title: Simultaneous and sensitive detection of human immunodeficiency virus type 1 (HIV) drug resistant genotypes by multiplex oligonucleotide ligation assay.
abstract: Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ≥2-5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral (ARV) drugs used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ≥2-5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B- and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance.
authors: Seattle Children's Research Institute, 1900 9th Avenue, Mailstop C9S-8, Seattle, WA 98101, USA.
authors: Giovanina M
authors: GM
authors: Ellis
authors: Tatyana A
authors: TA
authors: Vlaskin
authors: Andrew
authors: A
authors: Koth
authors: Louise E
authors: LE
authors: Vaz
authors: Sandra E
authors: SE
authors: Dross
authors: Ingrid A
authors: IA
authors: Beck
authors: Lisa M
authors: LM
authors: Frenkel
mesh_terms: D005838:Genotype; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D017354:Point Mutation; D012680:Sensitivity and Specificity
chemical_list: D015345:Oligonucleotide Probes
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