PCR-based methodology for detecting multidrug-resistant strains of Mycobacterium tuberculosis Beijing family...

The Beijing genotype of Mycobacterium tuberculosis has been identified in 40-50% of the clinical isolates studied in Russia during the last decade. This genotype has been reported to be associated with multiple drug resistance and possesses some significant pathogenic properties. Therefore, early identification of such strains is of extreme importance in the timely detection of drug resistance. The present study was performed on 354 strains isolated in Russia from 1996 to 2002 and previously characterised by IS 6110-restriction fragment length polymorphism (RFLP) typing and spoligotyping. These strains included 198 Beijing family strains and 156 strains of other genotypes (IS 6110-RFLP profiles). A subsequent polymerase chain reaction (PCR) analysis with IS 6110-derived outwardly oriented primers (IS 6110-PCR) easily discriminated the Beijing strains from non-Beijing strains. The multiplex allele-specific (MAS)-PCR assays were further used to detect mutations in katG315 and rpoB531, associated with resistance to isoniazid and rifampin, respectively. The katG315 and rpoB531 mutations were found to be more prevalent among Beijing (96.8% and 77.3%) than among non-Beijing strains (85.7% and 28%). Consequently, we propose a two-step methodology based on routine PCR and simple agarose gel electrophoresis in order to detect (i) a Beijing family strain using IS 6110-PCR, and, (ii) its possible resistance to the major anti-tuberculosis drugs using specific MAS-PCR assays.