INVESTIGATION OF RANGES AND FREQUENCY OF MUTATIONS IN THE embB GENE IN MYCOBACTERIUMTUBERCULOSIS ASSOCIATED WITH RESISTANCE TO ETHAMBUTOL USING REAL-TIME POLYMERASE CHAINREACTION

Abstract

Based on real-time allele-specific polymerase chain reaction, the ranges of potential mutations in codons of 306 and 405 of the embBgene in Mycobacterium tuberculosis associated with resistance to ethambutol were investigated. 5 different mutations were detected in codon 306 and 3 mutations were found in codon 406 of the embB gene. The detected mutations were confirmed by sequencing and mass spectrometry. By analyzing the frequency of detected mutations of , the set of reagents was developed for rapid testing of susceptibility tuberculous mycobacteria to ethambutol by multi-competitive allele-specific real-time PCR. Out of 107 tested specimens of clinical isolates, mutations of the embB gene of M. tuberculosis were detected in 49 (45.8%) specimens, and no mutations were found in 58 (52.2%) specimens. 39 (36.4%) specimens had mutations in codon 306 of the embB gene, and 9 (8.4%) specimens had a mutation in codon 406, and 1 (0.9%) specimen had mutations in both codons 306 and 406. The high level of agreement in the results of molecular genetic and bacteriological tests (84%) proved the significance of mutations in codons 306 and 406 of the embB gene in M. tuberculosis and the need for their identification in order to detect ethambutol resistant strains of M. tuberculosis. When using molecular genetic tests, the sensitivity level made 75.8%, while the specificity of standard culture-based methods makes 95.6%.

Keywords

• микобактерии туберкулеза
• лекарственная устойчивость
• этамбутол
• аллель-специфичная пцр в реальном времени