RESUMEN
Recently in Russia biochips for rifampin resistance detection of M. tuberculosis were developed. To investigate the conformity between rifampin resistance results determined both by the routinely used absolute concentration method and USING the biochips, 272 DNA samples of M. tuberculosis isolated from TB patients at Novosibirsk and Tomsk regions in 2000-2005 were analyzed. The biochip can detect 30 mutations in rpoB gene. The mutations were also tested using the single stranded conformational polymorphism method (SSCP). In addition, 60 DNAs were randomly sampled and sequenced. The results of rifampin resistance detection using biochip and absolute concentration methods were congruent in 86% cases, and were different when analyzed samples consisted of the susceptible and resistant strains of M. tuberculosis mixture. The most frequent mutations in the rpoB gene were S531 (76.2%), H526 (7%), D516 (5.6%), and L511 (5.6%). In 94% of rifampin resistant strains, there was also resistance to isoniazid. Therefore, in Siberia the rifampin resistance is the reliable marker for MDR strains of M. tuberculosis, and biochips can be used also for their detection. To hybridize with biochip the fluorescent-labeled single-stranded DNAs were routinely synthesized by two PCR, and intermediary product after the first PCR should be transferred into another tube. The last stage included high risk of cross-contamination. To exclude the risk, primer concentrations and temperature-time profile of PCR reactions were improved, and both PCR were combined in one tube. The two methods were congruent in 100%. The one tube method would be especially attractive for the routine PCR laboratory.
Asunto(s)
Antibióticos Antituberculosos , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Rifampin , Análisis Mutacional de ADN , ARN Polimerasas Dirigidas por ADN , Humanos , Mycobacterium tuberculosis/aislamiento & purificación , SiberiaAsunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/diagnóstico , Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Tuberculosis/sangre , Tuberculosis/inmunologíaRESUMEN
The purpose of the work was to study rifampicin- and izoniazid-resistent strains of M. tuberculosis, circulating in Western Siberia, by VNTR and IS6110 typing. The authors also studied genetic causes of resistance to these antibiotics and undertook a search of new VNTR loci, displaying polymorphism in genomes of closely related clonally-disseminated variants of Mycobacterium tuberculosis in W-Beijing family model analysis.
