甲型肝炎病毒和戊型肝炎病毒双重荧光定量PCR方法的建立及应用

Development and Application of Multiplex Real-time PCR Assay for Hepatitis A Virus and Hepatitis E Virus Detection

目的建立甲型肝炎病毒(HAV)和戊型肝炎病毒(HEV)的双重荧光定量PCR检测方法并应用于临床样本的检测。方法根据参考文献分别选取HAV以及HEV基因组的保守区域设计合成特异性引物和探针,从而建立并优化双重荧光定量PCR反应体系,之后评价反应体系的特异性、敏感性和稳定性。结果本研究建立的HAV和HEV双重荧光定量PCR技术应用于检测HAV核酸时检测极限可以达到1个拷贝/反应,对同一样品重复测定5次获得的Ct值的变异系数(CV)最大为1.5%,同时该体系检测HEV的最低检测极限也达到10个拷贝/反应,重复测试5次后Ct值的变异系数(CV)最大为2.6%,临床样本测试结果显示该体系能够对甲型肝炎和戊型肝炎临床样本中的病毒进行定量。结论本研究成功建立了HAV和HEV双重荧光定量PCR检测方法,灵敏度高,稳定性好,可以同时准确定量HAV和HEV,为这两种病毒的分子病原学诊断提供了一种更加快速的手段。

Objective To establish a multiplex real-time PCR assay for hepatitis A virus and hepatitis E virus clinical samples detection.Methods The specific primers and probes in the conserved region of HAV and HEV genome were designed according to the references.Therefore,HAV-HEV multiplex real-time PCR was established and optimization followed by the evaluation of specificity,sensitivity and reproducibility.Results The detection limit could up to 1copy each reaction when HAV-HEV multiplex real-time PCR was applied for HAV detection.The same dilution of the RNA standard was analyzed in five times in parallel and the maximum coefficient of variation（CV）of Ct values was1.5%.The detection limit was 10 copies each reaction in HEV sensitivity evaluation and the maximum CV of Ct values was 2.6%in reproducibility analysis.Clinical tests suggested HAV-HEV multiplex real-time PCR assay was able to quantify HAV and HEV in clinical samples.Conclusion The HAV-HEV multiplex real-time PCR was established successfully.This detection method was sensitive,stable and it could be used for simultaneous quantification of HAV and HEV.It provided another rapid way for pathogenic diagnosis of these two viruses.