鉴别犬瘟热病毒疫苗株和野毒株RT-PCR-RFLP方法的建立及应用  

Development of RT-PCR-RFLP for detection and differentiation of wild-type and vaccine viruses of canine distemper virus

作　　者：张洪亮[1] 张传美[1] 王聪[1] 黄娟[1] 任颖超[1] 单虎[1] 

机构地区：[1]青岛农业大学山东省预防兽医学重点实验室,山东青岛266109

出　　处：《中国兽医学报》2013年第11期1647-1656,共10页Chinese Journal of Veterinary Science

基　　金：山东省自然科学基金(ZR2010CL023);科技基础性工作专项(2012FY111000);863计划(2011AA1006)0703

摘　　要：根据Onderstepoort株H基因序列,设计1对引物建立RT-PCR-RFLP检测方法,对不同宿主来源的疑似犬瘟热临床样品进行检测,并对检出的犬瘟热病毒野毒山东株PCR产物进行克隆和序列分析,验证RT-PCR-RFLP检测方法。结果RT-PCR扩增片段为1 921bp,产物经RFLP分析,野毒株的PCR产物能被NdeⅠ酶切为1 282、345和294bp 3个片段,弱毒疫苗株则不能被切开;病毒RNA的最小检出量为2.15ng。临床检测共检出19份样品为犬瘟热阳性,其中15份为野毒株感染,其H基因编码区全长为1 824bp,均在1 279和1 543处有NdeⅠ酶切位点,推导氨基酸与野毒株的同源性在92.9%～96.9%,与疫苗株的同源性在89.0%～90.8%,进化树分析显示这些毒株在基因型上属于Asia-1型,为野毒株谱系。该方法的建立为临床上犬瘟热病毒的鉴别检测和诊断提供了依据。In order to develop a differentiation method for detection of wild-type and vaccine viru- ses of canine distemper virus（CDV）,a pair of primers was designed according to the hemaggluti- nin（H） gene sequence of Onderstepoort strain,and RT-PCR-RFLP was developed and used to de- tect suspected clinical samples collected from different host-species in Shandong province during 2010--2011. PCR products of wild-type CDV were subjected to gene sequencing. The results showed that a fragment of 1 921 bp was amplified from wild-type CDV which could be digested with Nde II into three fragments,while the vaccine viruses could not be digested with Nde I ,the lowest detected concentration of RNA was 2.15 ng. Nineteen positive samples of CD were detec- ted by RT-PCR,among of which, 15 wild-type strains identified by RT-PCR-RFLP contained two Nde I recognition sequence at site 1 279 and 1 543 of 1 824 bp-length H gene coding region,the deduced amino acid of 15 field strains showed 92.9%-96.9% homology with those of refered wild- type strains,and only 89.0%-90.8% identity with those of refered vaccine virus,the genotype of 15 field strains revealed by phylogenetic analysis was Asia-I type which belongs to wild-strain pedigree. The developed RT-PCR-RFLP could be used to detect and differentiate wild-type CDV from vaccine strains.

关 键 词：犬瘟热病毒 鉴别方法 RT—PCR-RFLP 血凝素蛋白(H) 

分 类 号：S852.65[农业科学—基础兽医学]