目的 建立戊型肝炎病毒(hepatitis E virus,HEV)不同基因型RNA实时定量逆转录PCR(qRT-PCR)检测方法及其所需质粒标准品,以利于临床一线实验室展开应用.方法 选取HEV ORF3高度保守区设计可检测HEV不同基因型病毒株的引物和探针;将拟扩增的HEV RNA目的基因片段构建成质粒,制备HEV质粒DNA标准品,用于浓度标准曲线及一步法qRT-PCR检测方法的建立;同时检测并分析HEV核酸与抗原和抗体之间的相关性;采用逆转录-巢式PCR(RT-nPCR)进行对比验证,并对部分阳性标本进行测序,通过生物进化树分析其流行病学特点.结果 成功建立基于HEV质粒标准品的qRT-PCR检测方法,适用于血清及粪便标本,最低检测限可达25拷贝/test;HEV核酸与抗原的检测结果具有较好的相关性,两者一致性达77.8%;经生物进化树分析发现9位HEV RNA阳性患者均感染为基因4型,但分属4a、4d和4n三个不同的基因亚型.结论 成功建立基于HEV质粒标准品的qRT-PCR检测方法,为后续临床应用商品化提供技术支持.%Objective To establish a real-time quantitative reverse-transcription PCR ( qRT-PCR) method for detection of hepatitis E virus ( HEV) of different genotypes based on standard HEV DNA plasmid in order to promote its application in clinical laboratory. Methods Specific primers and probe of HEV were designed based on the conserved open reading frame 3 (ORF3) regions. HEV DNA plasmids were construc-ted and 10-fold serial dilutions of the plasmids were prepared and used as standards to establish one-step qRT-PCR. The established method was compared with HEV antigen, antibody and RT-nPCR assays. Some positive samples were sequenced and analyzed by evolutionary tree. Results The one-step qRT-PCR meth-od for HEV detection in serum or feces samples was successfully establish. It could reach a sensitivity of 25 copies/test and 77. 8% of its results were consistent with those by HEV antigen assay. Nine patients were infected with HEV of genotypes 4a, 4d or 4n as indicated by evolutionary tree. Conclusion The HEV qRT-PCR method based on its standard plasmid is successfully established, which paves the way for commercial-ization of clinical applications.
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