| | | HIV-1重组毒株gag区快速基因分型方法
| | 医学实践论文【关键词】 人类免疫缺陷病毒1型；基因型；聚合酶链反应
摘要： 目的医学实践论文 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重组型的医学实践论文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
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.
Key words： human immunodeficiency virus type 1(HIV-1）；genetic subtype；polymerase chain reaction (PCR)
人类免疫缺陷病毒-1（HIV-1）高度变异，不同亚型毒株其生物学特性及；分子流行病学特征均有所不同〔1〕，若能快速、准确地对HIV进行基因分型不仅有助于了解HIV-1转播规律，而且可为阐明HIV基因型与生物表型关系、药物耐药性等研究提供资料。近年来，我国学者〔2〕建立了一种多重巢式PCR法对我国HIV-1主要流行株进行鉴定亚型。然而，由于HIV-1的基因变异度极高，在传播过程中可产生许多具有相对独立的基因序列型别，使得不同地区各亚型间具有其自身的特异性。广西壮族自治区为国内HIV感染的高发区，大部分感染发生在经济尚不发达地区的人群中。因此，建立广西HIV-1主要流行株快速基因分型方法十分必要。目前，广西的优势流行株为CRF01-AE和CRF08-BC重组毒株〔3,4〕。为此，本研究针对这2种重组毒株gag基因区建立了一种亚型特异性引物PCR法的快速基因分型法。
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