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
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Published in |
Journal of Clinical Microbiology, August 2013
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DOI | 10.1128/jcm.01669-13 |
Pubmed ID | |
Authors |
Guoqing Zhang, Fangping Cai, Zhiyong Zhou, Joshua DeVos, Nick Wagar, Karidia Diallo, Isaac Zulu, Nellie Wadonda-Kabondo, Jeffrey S. A. Stringer, Paul J. Weidle, Clement B. Ndongmo, Izukanji Sikazwe, Abdoulaye Sarr, Matthew Kagoli, John Nkengasong, Feng Gao, Chunfu Yang |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 45 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Researcher | 9 | 20% |
Student > Ph. D. Student | 7 | 16% |
Student > Bachelor | 5 | 11% |
Other | 4 | 9% |
Student > Postgraduate | 3 | 7% |
Other | 8 | 18% |
Unknown | 9 | 20% |
Readers by discipline | Count | As % |
---|---|---|
Medicine and Dentistry | 12 | 27% |
Agricultural and Biological Sciences | 9 | 20% |
Biochemistry, Genetics and Molecular Biology | 6 | 13% |
Immunology and Microbiology | 4 | 9% |
Engineering | 3 | 7% |
Other | 3 | 7% |
Unknown | 8 | 18% |