甲肝病毒特异性TaqMan荧光定量PCR法检测甲肝病毒核酸的研究

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

目的建立甲肝病毒（HAV）特异性TaqMan荧光定量PCR检测方法，并对血清等样品中的HAV进行检测。方法根据参考文献，选取HAV基因组保守区5’-NCR区引物和探针，建立特异性强、敏感度高、稳定性好的TaqManHAVReal—timeRT-PCR检测体系并应用于甲肝患者血清等病毒核酸的检测。结果本研究建立的方法能够特异检测出样品中的甲肝病毒，其灵敏度介于每反应0．1CCID50至0．01CCID50之间。同一样本重复检测3次，批内样本Ct值的变异系数最大2．O％，批间样本D值的变异系数最大2．6％。急性期甲型肝炎患者血清中甲肝病毒为5．18×102～4．93×107拷贝／ml。结论本实验建立的HAVReal—timeRT—PCR检测方法具有特异、灵敏、快速等优点，可成功应用于临床样本等甲肝病毒的病原学检测。

Objective To establish a specific TaqMan-based Real-time PCR assay for the detection of hepatitis A virus in serum samples. Methods According to the references, primers-probe sets which were located in 5＇-NCR, the most conservative part of HAV genome were designed and therefore we established a TaqMan real-time RT-PCR assay with great performance of specificity, sensitivity and reproducibility. And then it was used in the detection of HAV RNA in serum from HAV patients. Results The HAV Real-time RT-PCR assay established in this study were able to detect HAV RNA and its detection limit ranged from 0. 1CCID50/reaction to 0.01CCID50/reaction. When the detection of a same sample was repeated for three times, coefficients of varistion （CV） of intra- and inter-assay were calculated and they were all less than 2.0% and 2.6% respectively. Our data suggested that there were 5.18 × 102 -4.93×l07 RNA copies in lml of the serum from acute HAV patients. Conclusion The TaqMan-based Real-time PCR assay established in this study was specific and precise for the rapid detection of HAV RNA. It was applied successfully in the pathogen detection of clinical samples.