鉴别犬瘟热病毒强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法的建立

Development of a SYBR GreenⅠReal-time RT-PCR assay for detection and identification wild-type and vaccine strain of canine distemper virus

为建立犬瘟热强弱毒株SYBR Green Ⅰ荧光定量RT-PCR检测方法,根据Gen Bank公布的犬瘟热毒株的全基因序列并参考相关文献,选择M基因保守区域合成了1对通用引物M1/M4及鉴别犬瘟热强弱毒株的特异引物M2和M3。试验证明,该方法重复性良好;检测强弱毒株的最低检出限度在10拷贝;对新城疫病毒、水貂肠炎病毒、阿留申病毒、犬腺病毒、犬细小病毒5种病毒的特异性检测均为阴性;对175份犬瘟热疑似病料进行荧光定量RT-PCR,检出127份阳性样品,其中强毒株98份,弱毒疫苗株29份,常规RT-PCR检出112份阳性样品。结果表明,该方法的敏感性高于常规RT-PCR检测,并能有效的鉴别强弱毒株。

SYBR GreenⅠReal-time RT-PCR was developed for the detection and identification of wild-type and vaccine strains of canine distemper virus（ CDV）. According to the differences of whole gene sequence of M gene in the strains of CDV in Genebank,two pairs of primers,universal primer M1 and M4 and specific primers M2 and M3 were designed to differentiate wild-type and vaccine strain of CDV. The developed RT-PCR could detect minimum 10 copies for wild-type and vaccine strains and specifically differentiate NDV,MEV,ADV,CAV,and CPV. SYBR GreenⅠReal-time RT-PCR and normal PCR detected 127 positive samples and 112 positive samples,respectively. Twenty-nine positive samples were vaccine strain in 127 positive samples detected by SYBR GreenⅠReal-time RT-PCR. This results show that the sensitivity of this assay is higher than normal RT-PCR,which can also differentiate wild-type and vaccine strains of CDV.