title: Rapid and sensitive oligonucleotide ligation assay for detection of mutations in human immunodeficiency virus type 1 associated with high-level resistance to protease inhibitors.
abstract: 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.
authors.affiliation: Department of Pediatrics, Laboratory Medicine, University of Washington, Seattle, Washington, USA.
authors.forename: Ingrid A
authors.initials: IA
authors.lastname: Beck
authors.forename: Madhumita
authors.initials: M
authors.lastname: Mahalanabis
authors.forename: Gregory
authors.initials: G
authors.lastname: Pepper
authors.forename: Amy
authors.initials: A
authors.lastname: Wright
authors.forename: Shannon
authors.initials: S
authors.lastname: Hamilton
authors.forename: Erika
authors.initials: E
authors.lastname: Langston
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Frenkel
mesh_terms: D001483:Base Sequence; D011088:DNA Ligases / Q000378:metabolism*; D024882:Drug Resistance, Viral / Q000235:genetics*; D015684:Gene Products, pol / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D012367:RNA, Viral / Q000032:analysis / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013997:Time Factors
chemical_list: D015684:Gene Products, pol; D017320:HIV Protease Inhibitors; D009841:Oligonucleotides; D012367:RNA, Viral; D011088:DNA Ligases