Abstract：
Allele-Specific Polymerase Chain Reaction (ASPCR) is an affordable point-mutation assay whose validation could improve the detection of HIV-1 drug resistance mutations (DRMs) in resource-limited settings (RLS). We assessed the performance of ASPCR onforty-four non-B HIV-1 plasma samples from patients who were ARV treated in failure in N\'Djamena-Chad. Viral RNA was reverse-transcribed and amplified using LightCycler® FastStart DNA MasterPLUS SYBR Green I. Detection of six major DRMs (K70R, K103N, Y181C, M184V, T215F, T215Y) was evaluated on Roche LightCycler®480 automated system (with dilutions 0.01-100%). ASPCR-results were compared to Sanger-sequencing (gold-standard). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). Correlations of mutation curves were excellent (R2 >0.97); all DRMs were detected with desirable mutant/wild-type threshold differences (ΔCt≥9) except K70R(ΔCtK70R=6; ΔCtK103N=13; ΔCtM184V=9; ΔCtT215F=12; ΔCtT215Y=12; ΔCtY181C=9) and positive controls were below required thresholds. Also, ASPCR reproducibility on DRMs was assessed by using dilutions of intra-assay and inter-assay coefficient of variations respectively with a threshold of less than 50(i.e.<0.50 variation) which are;: K70R (0.02-0.28 vs. 0.12-0.37), K103N (0.08-0.42 vs. 0.12-0.37), Y181C (0.12-0.39 vs. 0.31-0.37), M184V (0.13-0.39 vs. 0.23-0.42), T215F (0.05-0.43 vs. 0.04-0.45) and T215Y (0.13-0.41 vs. 0.19-0.41). DRM detection-rate by ASPCR vs Sanger was respectively: M184V (63.6% vs. 38.6%); T215F (18.1% vs. 9.1%); T215Y (6.8% vs. 2.3%); K70R (4.5% vs. 2.3%). K103N (22.7% vs. 13.6%); Y181C (13.6% vs. 11.4%). ASPCR appears more efficient for detecting DRMs on diverse HIV-1 non-B circulating in RLS like Chad.
摘要：
等位基因特异性聚合酶链反应（ASPCR）是一种负担得起的点突变测定，其验证可以改善资源有限环境（RLS）中HIV-1耐药性突变（DRM）的检测。我们评估了来自N'Djamena-Chad抗逆转录病毒治疗失败患者的44例非BHIV-1血浆样本的ASPCR性能。使用LightCycler®FastStartDNAMasterPLUSSYBRGreenI.检测六种主要DRM（K70R，K103N,Y181C,M184V,T215F,T215Y）在RocheLightCycler®480自动化系统（稀释度0.01-100％）上进行评估。将ASPCR结果与Sanger测序(金标准)进行比较。突变曲线的相关性非常好（R2>0.97）；除K70R（ΔCtK70R=6；ΔCtK103N=13；ΔCtM184V=9；ΔCtT215F=12；ΔCtT215Y=12；ΔCtY181C阈值低于阳性对照）外，所有DRM均具有理想的突变/野生型阈值差异（ΔCt≥9）。此外，通过分别使用阈值小于50（即<0.50变化）的测定内和测定间变异系数的稀释度来评估DRM的ASPCR可重复性；：K70R（0.02-0.28vs.0.12-0.37),K103N(0.08-0.42vs.0.12-0.37),Y181C（0.12-0.39vs.0.31-0.37），M184V（0.13-0.39vs.0.23-0.42），T215F（0.05-0.43vs.0.04-0.45）和T215Y（0.13-0.41与0.19-0.41）。ASPCR与Sanger的DRM检出率分别为：M184V（63.6％与38.6%）；T215F（18.1%与9.1%）；T215Y（6.8%与2.3%）；K70R（4.5%与2.3%）。K103N（22.7%与13.6%）；Y181C（13.6%与11.4%）。ASPCR对于检测在RLS如乍得中循环的多种HIV-1非B上的DRM似乎更有效。
