Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors
Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors
复制标题
DOI：
10.1128/jcm.40.4.1413-1419.2002
复制
发表时间：
2002-04-01
影响因子：
9.4
通讯作者：
Frenkel, LM
中科院分区：
文献类型：
--
作者：
Beek, IA;Mahalanabis, M;Frenkel, LM
开发并评价了一种灵敏、特异、高通量的寡核苷酸连接试验（奥拉），用于检测基因型人类免疫缺陷病毒1型（HIV-1）对美国食品药品监督管理局批准的蛋白酶抑制剂的耐药性。这种基于连接的测定使用特异性针对野生型或突变体序列的差异修饰的寡核苷酸，允许在微量滴定板的单个孔中灵敏且简单地检测两种基因型。设计寡核苷酸检测与安普那韦、奈非那韦、茚地那韦、利托那韦、沙奎那韦和洛匹那韦高水平耐药相关的原发突变，包括氨基酸取代D30 N、I50 V、V82 A/S/T、I84 V、N88 D和L90 M。采用逆转录-聚合酶链反应（RT-PCR）扩增54例HIV-1感染者血浆RNA，并用双脱氧核苷酸链终止子测序，以评价与耐药相关的突变。通过奥拉在位置30、50、82、88、84和90对这些相同的扩增子进行基因分型，总共312个密码子。在奥拉中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），而在共有测序中为92.2%（90个突变密码子中的83个），可能是由于奥拉的灵敏度增加。奥拉检测基因亚群的频率高于测序，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，而DNA测序检测到15种。通过分析野生型和突变型质粒混合物，观察到突变体和野生型基因组的奥拉的可重复性和半定量检测，所述野生型和突变型质粒混合物在相反基因组的背景中含有低至5%的任一基因型。这种快速，简单，经济，高灵敏度的检测方法提供了一个实用的替代双脱氧测序的基因型评价HIV-1抗逆转录病毒药物的耐药性。
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 highlevel 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.