多重PCR归化法平行检测HBV和HCV的研究

Multiplex PCR normalization and parallel detection of HBV and HCV

目的 建立一种多重PCR归化法并应用于对HBV、HCV平行检测。方法 利用PCR反应 5′端允许添加非互补序列的原理 ,运用内外两对引物 ,经过 2轮扩增 ,使目的产物均带上共有序列 ,再以共有序列为引物进行扩增 ,实现多重扩增。比较和筛选四种核酸提取方法。运用正交优化法 ,优化并确定最佳扩增条件。对 2 8份血标本进行对比试验 ,并进行质量评价。结果 归化多重PCR方法对于HBV、HCV病毒合并感染患者的诊断敏感性为 83 .3 % ,诊断特异性为 70 .0 % ,诊断指数为15 3 3 % ,诊断效率为 72 .2 % ;对HBsAg阳性患者的HBVDNA的诊断敏感性为 78.6% ,诊断特异性为80 0 % ,诊断指数为 15 8.6% ,诊断效率为 79.2 %。对抗 HCV阳性患者的HCVRNA的诊断敏感性为75 0 % ,诊断特异性为 90 .0 % ,诊断指数为 165 .0 % ,诊断效率为 83 .3 %。结论 多重PCR归化法在多基因扩增或多种病原体的同时平行检测领域具有较大应用潜力。该方法实用、准确、可靠 。

Objective To design and establish a method of multiplex PCR normalization and parallel detection of HBV and HCV. Methods Using two pairs of primers, one inner and the other outer, each having a 20 bp common sequence, the authors amplified target DNA for two rounds. All products would have this common sequence. Using this common sequence as primer the authors performed further amplification. Finally, multiplex PCR was normalized to a single PCR style and eliminated multiple factors′ disturbance. Four kinds of nucleic acid extraction method were compared. Multiplex PCR normalization was established and optimized by using orthogonal design by analysing 6 kinds of key factors. The method was evaluated by detecting 28 samples of HBV and HCV.Results The sensitivity, specificity, diagnostic idex and efficiency for HBsAg, HCV antibody positive patients were 83 3%, 70.0%, 153.3%, 72.2% respectively. The sensitivity, specificity, idex and efficiency for HBsAg positive patients were 78.6%, 80.0%, 158.6%, and 79.2%, respectively. The sensitivity, specificity, idex and efficiency for HCV antibody positive patients were 75.0%, 90.0%, 165.0% and 83 3%,respectively. Conclusion The multiplex PCR normalization method may have potential applicability in parallel amplification of multiple genes of pathogens.