SNaPshot技术检测乙型肝炎病毒基因组P基因区单核苷酸多态性的研究  

SNaPshot technique for detection of single-nucleotide polymorphisms (SNPs) in HBV polymerase gene region of HBV gene

作　　者：陈勇[1] 余亚新 刘兴祥[1] 魏来[2] 

机构地区：[1]江苏省淮安市第四人民医院淮安市肝病研究所,223300 [2]北京大学人民医院肝病研究所

出　　处：《中华实验和临床病毒学杂志》2005年第2期162-164,i002,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：江苏省卫生厅重点科研课题 (H2 0 0 118) ;淮安市科技局重点科研课题

摘　　要：目的　建立SNaPshot技术对乙型肝炎病毒(HBV)基因组P基因区单核苷酸多态性(SNP)的分析。方法　针对HBVYMDD变异位置的上、下游设计引物,用PCR方法进行DNA扩增,产物直接测序或克隆测序。再针对变异位点74 1A G变异型(YVDD)和74 3G T变异型(YIDD)的紧邻上游设计高度特异的SNaPshot检测引物,用不同荧光标记的ddNTP对PCR产物进行一步延伸,然后置于310型DNA测序仪上观察荧光信号,可直观的检测上述两位点的单核苷酸多态性。结果　在拉米夫定治疗的慢性乙型肝炎患者中,经测序证实P基因区不仅存在74 1、74 3位点的变异,还存在5 14C A、5 2 3C A、5 6 2T A、6 6 7C A等位点的变异。对已证实P基因区变异的13例患者血清用SNaPshot技术检测YMDD结果与测序结果完全相同,显示SNaPshot技术高度的特异性。结论　SNaPshot技术检测HBV基因组SNPs具有快速、简便、准确特异的特点,还检测到野生株和变异株的混合存在。Objective To establish a rapid specific method to identify the single-nucleotide polymorphisms ( SNPs) of HBV polymerase gene region which are the methionine residue of the conserved YMDD motif. Methods Two specific primers were designed to amplify interested gene region involved in SNPs which were also used as HBV DNA identification. Specific primers of SNaPshot were designed to detect 741A-G (YVDD), 743G-T (YIDD). The different fluorescent dye labeled ddNTP was used to further extend the strand of PCR product and was detected by ABI PRISM 310 Genetic Analyzer. Sera from 13 patients with chronic hepatitis B after lamivudine treatment were analyzed. Results Aside from mutation of YMDD, there were mutations of 514C-A, 523C-A, 562T-A, 667C-A. The 13 samples were simutaneously tested with SNaPshot and DNA sequencing, the same results were obtained. The method of SNaPshot showed high specificity. Conclusion Mutation of YMDD results in the changes of ATG codon, and there are new ATG codon in the upper strand of YMDD. SNaPshot technique is rapid, specific and accurate for the SNPs monitoring of HBV DNA mutation during lamivudine therapy. Two samples were determined by SNaPshot technique,identifying the co-existence of the mixed wild type and mutant type HBV infection.

关 键 词：乙型肝炎病毒基因组 技术检测 单核苷酸多态性(SNP) 乙型肝炎病毒(HBV) 慢性乙型肝炎患者 DNA测序仪 拉米夫定治疗 P基因区变异 YMDD DNA扩增 PCR方法 PCR产物 设计引物 克隆测序 直接测序 变异位点 荧光标记 SNPs 变异型 

分 类 号：R446.5[医药卫生—诊断学]