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 drugs (ARV) 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.

Geographic and Temporal Trends in the Molecular Epidemiology and Genetic Mechanisms of Transmitted HIV-1 Drug Resistance: An Individual-Patient- and Sequence-Level Meta-Analysis

“…In addition, the infrastructure in many LMICs does not support the expansion in the number of these laboratories or the rapid transportation of samples to these laboratories. The finding that a few mutations were responsible for 80% of NNRTI-associated TDR in all regions and subtypes should motivate the development of inexpensive point-of-care point mutation assays for use in LMIC regions [ 46 , 47 ]. Even in the context of a public health approach to ARV therapy, where few standardized regimens are available at the population level, a reliable point-of-care genotypic resistance test could identify which patients should receive standard first-line therapy and which should receive a PI-containing regimen.…”

Future technologies for monitoring HIV drug resistance and cure

“…However, large-scale implementation of PMAs has stalled due to issues with primer binding site polymorphisms and mutant codon variants, such as E138E/A/G/K that compromise assay specificity and increase assay complexity and cost [25,26]. Although recent advances in PMAs can accommodate the simultaneous detection of multiple mutations, analysis of mutation combinations, such as thymidine analog mutations for zidovudine resistance, remains challenging [27].…”

A Simple Microfluidic Assay for the Detection of Ligation Product

“…Various methods of detecting single nucleotide base-pair mutations exist today [1][2][3][4]. A prominent mechanism used to determine the presence of point mutations is through DNA ligation at the position of the mutation [1,3,5,6].…”

“…A prominent mechanism used to determine the presence of point mutations is through DNA ligation at the position of the mutation [1,3,5,6]. DNA ligases facilitate the joining of nicks on doublestranded DNA duplexes.…”