[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].

ABSTRACT

OBJECTIVE: To explore the feasibility of utilizing polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis combined with Southern blot to detect ofloxacin (OFLX)-resistant Mycobacterium tuberculosis, and figure out the boundary between the OFLX-susceptible and resistant Mycobacterium tuberculosis. METHODS: OFLX-resistant Mycobacterium tuberculosis bacilli were induced in vitro and their minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC) were determined. The 320 bp segments of gyrA gene were amplified by PCR and their polymorphism was tested by SSCP from H(37)Rv, H(37)Ra and those induced OFLX-resistant strains. To prove the repeatability of PCR-SSCP combined with Southern blot, 36 clinical isolated OFLX-resistant strains were tested. RESULTS: All of the 85 OFLX-resistant mutations involved in this study were identified to have gyrA mutations. The results showed that there was an overlap if OFLX 10 microg/ml was used as a criteria for discriminating susceptible and resistant strains. Compared with the H(37)Ra, the similar single-stranded shift caused by the single-stranded conformation change was viewed in the 36 clinical isolated OFLX-resistant strains by PCR-SSCP combined with Southern hybridization. CONCLUSIONS: PCR-SSCP analysis combined with Southern blot can clearly distinguish OFLX-susceptible from OFLX-resistant strains. The results suggest that as a cut-off point between OFLX-susceptible and low-level OFLX-resistant to Mycobacterium tuberculosis, OFLX concentration of 10 microg/ml is considered to be a better one.