基于pJFH-1的HCV全基因组扩增方法的建立

Amplification of full-length hepatitis C virus genome based on plasmid pJFH-1

目的基于pJFH-1建立能稳定扩增HCV全基因组的长链PCR方法。方法以pJFH-1为测试模板,通过优化PCR扩增中各个重要环节,包括引物的选择、甘油和/或DMSO最适浓度的筛选、循环条件的摸索等,建立能稳定扩增HCV全基因组的长链PCR方案。结果高Tm值(>65℃)的引物更有利于HCV全基因组的扩增;5%、10%甘油或5%DMSO可显著提高PCR扩增的特异性和扩增效率,且甘油的促进作用优于DMSO;双温法较三温法能获得更高产量的PCR产物。结论通过优化长链PCR反应体系及条件,成功实现HCV基因全长的扩增。

Objective To optimize the protocols of long-PCR for amplifying full-length HCV genome based on plasmid pJFH-1.Methods Optimization of long-PCR strategies was performed by testing a series of primers,adding various concentrations of glycerol,DMSO or both,using different cycle systems to select the optimal long PCR conditions.Results Primers that have higher melting temperatures（65 ℃）could improve the efficiency of amplification.Glycerol of 5% and 10% or DMSO of 5% improved the specificity and efficiency of PCR amplification of full-length HCV genome.The promoting effect of glycerol was better than that of DMSO.Compared to three-stage temperature method,two-step temperature produced more PCR product.Conclusion Through the optimization of long-PCR protocol,full-length HCV genome is successfully achieved.