戊型肝炎病毒通用性PCR引物的设计及其基因分型的研究  

Design and Application of a Set of Universal PCR Primers for Genotyping of Hepatitis E Virus

作　　者：李峰[1] 孟继鸿[1] 董晨[1] 戴星[2] 杨义贵[3] 周镇先[3] 

机构地区：[1]东南大学医学院 [2]东南大学附属中大医院,南京210009 [3]南京市第二医院检验科,南京210037

出　　处：《病毒学报》2009年第1期9-16,共8页Chinese Journal of Virology

基　　金：江苏省自然科学基金资助项目(BK2006098);国家863高技术项目(2006AA02A235);国家自然科学基金资助项目(30671858);江苏省高技术项目(BG2006607)的部分资助

摘　　要：针对戊型肝炎病毒(HEV)基因分型尚无统一标准的现状,本研究通过分析GenBank中现有的82株HEV基因组全序列,设计一组HEV通用性PCR引物(HEVuPrimer)用于扩增不同基因型HEV可测序长片段,分别基于全基因序列、HEVuPrimer扩增序列和较常用的MXJ引物扩增序列对82株HEV进行基因分型,再以HEVuPrimer扩增1～4型HEV参考株和HEVIg M抗体阳性的临床标本,进行HEV基因分型的研究。研究结果表明HE-VuPrimer扩增区段与全基因序列对82株HEV的基因分型结果完全一致;HEVuPrimer与HEV序列的匹配程度明显高于MXJ引物,且HEVuPrimer区段的基因分型结果较MXJ区段的更为准确。HEVuPrimer可同时扩增出不同基因型HEV基因片段。124份临床标本中,60份测出特异性HEV RNA,阳性率为48.4%,基因分型结果均为4型HEV,但分属于4个不同的基因亚型,核苷酸同源性为80.0%～99.9%,其中有6例近期分离的HEV毒株形成一新的基因亚型。因此,基于HEVuPrimer扩增区段的HEV基因分型方法具有较高的可靠性和可信度,为建立统一、可行的HEV基因分型标准提供了新的思路。To improve the reliability and credibility of genotyping hepatitis E virus （HEV） and to explore the possibility of unifying standards of HEV genotyping by designing HEV universal primers for amplification of a long genomic fragment of different HEV genotypes. A set of universal primers （HEVuPrimer） was designed based on conserved regions determined by alignment analysis of 82 HEV strains with complete genome in GenBank. HEVuPrimer was compared with a set of previously used primers （MXJ primers） for their sequence-matching to different HEV strains and applied to amplify HEV genomic fragments from HEV reference strains with known different genotypes and clinical serum samples with anti-HEV- IgM by RT-nPCR. HEV genotyping based on the fragments amplified with HEVuPrimer was compared and validated with that based on HEV full genome and fragments obtained with MXJ primers. HEV genotyping by the phylogenetic analysis supplemented with the percent of nucleotide identity of the HEVuPrimer-determined fragments showed good correspondence with that based on HEV full-length genome. In addition, HEVuPrimer was much better than MXJ primers in matching sequences of HEV strains available from GenBank, and was able to amplify all the reference HEV strains with different genotypes. Among 124 samples with anti-HEV-IgM, 60 were positive for HEV RNA determined by a 644bp amplicon of RT- nPCR with the HEVuPrimenr. All the positive isolates belonged to HEV genotype 4 with nucleotide homology of 80.0%99.9%, and could be further divided into 4 subgenotypes. Moreover, a novel subtype was identified with 6 HEV strains isolated very recently. The RT-nPCR using the HEVuPrimer and phylogenetic analysis of the amplified region provided strong evidences for its feasibility in HEV genetic classification. Our data have new implication for the consensus of genotype classification of HEV.

关 键 词：戊型肝炎病毒 基因分型 通用性引物 逆转录-套式聚合酶链反应 

分 类 号：R373.2[医药卫生—病原生物学]