HIV-1整合酶耐药基因型检测方法的建立

A genotypic assay for amplification and sequencing of HIV-1 integrase genes in China

目的随着整合酶抑制剂的使用,整合酶抑制剂耐药突变出现不可避免,为了更好地预防和控制艾滋病,根据中国1型艾滋病病毒(HIV-1)主要流行株建立HIV-1整合酶基因型耐药检测方法。方法根据国内HIV主要流行株(CRF01_AE、B、CRF07_BC、CRF08_BC、C)参考序列,用Oligo软件在整合酶保守区设计引物。挑选160例不同病毒亚型、病毒载量> 1 000拷贝/mL的样本,进行巢式荧光定量聚合酶链反应核酸扩增和测序。拼接后的序列输入斯坦福大学耐药网站,进行耐药突变位点分析。结果成功扩增出HIV-1整合酶全长(288个氨基酸),160个样品157个扩增成功(成功率为98.1%)。3名操作人员对15个样本进行扩增和测序,核苷酸序列一致性为(99.83±0.05)%。结论根据中国HIV-1主要流行株建立了整合酶耐药基因型检测方法,有助于优化艾滋病病人抗病毒治疗,有助于HIV-1的防控。

Objective It is likely that transmitted integrase strand transfer inhibitors(INSTIs) mutations may emerge with the recent commercial introduction of INSTIs in China. Until now, a few data are available on genotypic HIV-1 INSTIs resistance assay. This study aims to develop an integrase-resistant genotype assay based on Chinese prevailing HIV-1 strains. Methods Primers were designed and analyzed by Oligo software at most conserved sites surrounding and within the integrase region of HIV-1 prevailing strains in China. HIV-1 subtype distributions of the study were CRF01_AE, CRF08_BC, B’, CRF07_BC, and C. 160 samples with viral loads above 1 000 copies/ml were chosen for nest PCR amplification. The sequences were assembled, error checked, and then sent to Stanford University HIV Drug Resistance Database(https://hivdb.stanford.edu/) to identify mutations. Results The full length of HIV-1 int gene was amplified successfully. Of 160 samples, 157 samples were amplified and sequenced successfully(the success rate was 98.1%). Reproducibility of repeat amplification and sequencing on 15 random clinical isolates by 3 different operators was 99.83±0.05%.Conclusion An integrase-resistant genotype assay has been established based on Chinese prevailing HIV-1 strains, which will be helpful for optimizing AIDS anti-viral treatment and for HIV-1 prevention and control.