适用于中国HIV-1分型的gag基因引物及其应用

Primers of gag gene for HIV-1 subtyping in China and application thereof in practice

目的对中国流行的主要HIV-1毒株进行遗传特征分析，确定适用于中国HIV分子流行病学调查的gag基因扩增引物。方法从HIV序列数据库下载CRF07_BC、CRF08_BC、C亚型及M组参考毒株的gag全长序列，经序列比对后设计gag基因扩增引物；获得的基因片段采用邻接法构建系统进化树，评价不同片段用于分析和确定HIV-1亚型的可靠性，并检测部分样本以评定效果。结果目前我国常用的gag基因306／c-gag引物扩增片段（HXB2836～1507）构建的系统进化树中，CRF07_BC、C亚型进化簇的Bootstrap值分别为70％和59％，其可靠性较低，难以形成较为稳定的进化簇用于病毒亚型判断。而利用设计的gag基因GUX／GDX引物扩增片段（HXB2781～1861）构建的系统进化树中，CRF07_BC、CRF08_BC和C亚型序列能够独立成簇，其Bootstrap值分别为99％、99％和77％，具有较高的可靠性。在实际应用中，新设计的引物具有较高的扩增阳性率和测序成功率，获得序列形成B、C亚型和CRF01_AE、CRF07_BC、CRF08_BC等5个大的进化簇，其Bootstrap值均超过80％。即使仅用下游引物GDX测序获得的较短片段亦能获得较为可靠的系统进化树。结论GUX／GDX引物扩增获得的gag基因片段可以构建较为可靠的系统进化树，用于区分和判断我国流行的主要HIV-1毒株亚型，尤其是BC重组毒株和C亚型毒株。该方法在我国HIV分子流行病学调查和研究中有广泛的应用价值。

Objective To design the appropriate primers of gag gene for HIV-1 subtyping in molecular epidemiology survey based on the genetic characteristics derived from the main HIV-1 strains prevailing in China. Methods The gag genes of HIV-1 CRF07_BC, CRF08_BC, and subtype C, together with subtyping reference sequences, were obtained from HIV sequence database. Referring to the alignments and genetic characteristics of HIV-1 full gag sequences, new primers of gag gene for amplification and subtyping were designed. The target fragment was used to construct neighbor-joining phylogenetic tree and evaluate its reliability. The newly designed primers （GUX/GDX） were used to amplify the plasma samples to evaluate their efficiency. Results The phylogenetic tree of 306/c-gag fragments （ positions 836-1507 of HIV-1 strain HXB2） showed that CRF07_BC and subtype C strains formed clusters with low bootstrap values （59% for CRF07_BC and 70% for subtype C）, and the phylogenetic tree could not distinguish the sequences of CRF07_BC, CRF08_BC, and subtype C very well. Whereas the sequences of CRF07_BC, CRF08_BC, and subtype C from GUX/GDX （positions 781-1861 ） were clustered separately with higher bootstrap values （99%, 99%, and 77% respectively）. In practice, a very good amplification and sequencing efficiency with over 90% positive results on average were obtained with GUX/GDX. Five clusters of subtype B, C, CRF01_AE, CRFO7_BC, and CRFO8_BC were formed with higher confidence （ Bootstrap values all above 80% ）. The reliable phylogenetic tree could be constructed based on the fragments sequenced only with antisense primer （GDX）. Conclusion Fragments obtained with GUX/GDX primers of gag gene can be used to reconstruct phylogenetic tree with high reliability to distinguish the HIV-1 strains circulating in China, especially for the major BC recombinant and subtype C strains, which provides a useful tool in HIV molecular epidemiologic research.