猪流行性腹泻病毒的RT-PCR检测及DY株的分离鉴定  

Detection by RT-PCR and isolation of porcine epidemic diarrhea virus DY strain

作　　者：任玉鹏[1,2] 陈弟诗[3] 颜其贵[1,4] 程渝[1] 郭玲[1] 杨绍林[1] 舒蕾[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]西南民族大学生命科学与技术学院,四川成都610041 [3]四川省动物疫病预防控制中心,四川成都610041 [4]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2013年第7期672-677,共6页Chinese Veterinary Science

基　　金：教育部“长江学者和创新团队发展计划”项目(IRT0848)

摘　　要：采集四川省某猪场疑似感染猪流行性腹泻病毒(PEDV)的粪便样品进行病原分离鉴定,并进一步研究了其分子生物学特性。参照NCBI上登录的CV777株M基因全基因序列设计引物,用RT-PCR法初步鉴定样品阴阳性,用Vero细胞对病毒进行连续带毒传代,将分离得到的阳性毒株命名为DY株,并克隆测定其M基因序列,分析结果表明,该株PEDV与NCBI上10个毒株的推导氨基酸序列同源性均高于95%,遗传进化树分析结果表明该毒株与GDYE9遗传距离最近;观察DY株引起的细胞病变,同时应用电镜技术对病毒形态进行观察;通过TCID50测定、中和试验以及核酸类型的鉴定,综合判定病毒分离成功。用分离的细胞毒进行乳猪回归试验,3只试验猪均出现腹泻症状。试验结果证实PEDV DY株被成功分离。Based on the published complete sequence of M gene of porcine epidemic diarrhea virus （PEDV） CV777 strain in GenBank,a pair of specific primers was designed. Fecal samples from the suspec- ted pigs with diarrhea in Sichuan were tested by RT-PCR. The complete M gene was amplified from posi- tive materials by RT-PCR and sequenced after being cloned into the pMD19-T Simple vector. The deduced amino acid sequence of the amplified M gene was compared with published PEDV M genes（10 strains）. Their homology was more than 95 %. Phylogenetic analysis of the M amino acid sequences showed the posi- tive strain DY was close to the GDYEg. Virus was continually inoculated onto Vero cells and induced a typical cytopathogenic effect, and the PEDV was observed by electron microscopy. The TCIDs0 of the DY strain was 10-14/0. 2 mL and the virus was insensitive to 5-bromodeoxyuridine. The purified virus was reinoculated into piglets,and then the piglets showed similar symptoms. The results indicated that the DY strain was PEDV.

关 键 词：猪流行性腹泻病毒 DY株 RT-PCR 分离鉴定 

分 类 号：S852.659[农业科学—基础兽医学]