广西HIV-1 CRF01-AE env和gag区快速基因分型方法的建立  

DEVELOPMENT OF A RAPID SUBTYPE-SCREENING ASSAY FOR THE ENV AND GAG REGION OF HIV-1 CRF01-AE STRAINS IN GUANGXI

作　　者：罗皓[1] 梁浩[2] 邵一鸣 张志勇[2] 刘伟 邢辉[3] 沈菁[2] 

机构地区：[1]广东医学院公共卫生学院,东莞523808 [2]广西医科大学 [3]中国疾病预防控制中心性病艾滋病预防与控制中心病毒免疫室 [4]广西区疾病预防控制中心

出　　处：《现代预防医学》2010年第7期1333-1335,共3页Modern Preventive Medicine

基　　金：广西科学基金项目(桂科自0447049)

摘　　要：[目的]建立一种快速简便的基因分型方法,对广西HIV-1 CRF01-AE毒株env和gag基因区进行亚型鉴定。[方法]从HIV阳性样品中提取核酸,使用HIV-1M组通用引物对env和gag基因区进行第一轮扩增,第二轮则使用CRF01-AE亚型的特异性引物进行扩增,根据扩增的目的带位置来判断亚型。将通用引物扩增出的所有样本进行基因测序以验证结果。[结果]43份CRF01-AE样本中,经env基因区亚型特异性引物PCR法检测得出39份(90.70%),灵敏度为90.70%,而gag基因区亚型特异性引物PCR法检测得出42份(97.34%),灵敏度为97.34%。将基因测序法和亚型特异性引物PCR法的检测结果进行比较,经差异性检验显示两种方法检测结果在两基因区中差异均无统计学意义,env基因区两方法检测结果结果一致者占90.70%。而gag基因区则高达97.34%。重复实验显示env基因区平均重复性为93.80%(61/65),gag基因区为98.30%(59/60)。[结论]该方法是一种简便、快速、低成本,具有高度灵敏性和特异性的HIV-1 CRF01-AE env和gag基因区分型法,能够直接对广西HIV-1 CRF01-AE重组毒株进行鉴定。[Objective] To developed a simple and rapid subtype-screening assay for the env and gag region of CRFO1- AE of human immunodeficiency virus type 1 （HIV-1） in Guangxi. [Methods] Proviral DNA from HIV-1 positive samples were extracted and subjected to the first round PCR with universal primers for the env and gag region that can detect HIV-1 M group isolates. In the second round PCR, one pairs of subtype-specific primers that was designed to detect subtype CRF01-AE was added into one tube. The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis. Addition- ally, all of these samples were sequenced. [Results] Detection of the subtype-specific primer sets of the 43 samples revealed that the env region of 39 were CRFO1-AE （90.70%）, with an adequate sensitivity （90.70%）. While the gag region of 42 were CRF01-AE （97.34%）, with an adequate sensitivity （97.34%）. Non-specific bands occasionally appeared but did not interfere with interpretation of the results. The phylogenetic analysis was consistent with subtype-specific primer sets and the consistent rate was 90.70% in the env region, while it was 97.34% in the gag region. The average reproducibility of the env region was 93.80%, while the gag region was 98.30%. [Conclusion] A simple, rapid and low cost assay is developed for subtypescreening of CRF01-AE in Guangxi.

关 键 词：人类免疫缺陷病毒1型 基因型 聚合酶链反应 

分 类 号：R512.91[医药卫生—内科学]