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。