Studies Of Molecular Mechanism Of Quinolones Resistance And Detection Methods Of Gene Mutation In Mycobacterium Tuberculosis

In order to study molecular mechanisms of quinolones resistance and detection methods of gene mutation in M tuberculosis, 30 quinolones-sensitive isolates and 47 quinolones-resistant isolates were studied. Firstly, their species were identified with 16S rRNA gene by polymerase chain reaction - single-stranded conformation polymorphism ( PCR-SSCP ) . Among them, 75 isolates had the same 16S rRNA SSCP profiles as that of the standard strain of M. tuberculosis, the other 2 quinolones-resistant isolates were non-tuberculous Mycobacteria.Products of gyrA-PCR amplification were 285 bp fragment. The sensitivity of the primers was Ipg. 19 Mycobacteria strains and 5 non-mycobacteria strains were amplified with gyrA primers respectively, the formers had positive products and the latters had nothing.The standard strain of M. tuberculosis and BCG were used as the control groups, 75 isolates of M. tuberculosis were analyzed by PCR-SSCP. Among 30 quinolones-sensitive isolates, 11 had the same profiles as that of H37Rv, 19 had the same profiles as that of BCG. Of 45 quinolones-resistant isolates, 34(75.6%) displayed different gyrA SSCP profiles from those of either H37Rv or BCG, except for 4 with the H37Rv profile and 7 with the BCG profile. Meanwhile, DNA sequencing of gyrA gene was performed in 5 quinolone-susceptible and 45 quinolones-resistant isolates of M. tuberculosis. The results suggested that the isolates with H37Rv gyrA SSCP profiles had AGC at condon 95, those with BCG gyrA SSCP profiles had ACC at condon 95. The mutation at codon 90 of gyrA occurred in 10 quinolones-resistant isolates, the mutation at codon 94 of gyrA occurred in 24 quinolones-resistant isolates, the results were identical completely between PCR-SSCP and DNA sequencing.The minimal inhibitory concentrations (MICs) of ofloxacin and levofloxacin were also measured in 45 quinolones-resistant M. tuberculosis isolates, At the same time, the correlation of gyrA mutations with ofloxacin and levofloxacin MICs was studied. The results showed that the MICs of ofloxacin were between 4ug/ml and 32ug/ml, the MICs of levofloxacin were between 2ug/ml and 16ug/ml in all mutational isolates. The mutational rates of both ofloxacin-resistant isolates with MICs over 4ug/ml and levofloxacin-resistant isolates with MICs over 2ug/ml wereall 100%.Many factors can affect the results of PCR-SSCP. It was found that when 8% gel (29:1) was used under the action of 120 voltages for 6 hours, the effects were more ideal.The mutations at codon 90 and 94 of gyrA were important molecular mechanism in quinolones-resistance of M. tuberculosis. PCR-SSCP can rapidly detect the quinolones-resistant isolates of M, tuberculosis. The data were identical to DNA sequencing, but PCR-SSCP had the advantages such as simple, rapid and suitable for clinical apply. PCR-SSCP might become a simple and rapid diagnostic test for quinolones-resistant M. tuberculosis. This method was expected to be applied to detect drug susceptibility of clinical specimens.