Study On The Relationship Between MIRU-VNTR And Drug Resistance Gene Mutation And Drug Resistance Of Mycobacterium Tuberculosis

Objective:To investigate the mutation characteristics of rifampicin and isoniazid resistant genes,and to explore the relationship between MIRU-VNTR gene copy number polymorphism and tuberculosis resistance,so as to facilitate the early diagnosis of drug-resistant patients and provide theoretical support for reducing the failure rate of treatment.Methods:clinical Mycobacterium tuberculosis strains were collected and drug sensitivity test was carried out,including first-line drugs:isoniazid(INH)and rifampicin(RFP),second-line drugs:kanamycin(km)and ofloxacin(ofx);Primers were designed according to MIRU-VNTR gene and drug resistance gene,and the target gene was sequenced after PCR amplification.Logistic regression model was used to analyze the relationship between MIRU-VNTR copy number polymorphism,three common drug resistance gene mutations and drug resistance.Results:(1)Results of drug resistance related basic information:this study collected 332 tuberculosis patients in Anqing City,including 259(78.01%)males and73(21.99%)females,with an average age of(57.16±37)years old;The results of DST test showed that there were 57 drug-resistant strains,and the drug resistance rate was 17.17%(57/332).There were 51 strains resistant to INH and 27 strains resistant to RFP.There were 24 strains(7.23%,24/332)of MDR-TB and 1 strains(0.30%,1/332)XDR-TB.(2)The drug resistance gene mutation characteristics:the results showed that 36(70.59%)strains of kat G gene and 7(13.73%)strains of inh A gene were identified by PCR amplification,20(6.02%)strains of rpo B gene and 48(84.21%)strains were identified by PCR amplification;Compared with the wild-type kat G gene,the main amino acid mutation sites were 315(52.94%;27/51)and 251(1.96%;1/51),while 7(13.73%)strains of inh A gene were mutated at C15T.In addition,the main mutation sites of rpo B gene were 445 in 6(22.22%)strains,450 in 6(22.22%)strains and 452in 3(11.11%)strains,and 2(9.10%)strains were found to be double site mutations.(3)Relationship between mutations of different drug resistance genes and drug resistance:the patients were divided into two groups according to whether they were resistant to isoniazid or rifampicin.There were 43 males(84.30%)and 8 females(15.70%)in the INH resistant group,21 males(77.80%)and 6 females(22.20%)in the RFP resistant group.There was no significant difference in the composition of males and females between the two groups(P>0.05).There were significant differences in age(χ~2=2.026,P=0.044),kat G gene mutation rate(χ~2=194.229,P<0.001),inh A gene mutation rate(P<0.001)and rpo B gene mutation rate(P<0.001)between INH resistant and non INH resistant groups;The difference of kat G gene and rpo B gene mutation rate between RFP resistant and non RFP resistant groups was statistically significant;Multivariate logistic regression analysis showed that INH resistance was associated with kat G gene(OR=166.776,95%CI:29.759-934.636,P<0.001)and rpo B gene(OR=18.696,95%CI:1.910-182.992,P=0.012),while RFP resistance was also associated with kat G gene(OR=9.899,95%CI:1.166-84.075,P=0.036)and rpo B gene(OR=208.593,95%CI:24.336-1787.964,P<0.001).(4)Relationship between MIRU-VNTR gene copy number polymorphism and drug resistance:the MIRU-VNTR gene copy number range from 0 to 6,INH and RFP drug resistance as dependent variables,copy number of each gene as independent variables,univariate and multivariate logistic regression analysis showed that MIRU40 gene copy number was negatively correlated with isoniazid resistance(OR=0.618,95%CI:0.443-0.862,P=0.005),There was no statistical significance between other gene loci and drug resistance.(5)The effect of drug resistance gene mutation and MIRU-VNTR copy number on drug resistance:multivariate logistic regression analysis was performed with INH and RFP resistance as dependent variables,mutation of three common drug resistance genes and copy number of three MIRU-VNTR genes as independent variables,the results showed that INH drug resistance was still associated with kat G gene and rpo B gene mutation(P<0.001),RFP resistance was also associated with kat G and rpo B gene mutations(P<0.001);In addition,only INH sensitive group and drug-resistant group may be associated with MIRU20 gene copy number(P=0.051).Conclusion:(1)The prevalence of overall multidrug resistance rate(MDR-TB)in this area is still very high,and the types of drug resistance gene mutations are quite different.(2)It was confirmed for the first time that the gene loci highly associated with INH resistance were kat G gene 315 and Inh A promoter C15T,while rpo B gene loci531 and 526 were closely associated with RFP resistance.(3)Using MIRU-VNTR genotyping technology,we successfully verified the previous results that MIRU40 gene copy number is related to INH resistance.