HCV非结构蛋白5B复合酶切分型方法的建立及应用

Establishment and application of HCV genotyping based on NS5B region

目的建立灵敏有效的HCV6a型非结构蛋白(non-structural protein,NS)5B复合酶切分型方法,探讨NS5B区与5'-端非编码区(5'-noncoding region,5'-NCR)的分型是否一致。方法应用5'-NCR复合酶切分型方法对1093份HCVRNA阳性血清样本进行分型,并筛选基因6a型样本进行NS5B区的扩增,对第2轮PCR产物进行复合酶切分型和测序,并经NCBI Genoty ping证实基因分型。结果 5'-NCR分型法检出10份HCV6a型样品,序列分析显示均存在第145位CA碱基插入和第139位C碱基插入特征。用NS5B分型法检测此10例,均为6a型,序列分析证实存在EaeⅠ酶切位点。结论 NS5B区与5'-NCR区复合酶切分型方法对HCV6a分型具有良好的一致性,以EaeⅠ切点为主的NS5B区复合酶切分型方法可供临床应用。

Objective To establish a sensitive and efficient method for differentiation of HCV genotype 6a based on non-struc- tural protein （NS） 5B region, and to investigate whether HCV genotyping based on NS5B region is consistent with that based on 5＇- noncoding region （NCR）. Methods HCV genotyping based on 5＇-NCR was performed on 1093 HCV RNA positive serum samples, and then the NS5B region of HCV genotype 6a serum samples was amplified. Enzyme analysis and sequencing were performed on the second PCR products and the genotype was verified by NCBI Genotyping. Results Ten HCV genotype 6a serum samples were obtained by HCV genotyping based on 5＇-NCR, and sequence analysis showed CA insertion at position 145 and C insertion at posi- tion 139 existed in this region. The 10 HCV genotype 6a serum samples were verified by HCV genotyping based on NS5B region, and sequence analysis confirmed the existence of Eae I restriction site in NS5B region. Conclusions HCV genotyping for genotype 6a based on NS5B region is consistent with that based on 5＇-NCR. HCV genotyping based on Eae I restriction sites of NS5B region is available for clinical practice.