HIV-1重组毒株gag区快速基因分型方法
人类免疫缺陷病毒1型；基因型；聚合酶链反应,,人类免疫缺陷病毒1型；基因型；聚合酶链反应,1材料与方法,2结果,3讨论,参考文献
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(741%), CRF01-AE样本46份(8518%), 4份(741)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(9783%) 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>005，差异无统计学意义，结果一致性高达9815%。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983% (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。关键词：人类免疫缺陷病毒1型；基因型；聚合酶链反应
Rapid identification for gag region of circulating recombinant form of humanimmunodeficiency virus type 1
LUO Hao,LIANG Hao，SHAO Yiming,et al.
AIDS Research Center of Guangxi Medical Univeristy(Nanning 530021,China)
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. ......
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