Simultaneous Detection of Major Drug Resistance Mutations in the Protease and Reverse Transcriptase Genes for HIV-1 Subtype C by Use of a Multiplex Allele-Specific Assay

High-throughput, sensitive, and cost-effectiveHIVdrug resistance (HIVDR) detection assays are needed for large-scalemonitoring of the emergence and transmission ofHIVDR in resource-limited settings. Using suspension array technology, we have developed amul-tiplex allele-specific (MAS) assay that can simultaneously detectmajorHIVDRmutations at 20 loci. Forty-five allele-specific primers taggedwith unique 24-base oligonucleotides at the 5 = endwere designed to detectwild-type andmutant alleles at the 20 loci ofHIV-1 subtypeC. TheMAS assaywas first established and optimizedwith three plasmid templates (C-wt, C-mut1, andC-mut2) and then evaluated using 148 plasma specimens fromHIV-1 subtypeC-infected individuals. All thewild-type andmutant alleleswere unequivo-cally distinguishedwith plasmid templates, and the limits of detectionwere 1.56 % forK219QandK219E, 3.13 % for L76V, 6.25 % for K65R,K70R, L74V, L100I, K103N,K103R,Q151M,Y181C, and I47V, and 12.5 % forM41L,K101P,K101E, V106A,V106M,Y115F, M184V, Y188L,G190A,V32I, I47A, I84V, and L90M.Analyses of 148 plasma specimens revealed that theMAS assay gave 100%concor-dancewith conventional sequencing at eight loci and>95 % (range, 95.21 % to 99.32%) concordance at the remaining 12 loci. The dif-ferences observedwere causedmainly by 24 additional low-abundance alleles detected by theMAS assay.Ultradeep sequencing analy