肠道病毒71型的RT-PCR诊断及基因特征  

Identification of Enterovirus Type 71 by RT-PCR and the Gene Characterization

作　　者：崔爱利[1] 许文波[1] 李秀珠[2] 胡家瑜[2] 凌华[3] 唐伟[2] 杨智宏[4] 张燕[1] 陈立[1] Hiroyuki Shimizu 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052 [2]上海市疾病预防控制中心,上海200336 [3]重庆市疾病预防控制中心,重庆400042 [4]复旦大学附属儿科医院,上海200032 [5]日本国立卫生研究院

出　　处：《病毒学报》2004年第2期160-165,共6页Chinese Journal of Virology

基　　金：WHO资助;项目编号(WP/CHN/IVD/001/RB02)

摘　　要：2002～2003年从上海市和重庆市手足口病患儿的疱液标本中分离到10株病毒,其中上海9株,重庆1株。利用两对分别针对EV71和Cox.A16病毒VP1区的特异性引物,用RT PCR方法对病毒进行初步鉴别。根据PCR所用引物及PCR扩增出的产物片段大小不同,可初步判定出EV71和Cox.A16病毒的血清型别。RT PCR结果提示,上海分离到的9株病毒中,有2株为EV71,其余7株病毒为Cox.A16;重庆分离到的1株病毒为EV71。10株病毒的RT PCR产物经序列测定和分析证实,PCR定型结果正确,说明PCR法具有很高的特异性,可作为EV71初步鉴定的首选方法。对所分离的3株EV71病毒进行VP1区编码基因全序列的测定和遗传学分析,通过同源性比较和构建系统发生树发现,此3株EV71病毒和中国大陆已发表的7株EV71病毒(SHZH03、SHZH98、SH F1、SH F2、SH H25、SH H26和CHN 87)全部属于C基因型,与该型代表株比较,同源性为89 3%～94 6%;与A、B基因型代表株比较,同源性为81 3%～84 0%,差异较大。在C基因型中,此3株EV71病毒和中国大陆先前分离的6株病毒(SHZH03、SHZH98、SH F1、SH F2、SH H25、SH H26)的同源性较高,在94 5%～100%范围内。在系统发生树上,这9株病毒形成一个较独立的分支。与CHN 87株的同源性在92 1%～93 6%之间。与已知的C1、C2、C3亚型代表株比较,同源?10 virus strains were isolated from clinical specimens of children with hand-foot-and-mouth disease (HFMD), 9 isolates from Shanghai and 1 isolate from Chongqing. All of the 10 isolates were tested by RT-PCR assay with two specific primer pairs for VP1 genes of enterovirus type 71 (EV71) and Coxsackie virus A16 (Cox. A16) respectively. The enterovirus serotype 71 and Cox. A16 were primarily identified depending on the size of PCR products and the primers used. The RT-PCR results indicated that 2 EV71 isolates were from Shanghai, 1 was from Chongqing and 7 Cox. A16 isolates were from Shanghai. All of the 10 PCR products were sequenced, the sequence analysis confirmed that PCR identified results was 100% correlative to virus sequencing, so RT-PCR assay is highly specific and probably may be the first choice for identification of EV71 and Cox. A16. 891 nucleotides of VP1 coding genes of 3 EV71 isolated strains were sequenced and compared with that of previously isolated 7 EV71 Chinese isolates available from GenBank (SHZH03?SHZH98?SH-F1?SH-F2?SH-H25?SH-H26 and CHN-87) by homogeneity and phylogenetic tree analyses. The homogeneity of these 10 Chinese strains with the representative isolates of C genotype of EV71 was between 89.3%-94.6%; with the representative isolates of A and B genotypes was between 81.3%-84.0%. The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C except CHN-87, which was untyped. The homogeneity of the 3 EV71 isolated strains and 6 previously isolated strains (SHZH03?SHZH98?SH-F1?SH-F2?SH-H25?SH-H26) were between 94.5%-100%, that formed a single branch in the phylogenetic tree. There were only 89.3%-92.9% homology among these 9 strains and the representative strains of C1?C2?C3 sub-genotypes of EV71, this suggested that these 9 Chinese isolates and the TAI-98 composed a new sub-genotype, the C4 sub-genotype, of the C genotype. EV71 of sub-genotype C4 distributed in Shenzhen, Chongqiang and Shanghai from 1988-2003. It is much helpful to develop EV71 diagnosis, virus surv

关 键 词：肠道病毒71型 RT-PCR 诊断 基因 分子流行病学 

分 类 号：R373.2[医药卫生—病原生物学]