| Objective: The purpose of this study is to analyze gene mutations related with isoniazid and rifampicin resistance of Mycobacterium tuberculosis(MTB) in Guiyang area and to investigate the genotype distribution of Mycobacterium tuberculosis clinical isolates from Guiyang area with the multiple locus variable number tandem repeats analysis. Methods: 1. MTB strains were isolated by Lowenstein-Jensen method. Drug sensitivity of MTB isolates was detected by conventional ratio method. 2.The rpo B,kat G, inh A, oxy R-aph C gene fragments of Mycobacterium tuberculosis was amplified and sequenced. These strains included seventeen multi-drug resistant clinical isolates and fourteen sensitive clinical isolates. 3.The rpo B,kat G, inh A, oxy R-aph C gene sequences of these strains and sequences of H37 Rv downloaded from Gen Bank were analyzed by CLC Free Workbench4.5. 4.Eighty-six Mycobacterium tuberculosis were genotyped by MIRU method.Polymerase chain reaction was carried out for amplification of 12 known loci containg MIRU.The resulatant PCR product was fractionated electrophoretically in 2.0% agarose gels.Electrophoresis was performed at 100 V for 2.5h using a 100 bp ladder as the molecular marker.The gel was observed and photographed by Gel imager.5.The cluster analysis results were obtained by NTsys2.10 e. 6.The difference of age,sex and genetype between multi-drug resistant and sensitive MTB were all statistically analyzed by chi-squrae test.Results: 1. The mutations of rpo B gene were found in 12 multi-drug resistant clinical isolates,including rpo B531(TCGâ†'TTG) in 7 isolates, rpo B526(CACâ†'TAC) in 2 isolates, rpo B533(ATCâ†'TTC) in 1 isolate,and rpo B565(ATCâ†'TTC) in one isolate. rpo B530(CTGâ†'ATG) combined with 531(TCGâ†'TTC) was found in one isolate.rpo B530 and 565 were out of gene chip 2.The mutations of kat G gene were found in 16 multi-drug resistant clinical isolates. 70.5%(12/17) of drug-resistant strains showed kat G315(AGCâ†'ACC) mutation. kat G463(CGGâ†'TGG) was found in 11 susceptible clinical isolates and 4 multi-drug resistant clinical isolates. The mutation rate of this type of mutation in sensitive strains and multi-drug resistant strains was 78.6%( 11/14) and 23.5%(4/17) respectively. There were no significant difference of this rate between sensitive strains and resistant strains(χ2=6.48,P>0.05). Double mutations at two separate loci of kat G were detected in 12 multi-drug resistant isolates, including kat G315(AGCâ†'ACC) combined with 463(CGGâ†'TGG) in 10 strains, 463(CGGâ†'TGG) combined with 299(GGCâ†'AGC) in one isolate and 463(CGGâ†'TGG) combined with 419(GACâ†'CAC) in one isolate. No mutation in inh A was found in this study. G32 A mutation of oxy R-aph C gene was found in 2 multi-drug resistant strain. kat G299,kat G419 and –G32A of oxy R-aph C gene were out of gene chip.3.86 strains were divided into 4 genotype groups(I,II,III,IV) and 75 genotypes by MRIU. The percentage of cluster I was 74.4%, including 36 of multi-drug resistant strains and 28 of sensitive strains. 4.The cluster rate is 18.6%. 5.In this sample collection, 12 MIRU loci showed different discriminative according to their allelic diversity. Locus 26 and 31 showed highly discriminative(h>0.60), while locus 10,39,16,40,23, and 2 showed moderately discriminative(h≥0.30). Locus 4,20,27,24 showed low discriminative(h < 0.30). 6.The difference of age,six or genetype showed no significance between sensitive strains and resistant strains by Chi-square test(χ2=6.48,6.48,10.23, respectively,P>0.05) Conclusion:1. rpo B531 and rpo B526 are the dominant gene mutation related with rifampicin resistant Mycobacterium tuberculosis in Guiyang. 2. kat G315 is the dominant gene mutation related with isoniazid resistant Mycobacterium tuberculosis in Guiyang. 3. Some of the mutations related with multi-drug resistant strains could not be detected by ready-made gene chips.4.MTB in Guiyang area showed genetic diversity and epidemic flora was the cluster I. 5.In Guiyang area,Locus 26 and 31 showed highly discriminative and locus 4,20,27,24 showed low discriminative. 6.There were no significant difference of age,six,genetype between sensitive strains and resistant strains. |
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