【摘要】 目的建立一种简便、快速基因分型方法,对广西人类免疫缺陷病毒(HIV-1)重组毒株gag基因区进行亚型鉴定。方法从HIV阳性样本中提取核酸,使用HIV-1 M组通用引物对gag区进行第1轮扩增,第2轮使用分别检测C亚型和CRF01-AE重组型的二套特异性引物放入同一反应管中进行扩增,根据不同亚型扩增的目的带位置不同判断亚型。另外设计了一套引物,专门用于检测B’/C重组毒株。扩增出的所有样本均进行基因测序和系统树分析以验证结果。结果54份样本中,经基因测序和系统树分析证实CRF08-BC样本4份(7.41%),CRF01-AE样本46份(85.18%),4份(7.41)无法确定亚型。经亚型特异性引物PCR法检测出4份(100%)B’/C重组毒株,45份(97.83%)CRF01-AE重组毒株,灵敏度为98%,特异性为100%。2种方法检测结果经差异性检验显示,P>0.05,差异无统计学意义,结果一致性高达98.15%。与基因分析结果吻合。重复实验显示,B’/C的平均重复性为100%(20/20),CRF01-AE为98.3%(59/60)。结论该方法具有简便、快速,高度灵敏度和特异性的特点,可直接对广西HIV-1重组毒株CRFO 1-AE gag基因区进行分型。更多还原

【Abstract】 Objective To develope a simple and rapid subtype-screening assay for the gag region of the circulating recombinant form(CRF) of human immunodeficiency virus type 1(HIV-1)in Guangxi.Methods Proviral DNA from HIV-positive samples were extracted and subjected to the first round PCR with universal primers for the gag region that can detect HIV-1 M group isolates.In the second round PCR,two pairs of subtype-specific primers that were designed to detect subtype C and CRF01-AE respectively were added into one tube.The PCR products of different subtypes could be distinguished in agarose-gel electrophoresis.Another pair of subtype-specific primers exclusively detecting the the prevalent recombinantstrains CRF07-BC and CRF08-BC was designed and used.Additionally,all of these samples were sequenced and analyzed phylogenetically.Results DNA sequencing and phylogenetic analysis of the gag region of the 54 samples showed that 4 samples(7.41%) were infected with CRF08-BC,46(85.18%) with CRF01-AE and 4(7.41%) remained unclassifiable.Detection of the subtype-specific primer sets revealed that 4 were B’/C(100%),and 45 were CRF01-AE(97.83%),with an adequate sensitivity(98%) and a high specificity(100%).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 98.15%.The average reproducibility was 100% for B’/C samples and 98.3% for CRF01-AE samples.Conclusion A simple,rapid and low cost assay is developed for subtype-screening of CRFO1-AE in Guangxi.For the B’/C strains in Guangxi,it needs to be verified further by increasing samples.更多还原