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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 establish a long-chain PCR method for stable amplification of HCV genome based on pJFH-1. Methods The pJFH-1 was used as a template to optimize the amplification of HCV genome by optimizing the PCR amplification, including the selection of primers, screening of optimal concentration of glycerol and / or DMSO, exploring the cycling conditions and so on. Long-chain PCR program. Results Primers with high Tm values (> 65 ℃) were more favorable for HCV genome wide amplification. 5%, 10% glycerol or 5% DMSO could significantly improve the specificity and amplification efficiency of PCR amplification and the promotion of glycerol Which is better than that of DMSO. Compared with the three-temperature method, the dual-temperature method can obtain higher yield PCR products. Conclusion The full-length amplification of HCV gene was successfully achieved by optimizing the system and conditions of long-chain PCR reaction.