PCR结合单酶切对戊型肝炎病毒进行基因分型的研究  

Genotyping of hepatitis E virus by PCR combining with single restriction endonuclease analysis

作　　者：潘宁[1] 戴星[2] 孟继鸿[1] 刘社兰[3] 

机构地区：[1]东南大学医学院病原生物学与免疫学系,南京210009 [2]东南大学医学院附属中大医院皮肤科 [3]江苏省疾病预防控制中心急性传染病科

出　　处：《中华实验和临床病毒学杂志》2005年第2期179-181,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金 (3 0 2 712 3 1) ;江苏省自然科学基金(BK2 0 0 2 0 5 3 )

摘　　要：目的　建立PCR结合单酶切对戊型肝炎病毒(HEV)进行基因分型的方法,并将其应用于HEV基因型分布的研究。方法　采用简并引物扩增1～4型HEVORF1片段,1、2型HEVPCR产物为2 75、2 6 9bp ,3、4型PCR产物为317、314bp ,分别应用NaeⅠ、NotⅠ消化两种长度的PCR产物,根据酶切结果区分4种基因型。结果　采用此方法对4种基因型的HEV标准株分型,结果与预期一致;4 3份HEVIgM阳性的临床标本中,19份PCR阳性,分型结果均为4型HEV。结论　此方法可以快速简便地区分HEV 4种基因型。南京地区散发戊型肝炎大多由4型HEV所致。Objective To develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area. Methods Twenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis. Results The PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae Ⅰ, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not Ⅰ, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing,19 were HEV PCR-positive and identified as genotype 4. Conclusion A simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

关 键 词：戊型肝炎病毒 基因分型 酶切 PCR产物 散发戊型肝炎 HEV 基因型分布 IGM阳性 引物扩增 临床标本 分型结果 南京地区 4型 Nae Not 标准株 区分 

分 类 号：R373.2[医药卫生—病原生物学]