Specific, sensitive, and rapid assay for human immunodeficiency virus type 1 pol mutations associated with resistance to zidovudine and didanosine.
The effectiveness of antiretroviral therapy may be limited by the development of human immunodeficiency virus type 1 (HIV-1) resistance. Monitoring for resistance will perhaps allow changes in therapy prior to deterioration in the patient's clinical or immunologic status. Our objective was to develop a rapid, specific, and sensitive genotypic assay for HIV-1 resistance to zidovudine (ZDV) and didanosine (ddI) which is simple to perform. In our assay the DNA of HIV-1 pol was amplified by PCR using two sets of nested oligonucleotide primers. Mutations of reverse transcriptase (RT) encoding amino acids (aa) 74 and 41, 70, and 215 which have been associated with HIV-1 resistance to ddI and ZDV, respectively, were detected with a ligase detection reaction (LDR) and indicated colorimetrically. The RT genotypes of 35 patient specimens (140 codons) blindly assessed for these mutations were in agreement by PCR-LDR and by dideoxynucleotide sequencing. To evaluate the limits of the assay, other specimens with mutations close to the ligation site were evaluated by PCR-LDR. The assay was sensitive and specific for all specimens except when mutations occurred within 2 bases on either side of the ligation site. In summary, this PCR-LDR assay specifically, sensitively, and rapidly detected pol mutations (RT aa 74, 41, 70, and 215) associated with HIV-1 resistance to ddI and ZDV.
Frenkel
L M
LM
Department of Pediatrics, University of Rochester, New York.
Wagner
L E
LE
Atwood
S M
SM
Cummins
T J
TJ
Dewhurst
S
S
D001483:Base Sequence; D011088:DNA Ligases; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D016049:Didanosine / Q000494:pharmacology; D004352:Drug Resistance, Microbial / Q000235:genetics; D005069:Evaluation Studies as Topic; D015751:Genes, pol; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013997:Time Factors; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data; D015215:Zidovudine / Q000494:pharmacology
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D015215:Zidovudine; D011088:DNA Ligases; D016049:Didanosine