Mutagenically separated PCR assay for rapid detection of M41L and K70R zidovudine resistance mutations in CRF01_AE (subtype E) human immunodeficiency virus type 1.
A rapid zidovudine (ZDV) resistance genotypic assay was developed based on the mutagenically separated PCR (MS-PCR) technique to detect two ZDV-resistant mutations, M41L and K70R in CRF01_AE (subtype E). Endpoint dilution analysis revealed that the newly constructed MS-PCR assay could successfully detect three to nine copies of human immunodeficiency virus type 1 template RNA. The test against wild-type and mutant template mixtures in different ratios demonstrated that the assay could detect 10% minor population, at least. Fifty-one subtype E clinical samples were analyzed by the newly constructed MS-PCR assay and direct nucleotide sequencing. The concordance of the two assays was 92 and 100% in codons 41 and 70, respectively. The MS-PCR assay is a rapid, simple, and inexpensive assay that is highly sensitive in detecting mutant targets, including minor populations. Thus, it could be used as a powerful tool for epidemiological surveillance of drug-resistant mutations in developing countries.
Myint
Lay
L
AIDS Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
Ariyoshi
Koya
K
Yan
Hua
H
Frater
Alexander J
AJ
Auwanit
Wattana
W
Pathipvanith
Panita
P
Yamada
Kaneo
K
Matsuda
Masakazu
M
Chiba
Tomoko
T
Fujita
Kazunori
K
McClure
Myra
M
Weber
Jonathan N
JN
Sugiura
Wataru
W
D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000379:methods; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D015215:Zidovudine / Q000494:pharmacology*
D015215:Zidovudine