毒序列设计了1对引物,用来扩增包含水貂肠炎病毒NS1基因在内的2 013bp大小的片段。通过对国内疫苗毒MEVB株及其他细小病毒参考株的NS1基因序列比较发现,疫苗毒MEVB株NS1基因核苷酸1401位的碱基由T变为C,致使1399位～1404位的核苷酸构成由ATTGAT变异为ATCGAT,多了1个限制性内切酶ClaⅠ酶切位点,其他MEV分离毒株中均未出现此酶切位点。用特异性引物扩增出2 013bp的目的片段后,再用限制性内切酶ClaⅠ进行酶切分析,疫苗毒MEVB株被切割成1 404bp和609bp的2个片段,国内其他分离株均未出现上述片段。结果表明,所建立的PCR-RFLP方法可以区分疫苗毒MEVB株和野毒株。

Establishment of PCR-RFLP for differentiation of mink enteritis virus vaccine strain from wild strain

To establish a PCR-RFLP method for the differentiation of mink enteritis virus wild strain from vaccine strain,a pair of primers was designed according to the nucleotide sequence of mink enteritis virus available in the GenBank.The NS1 genes of mink enteritis viruses including wild and vaccine MEVB strain,other wild canine parvovirus and feline panleukopenia virus strains were amplified and analyzed.The result showed the substitution of T to C in 1401 site,and an additional restriction site of ClaⅠwas found in the vaccine MEVB strain,while it did not exist in other reference strains.The PCR product of NS1 gene of vaccine MEVB strain can be digested by ClaⅠ 2 fragments of 1 404 bp and 609 bp,respectively;however,the wild strains can not digested.Therefore,the method could be used to differentiate the mink enteritis virus wild strain from the vaccine strain.