title: 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.
abstract: High-throughput, sensitive, and cost-effective HIV drug resistance (HIVDR) detection assays are needed for large-scale monitoring of the emergence and transmission of HIVDR in resource-limited settings. Using suspension array technology, we have developed a multiplex allele-specific (MAS) assay that can simultaneously detect major HIVDR mutations at 20 loci. Forty-five allele-specific primers tagged with unique 24-base oligonucleotides at the 5' end were designed to detect wild-type and mutant alleles at the 20 loci of HIV-1 subtype C. The MAS assay was first established and optimized with three plasmid templates (C-wt, C-mut1, and C-mut2) and then evaluated using 148 plasma specimens from HIV-1 subtype C-infected individuals. All the wild-type and mutant alleles were unequivocally distinguished with plasmid templates, and the limits of detection were 1.56% for K219Q and K219E, 3.13% for L76V, 6.25% for K65R, K70R, L74V, L100I, K103N, K103R, Q151M, Y181C, and I47V, and 12.5% for M41L, K101P, K101E, V106A, V106M, Y115F, M184V, Y188L, G190A, V32I, I47A, I84V, and L90M. Analyses of 148 plasma specimens revealed that the MAS assay gave 100% concordance with conventional sequencing at eight loci and >95% (range, 95.21% to 99.32%) concordance at the remaining 12 loci. The differences observed were caused mainly by 24 additional low-abundance alleles detected by the MAS assay. Ultradeep sequencing analysis confirmed 15 of the 16 low-abundance alleles. This multiplex, sensitive, and straightforward result-reporting assay represents a new efficient genotyping tool for HIVDR surveillance and monitoring.
authors.affiliation: International Laboratory Branch, Division of Global HIV/AIDS, CGH.
authors.forename: Guoqing
authors.initials: G
authors.lastname: Zhang
authors.forename: Fangping
authors.initials: F
authors.lastname: Cai
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Zhou
authors.forename: Joshua
authors.initials: J
authors.lastname: DeVos
authors.forename: Nick
authors.initials: N
authors.lastname: Wagar
authors.forename: Karidia
authors.initials: K
authors.lastname: Diallo
authors.forename: Isaac
authors.initials: I
authors.lastname: Zulu
authors.forename: Nellie
authors.initials: N
authors.lastname: Wadonda-Kabondo
authors.forename: Jeffrey S A
authors.initials: JS
authors.lastname: Stringer
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Weidle
authors.forename: Clement B
authors.initials: CB
authors.lastname: Ndongmo
authors.forename: Izukanji
authors.initials: I
authors.lastname: Sikazwe
authors.forename: Abdoulaye
authors.initials: A
authors.lastname: Sarr
authors.forename: Matthew
authors.initials: M
authors.lastname: Kagoli
authors.forename: John
authors.initials: J
authors.lastname: Nkengasong
authors.forename: Feng
authors.initials: F
authors.lastname: Gao
authors.forename: Chunfu
authors.initials: C
authors.lastname: Yang
mesh_terms: D024882:Drug Resistance, Viral; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D046228:Microarray Analysis / Q000379:methods; D008826:Microbial Sensitivity Tests / Q000379:methods; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D017422:Sequence Analysis, DNA
chemical_list: C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D016333:HIV Protease