猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法的建立  

Development of RT-PCR-RFLP for Detection and Differentiation of Wild-type and Vaccine Viruses of Classical Swine Fever Virus

作　　者：胡继明[1] 杨培培[1] 王颖[1] 孙海芳[1] 黄娟[1] 陈俏俏 杨瑞梅[1] 张传美[1] 秦晓冰[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,青岛266109 [2]莱阳市畜牧局,莱阳265200

出　　处：《畜牧兽医学报》2012年第6期943-949,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家科技支撑计划(2011BAD34B00);科技基础性工作专项(SQ2012FY3260033);山东省生猪产业创新团队建设;青岛市公共领域科技支撑计划(11-2-3-24-nsh)

摘　　要：本研究旨在建立猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP鉴别检测方法。根据Shimen株设计1对特异性引物,建立猪瘟病毒RT-PCR-RFLP检测方法;对20份疑似猪瘟临床样品进行检测,并对检出的山东8株流行野毒株和2株疫苗株PCR产物进行克隆与序列分析,验证上述方法。结果RT-PCR扩增片段为825bp,产物经RFLP分析,野毒株的PCR产物能被ApaⅠ酶切为322bp和503bp 2个片段,兔化弱毒疫苗株则不能被酶切,检测出RNA的最低浓度为0.028 6μg.mL-1;8株流行野毒株都含GGGCCC序列(ApaⅠ酶切位点),2株疫苗株相应序列为GAGCCC,不能被ApaⅠ酶切;8株流行野毒株属于基因2群,2株疫苗株与HCLV遗传关系近,为基因1群。建立了可鉴别猪瘟病毒野毒株和兔化弱毒疫苗株RT-PCR-RFLP检测方法,为猪瘟的防控提供有效手段。A RT-PCR-RFLP assay was developed for the detection and differentiation between wild-type and rabbit attenuated vaccine viruses of classical swine fever virus （CSFV）. A pair of specific primers was designed based on the Shimen strain, and used to verify the assay. Eight epi- demic wild-type strains isolated from 20 clinical pathological samples of suspected swine fever and 2 vaccine strains were amplified by RT-PCR, cloned and sequenced. The results showed that a fragment of 825 bp was amplified from genomic RNA of CSFV wild-type which could be digested with Apa I into two fragments of 322 bp and 503 bp when analyzed with RFLP, while the vac- cine strain could not be digested with Apa I. The lowest concentration of RNA could be detected in the present assay is 0. 028 6 μg · mL^-1. Eight epidemic wild-type strains contained the Apa I recognition sequence GGGCCC while the two vaccine strains contained the sequence GAGCCC. Eight epidemic wild-type strains belonged to Genomic Group 2. On the contrary, two vaccine strains with close genetic relationship to HCLV belonged to Genomie Group 1. The developedRT-PCR-RFLP could be used to detect and differentiate wild-type CSFV from pig vaccinated with the vaccine viruses.

关 键 词：猪瘟病毒 野毒株 兔化弱毒疫苗株 RT-PCR-RFLP 

分 类 号：S852.651[农业科学—基础兽医学]