戊型肝炎病毒TaqManReal—timeRT—PCR法的建立及应用

Establishment and application of TaqMan real-time RT-PCR for the detection of hepatitis E virus

目的建立灵敏、特异、稳定的戊型肝炎病毒（HEV）TaqManReal—timePCR检测方法。方法根据GenBank中的HEV相关序列，选取HEV基因组ORF2的保守区域设计合成特异性引物和探针，建立TaqManHEVReal—timeRT—PCR检测体系，评价体系的特异性、敏感度和稳定性，并应用于临床样本的检测。结果本研究建立的HEVReal—timeRT—PCR检测体系最低检测极限达到10个拷贝／反应，重复性实验c￡值的变异系数（CV）最大为1．53％，并且该体系能特异检测出戊肝临床样本中的HEV，其拷贝数从1．87×104拷贝／ml到8．12×106拷贝／ml不等。结论成功建立特异性强、灵敏度高的HEVReal—timeRT-PCR检测方法，应用于临床样本检测时取得了良好效果，为HEV分子病原学诊断打下基础。

Objective To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis E virus （HEV）. Methods According to the references, primers-probe sets which were located in ORF2, the conservative part of HEV genome were designed and therefore we established a HEV TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HEV RNA in clinical samples. Results The HEV Real-time RT-PCR assay established in this study were able to detect HEV RNA with a detection limit of 10 copies/reaction. When the detection of a same sample was repeated for several times, coefficients of variation （CV） was all less than 1.53%. Our data also suggested that there were 1.87 × 106-8.12 × 109 RNA copies in lml of the clinical samples. Conclusion The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HEV RNA. It was applied successfully in the pathogen detection of clinical samples.