Evaluation of an oligonucleotide ligation assay for detection of mutations in HIV-1 subtype C individuals who have high level resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors.
The oligonucleotide ligation assay (OLA) has been proposed as an affordable alternative to sequence-based HIV-1 drug resistance testing in resource poor settings. The aim was to evaluate OLA for detecting mutations K103N, Y181C, K65R, Q151M, M184V and T215Y/F in subtype C. Forty-four subtype C and 8 subtype B HIV-1 positive individuals were analysed using the ViroSeqtrade mark HIV-1 genotyping assay (Applied Biosystems, Foster City, CA). A one-step RT-PCR and nested PCR were performed using subtype B specific primers from the OLA kit (NIH AIDS Research and Reference Reagent Program). Seventy-eight subtype C sequences were used to design subtype C specific primers. Ligation and detection steps were followed according to OLA kit protocol. For codons, K103N, Y181C, K65R, Q151M, M184V and T215Y/F, four or more mismatches compared to the probe or mismatches less than four bases from the ligation site were not tolerated. Results revealed accurate identification of mutations in 2/10, 4/9 3/9, 6/7, 2/7 and 6/7 VQA samples and 5/20, 4/17 0/20, 18/24, 5/24 and 13/24 subtype C positive individuals, respectively. It was concluded that the probes and primers in the NIH reference kit would need modification to optimize detection of mutations in subtype C individuals.
Wallis
Carole Lorraine
CL
Department of Molecular Medicine and Haematology, School of Pathology, University of the Witwatersrand Medical School, South Africa.
Mahomed
Imraan
I
Morris
Lynn
L
Chidarikire
Thato
T
Stevens
Gwynn
G
Rekhviashvili
Natela
N
Stevens
Wendy
W
D019380:Anti-HIV Agents / Q000008:administration & dosage / Q000494:pharmacology; D024921:Drug Resistance, Multiple, Viral; D015497:HIV-1 / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008967:Molecular Biology / Q000379:methods*; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D019380:Anti-HIV Agents; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D018894:Reverse Transcriptase Inhibitors