1种血清戊型肝炎病毒荧光定量 RT-PCR 快速检测方法的建立

摘要

目的：建立一种从血清样本中基于荧光定量 PCR 的快速检测戊型肝炎病毒的方法。方法（1）从 GeneBank 获得包含我国流行的3个基因型的100条戊型肝炎病毒序列。选择合适的序列设计合成荧光定量引物和 Tanqman 探针。（2）将引物从血清模板中 PCR 扩增片段在体外转录产物作为荧光定量 PCR 标准品，同时引入了微量核酸裂解液。（3）取10份临床戊肝阳性患者血清样品，用建立的方法检测，进一步验证该方法。结果该检测技术可以有效检测 I 型和 IV 型戊型肝炎阳性患者的血清样本，而对其他病毒传染病患者血清检测为阴性结果，证实该 RT-PCR 检测技术的特异度较高、可靠性较好。对10份临床血清样品的验证检测进一步证实了该方法快速、便捷、灵敏且重复性好。结论建立了一种适用于中国人群戊型肝炎病毒主要基因型检测的荧光定量 RT-PCR 检测技术。可满足戊型肝炎病毒临床早期快速诊断的需求。%Objective To establish a method for the rapid detection of hepatitis E virus (HEV)from serum samples based on fluorescence quantitative PCR.Methods (1 )One-hundred HEV sequences including our country popular three major genotypes were obtained from the GeneBank with the Vector NTI software.The proper sequence was selected to design and synthesize the primers of the fluorescence quantitation and the Taqman probe.(2)The amplification region PCR fragment was transcribed in vitro to synthesize cRNA standard,at the same time the trace serum virus lysate was introduced into a universal real-time TaqMan PCR assay.(3)10 clinical serum samples were collected from the patients with clinical hepatitis E and detected by using the established method for further verifying this method.Results This detection technique could effectively detect the serum samples in the pa-tients with genotype I and genotype IV hepatitis E positive,while the serum detection in the patients with other virus infectious dis-eases had the negative results,which verified that this RT-PCR detection technique had higher specificity and good reliability.The detection results from 10 clinical serum samples further verified that this method was rapid,convenient and sensitive with good re-peatability.Conclusion A fluorescence quantitative RT-PCR detection technique suitable for detecting main genotypes of HEV in China population is established,which can meet the demand of early and rapid diagnosis for HEV.