适用于中国HIV-1分型的gag基因引物及其应用

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(HXB2 836～1507)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(HXB2 781～1861)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.

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

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-I strain HXB2) showed that CRFOT_BC and subtype C strains formed clusters with low bootstrap values (59% for CRFO7_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, CRFO8_BC, and subtype C from GUX/GDX (positions 781-1861) were clustered separately with higher beotstrap 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, CRF07＿BC, and CRF08_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 swains circulating in China, especially for the major BC recombinant and subtype C swains, which provides a useful tool in HIV molecular epidemiologic research.