摘要
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.
出处
《中华实验和临床病毒学杂志》
CAS
CSCD
2012年第6期486-488,共3页
Chinese Journal of Experimental and Clinical Virology
关键词
肝炎病毒
戊型
逆转录聚合酶链反应
诊断
Hepatitis E virus
Reverse transcriptase polymerase chain reaction
Diagnosis