HIV-1重组毒株gag区快速基因分型方法

Rapid identification for gag region of circulating recombinant form of humanimmunodeficiency virus type 1

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。

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.