A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virological failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants containing subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt-curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets containing AAC mixed with targets containing AAA were above 98% when AAC was present at a proportion of at or above 10%. Multiplexed solid-phase melt-curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.
View details for PubMedID 31026601