HCV的C/E1区PCR扩增方法的改进

Methodological improvement for PCR amplification of the C/E1 region in HCV genome

目的通过对丙型肝炎病毒(HCV)基因组变异度较大的C/E1区兼并性引物的修改以提高该基因片段的扩增成功率,同时也为突变发生频繁的RNA病毒的核酸扩增提供方法上的借鉴。方法选用文献中针对C/E1区的多点兼并性引物("原引物")进行PCR扩增、测序,根据序列比对结果对原引物进行修改,没有突变的点进行去兼并还原,新突变点重新兼并,最终设计出特异性高适用性好的新引物。结果以44例HCV核酸阳性的艾滋病病毒(HIV)感染者为研究样本,原引物仅能成功扩增23例(52.3%),而改进后的新引物能成功而高效的扩增40例(90.9%)。结论新引物能显著提高HCV核酸的检出率和扩增效率,为HCV的基因分型提供新的检测策略。

Objective To improve the success rate of PCR amplification of the variable region C/E1 in HCV genome,the degenerate primers from reference were altered properly which could be a new idea in PCR amplification of the variable genes of RNA virus. Methods The C/E1 region was amplified and sequenced by degenerate primers from reference （original primers）. According to the alignment of sequences,the more universal and specific primers were created after some degenerate points were restored and new degenerate points were formed. Results 23 of 44（52.3%） HCV-DNA-positive samples of HIV-infected patients could be amplified by the original primers while it was 40 of 44（90.9%） if the modified primers （new primers） were applied. Conclusions The new primers were much better than the original ones in success rate and efficiency of PCR amplification,it provide a new strategy for genotype of HCV.