Novel enzyme-linked minisequence assay for genotypic analysis of human immunodeficiency virus type 1 drug resistance.
We constructed a novel tool for genotypic analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance by using an enzyme-linked minisequence assay (ELMA). ELMA is a combination of hybridization and a 1-base extension reaction, and we designed the assay to detect five mutations conferring nucleoside analogue resistance (M41L, D67N, K70R, T215Y, and M184V) and six mutations conferring protease inhibitor resistance (D30N, M46I, G48V, V82A, I84V, and L90M). At all detection points, ELMA demonstrated high sensitivity and specificity, sufficient for clinical use. Compared to that obtained by direct sequencing, the genotypic information obtained by ELMA is limited to the targeted loci for which it was designed. However, ELMA proves advantageous in several respects. The assay does not require expensive equipment, such as an autosequencer, and can be performed in regular clinical diagnostic laboratories. Therefore ELMA can be a candidate for a drug resistance monitoring assay to be introduced in developing countries. In addition, ELMA demonstrated higher sensitivity in the detection of minor resistant populations. We successfully detected a minor virus population (10%) by the assay. The high sensitivity and specificity of the assay recommend it as a first screening assay for drug resistance surveillance.
Sugiura
Wataru
W
AIDS Research Center, National Institute of Infectious Diseases, Tokyo 2080011, Japan. wsugiura@nih.go.jp
Shimada
Kazunori
K
Matsuda
Masakazu
M
Chiba
Tomoko
T
Myint
Lay
L
Okano
Aiko
A
Yamada
Kaneo
K
D001483:Base Sequence; D017931:DNA Primers; D024882:Drug Resistance, Viral / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005838:Genotype; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics*; D006801:Humans; D009154:Mutation; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology
D017931:DNA Primers; D017320:HIV Protease Inhibitors; D015345:Oligonucleotide Probes; D018894:Reverse Transcriptase Inhibitors