[Polymerase chain reaction-single strand conformation polymorphism analysis combined with nucleic acid hybridization to detect ofloxacin-resistant Mycobacterium tuberculosis mutation selected in vitro].
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.
Zhang
Jian-yuan
JY
Department of Bacteriology and Immunology, Institute of Tuberculosis & Thoracic Tumor, Beijing 101149, China.
Wang
Su-min
SM
Li
Chuan-you
CY
Tian
Miao
M
Zhang
Xu-xia
XX
Zhao
Bing
B
Li
Wei-min
WM
Zhao
Yan-lin
YL
Qiu
Yun-qing
YQ
Ma
Yu
Y
D000995:Antitubercular Agents / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D005798:Genes, Bacterial; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D015242:Ofloxacin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational
D000995:Antitubercular Agents; D015242:Ofloxacin