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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 amplification of this gene fragment by modification of the C / E1 region merger primer which has a high degree of variation in the genome of hepatitis C virus (HCV), and also provide nucleic acid amplification for RNA viruses with frequent mutation Methodological reference. Methods The multi-point annexing primer (“” primer ") targeting C / E1 region in the literature was used for PCR amplification and sequencing. According to the result of sequence alignment, the original primer was modified, and no mutation point was taken for detoxification. The new mutation points to re-merge, and ultimately designed a new high-specificity specific primers. Results 44 HIV-positive patients with HCV nucleic acid were used as experimental samples. Only 23 cases (52.3%) were successfully amplified by the original primers, and 40 cases (90.9%) were successfully and efficiently amplified by the improved primers %). Conclusion The new primers can significantly improve the detection rate and amplification efficiency of HCV nucleic acid and provide a new detection strategy for HCV genotyping.