Beck Ingrid A, Mahalanabis Madhumita, Pepper Gregory, Wright Amy, Hamilton Shannon, Langston Erika, Frenkel Lisa M
Department of Pediatrics, Laboratory Medicine, University of Washington, Seattle, Washington, USA.
J Clin Microbiol. 2002 Apr;40(4):1413-9. doi: 10.1128/JCM.40.4.1413-1419.2002.
A sensitive, specific, and high-throughput oligonucleotide ligation assay (OLA) for the detection of genotypic human immunodeficiency virus type 1 (HIV-1) resistance to Food and Drug Administration-approved protease inhibitors was developed and evaluated. This ligation-based assay uses differentially modified oligonucleotides specific for wild-type or mutant sequences, allowing sensitive and simple detection of both genotypes in a single well of a microtiter plate. Oligonucleotides were designed to detect primary mutations associated with high-level resistance to amprenavir, nelfinavir, indinavir, ritonavir, saquinavir, and lopinavir, including amino acid substitutions D30N, I50V, V82A/S/T, I84V, N88D, and L90M. Plasma HIV-1 RNA from 54 infected patients was amplified by reverse transcription-PCR and sequenced by using dideoxynucleotide chain terminators for evaluation of mutations associated with drug resistance. These same amplicons were genotyped by the OLA at positions 30, 50, 82, 88, 84, and 90 for a total of 312 codons. The sensitivity of detection of drug-resistant genotypes was 96.7% (87 of 90 mutant codons) in the OLA compared to 92.2% (83 of 90) in consensus sequencing, presumably due to the increased sensitivity of the OLA. The OLA detected genetic subpopulations more often than sequencing, detecting 30 mixtures of mutant and wild-type sequences and two mixtures of drug-resistant sequences compared to 15 detected by DNA sequencing. Reproducible and semiquantitative detection of the mutant and the wild-type genomes by the OLA was observed by analysis of wild-type and mutant plasmid mixtures containing as little as 5% of either genotype in a background of the opposite genome. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to dideoxy sequencing for genotypic evaluation of HIV-1 resistance to antiretrovirals.
我们开发并评估了一种灵敏、特异且高通量的寡核苷酸连接分析方法（OLA），用于检测1型人类免疫缺陷病毒（HIV-1）对美国食品药品监督管理局批准的蛋白酶抑制剂的基因型耐药性。这种基于连接的分析方法使用针对野生型或突变序列的差异修饰寡核苷酸，能够在微量滴定板的单个孔中灵敏且简单地检测两种基因型。寡核苷酸被设计用于检测与对安普那韦、奈非那韦、茚地那韦、利托那韦、沙奎那韦和洛匹那韦的高水平耐药相关的主要突变，包括氨基酸替代D30N、I50V、V82A/S/T、I84V、N88D和L90M。通过逆转录-聚合酶链反应（RT-PCR）扩增了54例感染患者的血浆HIV-1 RNA，并使用双脱氧核苷酸链终止剂进行测序，以评估与耐药相关的突变。通过OLA对这些相同的扩增子在第30、50、82、88、84和90位进行基因分型，共检测312个密码子。与一致性测序中92.2%（90个突变密码子中的83个）相比，OLA检测耐药基因型的灵敏度为96.7%（90个突变密码子中的87个），推测这是由于OLA的灵敏度更高。与DNA测序检测到的15种相比，OLA比测序更频繁地检测到遗传亚群，检测到30种突变型和野生型序列的混合物以及两种耐药序列的混合物。通过分析在相反基因组背景中含有低至5%任一基因型的野生型和突变型质粒混合物，观察到OLA对突变型和野生型基因组进行了可重复的半定量检测。这种快速、简单、经济且高度灵敏的分析方法为HIV-1对抗逆转录病毒药物耐药性的基因分型评估提供了一种替代双脱氧测序的实用方法。
