丙型肝炎病毒1b型NS3基因扩增方案的改进

摘要

Objective To optimize the amplification and sequencing of non structural protein 3 (NS3) gene in patients infected with hepatitis C virus (HCV) genotype lb. Methods The genotyping after extracting HCV RNA from the serum was detected with type specific primers assay. After designing three pairs of outside primers and three pairs of inner primers, HCV NS3 (HCV genotype lb) were amplified by a nested PCR assay (option I) with above primers and sequenced. In order to optimize effects of NS3 amplification and sequencing and to reduce the complicated operation, we improved the new nested PCR assay (option II) and redesigned the primers, including a pair of outside primers and a pair of inner primers. We amplified directly HCV NS3 (HCV genotype lb) with improved nested PCR assay and sequenced NS3 with three forward primers and a reverse primer. Results Compared to the original nested PCR assay (option I) , the improved nested PCR assay (option II) is simpler in operation and of higher efficiency. Conclusion The improved nested PCR assay (option II) is more effective and practical for amplification and sequencing of HCV NS3.%目的 优化扩增武汉地区HCV 1b型患者HCV非结构蛋白3(NS3)的cDNA,并进行测序.方法 从患者血清中提取HCV RNA,采用型特异性引物扩增分型法检测HCV患者基因分型.设计3对外侧引物和3对内侧引物,采用套式PCR技术(方案一)分别扩增HCV1b型患者的HCV NS3 3个区段,然后用3对内侧引物进行测序.为了优化NS3扩增和测序效果和减少操作繁琐,对引物进行改进,设计1对外侧引物和1对内侧引物,采用改进的套式PCR技术(方案二)直接扩增HCV 1b型患者的HCV NS3区,然后用3个正向引物和1个逆向引物进行测序.结果 改进的套式PCR技术(方案二)与套式PCR技术(方案一)相比,操作较为简单,而且扩增效率增高,扩增效果较好.结论 采用改进的套式PCR技术(方案二)扩增和测序HCV NS3区更迅速、有效、实用.