【摘要】 对已发表的戊型肝炎病毒(HEV)不同基因型毒株全序列进行分析,针对ORF3的保守区设计引物和探针优化建立了HEV荧光RT-PCR检测体系。分析表明该反应体系具有良好的扩增效率、特异性和稳定性,且检测灵敏度比普通RT-PCR方法高10～100倍。利用所建立的荧光定量PCR方法对采集自浙江及其周边上海、江苏等地区规模化猪场的样品以及HEV抗体阳性人血清样品进行了HEV核酸检测。其中,77份人血清样本仅有2份为HEV核酸阳性(2.6%)。而猪粪便样品阳性率则高达20.5%(56/273),且所调查的猪场均存在HEV感染。进一步的分子流行特征分析表明,浙江及周边地区的猪HEV毒株多为基因Ⅳ型,与其他地区的Ⅳ型HEV毒株同源性较高(83.4%～89.5%),但它们之间也存在较多的变异位点。猪源HEV毒株PJ-1则与多数Ⅲ型HEV毒株同源性较高(88%),进化分析也表明其为基因Ⅲ型。人血清样品中检测到的2份HEV毒株Human-1与Human-2与本地区Ⅳ型猪源毒株在分子进化关系上具有很高的亲源性,它们分布于同一分支内,而不构成独立分支。以上分析结果表明浙江及其周边地区猪HEV感染较为广泛,且以基因Ⅳ型毒株为主,但也存在基因Ⅲ型毒株的流行。更多还原

【Abstract】 A Taqman-based real time PCR assay was developed for detection of hepatitis E virus according to the ORF3 gene.The assay showed highly specificity and stability,as well as a 10-100 fold higher sensitivity as compaired with conventional RT-PCR.The carrier status of hepatitis E virus(HEV) in pig populations in different pig farms of Zhejiang province,as well as in HEV-positive human sera was examined by the assay.The results showed that only 2 as positive samples of 77 human sera were detected by real time PCR.However,out of 273 fecal samples from 18 pig farms with anti HEV antibodies,fifty-six were positive for HEV nucleic acids(recovery rate 25.5%).Further study revealed that most of the swine HEV isolates in this area belonged to genotype Ⅳ,which displayed high homologies to each other.Nevertheless,there were substantial substitutions in these isolates.Strikingly,another swine HEV isolate(PJ-1),which showed high identity of about 88% to genotype Ⅲ HEV isolates,was found to be distributed in genotype Ⅲ cluster by phologenetic analysis.Two human HEV isolates shared 83.4%-89.5% nucleic acid identity to those swine HEV isolates of genotype Ⅳ,and belonged to HEV genotype Ⅳ together with majority of the HEV isolates from other parts of China as well as those from Japan.It is evident that genotype Ⅳ HEV is of substantial prevalence in the pig populations in the area which were closely related to the human HEV isolate.更多还原