HIV-1包膜单基因Nest-PCR体系优化及包膜基因准种扩增  

Optimization of HIV-1 envelope gene PCR amplification system and amplification of envelope gene quasispecies

作　　者：全红[1] 刘松[1] 张昊天[2] 任彩云[1] 庄敏[1] 凌虹[1] 李妍[1] 

机构地区：[1]哈尔滨医科大学微生物学教研室 [2]哈尔滨市食品药品检验检测中心,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2012年第4期323-328,共6页Journal of Harbin Medical University

基　　金：艾滋病和病毒性肝炎等重大传染病防治科技重大专项(2008ZX10001-012);黑龙江省教育厅科学技术研究项目(12511184);黑龙江省卫生厅科研课题(2011-205)

摘　　要：目的确定HIV-1包膜(envelope,env)单基因扩增(single genome amplification,SGA)的最优反应体系,获得env单基因扩增产物。方法采用SGA及Nest-PCR扩增HIV-1 env全长基因,并对Nest-PCR反应体系中各成分量进行优化;在此基础上,适当稀释HIV-1感染者cDNA模板进行Nest-PCR,以获得PCR扩增产物阳性率不超过30%的模板稀释度。结果 SGA Nest-PCR扩增HIV-1 env全长基因,引物浓度为0.3μmol/L,dNTP浓度为0.25μmol/L时可获得特异性高的env全长基因;凝胶回收纯化及稀释第一轮PCR产物可有效提高特异性条带的产量。采用优化反应体系成功从一名HIV-1感染者体内扩增出18株env单基因序列。结论优化了HIV-1 env基因SGA Nest-PCR的反应体系,并从HIV-1感染者获得了病毒包膜基因准种,为后续进行准种分析奠定了基础。Objective To optimize the PCR conditions for si of HIV-1 envelope （ertv） gene. Methods The efficiency of ngle genome amplification （SGA） the SGA-PCR was assessed based on the amplification results of env gene using the non-diluted cDNA template, the optimal dilu- tion of cDNA template that may result in no more than 30% positive amplification was predic- ted. Results The elements of SGA and Nested-PCR such as the amounts of primers were 0. 3 μmol/L, dNTP were 0.25 μmol/L, purification of products of the first-round PCR reactions and diluting cDNA templates increased the amount of interest products effectively. One HIV-1 infected individual after the env gene SGA, 18 interest products confirmed by sequencing env sequence. Conclusion Optimization of the HIV-1 env gene the SGA Nest-PCR reaction sys- tem, and the viral subsequent analysis envelope gene quasispecies from HIV-1 infection, laid the foundation for of the quasispecies.

关 键 词：HIV-1 包膜 单基因扩增 Nest-PCR 

分 类 号：R512.91[医药卫生—内科学] R373[医药卫生—临床医学]