Rapid detection of ethambutol-resistant genes in Mycobacterium tuberculosis clinical isolates

Abstract

An oligonucleotide probe has been designed and compounded for detecting the ethambutol (EMB) resistance gene of Mycobacterium tuberculosis. It was placed on a nitrocellulose membrane for use in the reverse dot-blot hybridization assay, with the PCR product labeled with biotin. Analysis was carried out on 82 M. tuberculosis clinical isolates. The results indicate that among the 31 EMB-sensitive strains, the single-strand conformational polymorphism (SSCP) spectrum and reverse dot-blot (RDB) assay results on the embB gene in 26 strains were completely identical to those of the standard strain (H37Rv), testing positive for E1 hybridization; the SSCP spectrum of the remaining five strains indicated that migration had occurred. Among the 51 EMB-resistant strains, 24 strains tested positive for E1 hybridization; of the remaining 27 strains, 18 strains tested positive for E1b hybridization, two tested positive for E1c hybridization, five tested positive for E1d hybridization, one tested positive for E1e hybridization, and one tested positive for E1f hybridization. The lowconcentration (64.70%) drug-resistant strains did not have the mutation at the codon 306 of embB. Among the 18 highconcentration drug-resistant strains, 15 strains tested positive for E1b hybridization, two tested positive for E1c hybridization, and one tested positive for E1f hybridization. The mutation detection rate was 52.94%. The RDB technology may be an easy and rapid method for detecting the codon 306 mutation of embB gene in some M. tuberculosis clinical isolates. We conclude that the ATG→GTG and ATG→CTG mutations of codon 306 of embB are one of the main causes of stron g EMB drug resistance in M. tuberculosis.