应用TaqMan荧光定量RT-PCR检测猪肝中戊型肝炎病毒的研究

Application of TaqM an based real-time fluorescent quantitative RT-PCR assay in detection of hepatitis E virus in swine liver

目的建立适用于猪肝样本中戊肝病毒检测的荧光RT-PCR方法。方法采用匀浆处理直接提取法（方法一）与聚乙二醇处理法（方法二）两种处理方法对染毒猪肝样本进行病毒富集,针对4种型别戊肝病毒的基因组而设计的特异性引物和探针,建立一套Taq Man荧光定量RT-PCR方法,并评价该方法的准确性和灵敏性。采用新建荧光定量RT-PCR检测人工染毒的猪肝样本并比较两种方法的处理效率。结果所建立的Taq Man荧光定量RT-PCR标准曲线显示在10^2-10^8copies/μL范围内具有良好的线性关系,相关系数为0.999。批内和批间重复性实验结果表明,不同浓度的质粒标准品检测所获得的Ct值的变异系数分别在0.11%-3.75%、2.47%-6.62%间。2种方法均可以检测出10^2copies/μL的染毒猪肝中戊肝病毒拷贝数。结论所建立的Taq Man荧光定量RT-PCR方法具有灵敏度高、稳定性好、特异性强等特点,适用于HEV的定量检测。匀浆处理直接提取法更加快捷,适用于长期监测。

Objective To explore a suitable method for detection of hepatitis E virus（ HEV） in pig liver sample by TaqM an real time RT-PCR method. Methods Two methods,homogenized direct extraction method（ Method A） and polyethylene glycol precipitation method（ Method B）,were used for concentration of HEV from swine liver samples. Based on the specific primers and probes of the four genotypes of HEV,a TaqM an real-time RT-PCR method was developed and its accuracy and sensitivity were evaluated. Real time RT-PCR was used to detect HEV from swine livers with induced infection of HEV. Performances of the two methods were compared. Results The established TaqM an real-time RT-PCR standard curve showed a good linear relationship in range of 10^2-10^8 copies / μL. The correlation coefficient（ R^2）of the standard curves with plasmid DNA was 0. 999. The coefficients of variation values of the different diluted plasmid DNA were detected between 0. 11% and 2. 47% and between 6. 62% and 2. 69% in the same or different repeated experimental groups. The efficiency of Method A and Method B was similar with a detection limit of 10^2 copies / μL. Conclusion The TaqM an real-time RT-PCR assay was established with high sensitivity,good stability and strong specificity,suitable for quantitative detection of HEV. The homogenized direct extraction method is more rapid for HEV concentration from swine livers and suitable for long-term surveillance of HEV.