Preliminary Study On Genotyping Of Mycobacterium Tuberculosis Clinical Isolates From Gansu Province

Objective 215 Mycobacterium tuberculosis isolates were studied with MLVA and Spoligotyping to evaluate the application of the two technic of genotyping in molecular epidemiology study of tuberculosis and to explore the characteristic and distribution of their genotyping in order to provide the government with scientific measures to prevent and control tuberculosis disease.Methods M.tuberculosis strains isolated from the patients in Gansu,and typed by MLVA and Spoligotyping respectively to compare the results of both methods and access the application of both methods in epidemiological studies of tuberculosis.215 M.tuberculosis Complex was isolated from clinical confirmed patients at Lanzhou pulmonary disease Hospital and was genotyped by traditional 43-space spoligotyping and MLVA that selected 15 VNTR loci analyzed by PCR and agarose gel electrophoresis.The characteristics of DNA fingerprinting of 215 strains was analyzed by BioNumerics 4.5 software.The basic dates were analyzed by Spss 11.0.Results 215 Beijing family genotype was the most common genotype family,identified in 186/215(86.51%)cases,followed by T family4.19%(9/215),H family 1.86%(4/215)and MANU family 1.40%(3/215) respectively.Univariate analysis showed Beijing family genotype was not significantly associated with resistance to all of four drugs(RFP,IHN,EMB and SM), patient's gender,age,jobs,antituberculosis therapy history,regular of irregular therapy and new cases or relapses(p>0.05),while was significantly associated with regions of patients(p<0.05).Multivariate logistic regression analysis showed there were not statistically significant associations between the Beijing family genotype strains and resistance to all of four drugs(RFP,IHN,EMB and SM),patient's gender, age,job,regions of patients,antituberculosis therapy history,regular of irregular therapy and new cases or relapse cases.By MLVA method,a total of 127 different allele profiles(including 90 unique patterns)were identified by 15 VNTR loci for all 215 isolates fell into 7 clusters.74.88%(161/215)of the isolates were dominant in Gansu.Univariate analysis showed the clustered strains were not significantly associated with resistance to all of four drugs(RFP,IHN,EMB and SM),patient's gender,age,regular of irregular therapy and new cases or relapses(p>0.05),while was significantly associated with antituberculosis therapy history(p<0.05).Using HGDI,two VNTR loci,(MIRU26,MIRU21)were found to be "highly discriminatory" (DI≥0.6),four VNTR loci(ETR-A,ETR-E,MIRU16,MIRU40)were "moderately discriminatory"(DI:0.3-0.59),and the other nine loci(MIRU10,MIRU23,MIRU27, ETR-B,ETR-C,ETR-D,MIRU39,Mtub30 and Mtub39)were "poorly discriminatory" (DI<0.3).Compared with MLVA,215 Mycobacterium tuberculosis isolates from Gansu Province were grouped 22 clusters by Spoligotyping,but 127 clusters by MLVA.In these two PCR- based methods,the discriminative ability of MLVA is better than the Spoligotyping.Conclusion The strains of Mycobacterium tuberculosis isolated in Gansu Province present definitely gene diversity.Beijing family genotype is the most epidemic genotype in the region.Then other genotypes of Mycobacterium tuberculosis should be emphasized to prevent and control the spread of other spoligotypes strains.Beijing genotype was not recognized as the one transferred from some of the drug resistance strains.MIRU21,MIRU26,ETR-A,ETR-E,MIRU16,MIRU40 loci were the optimized loci with MLVA to genotype M.tuberculosis from Gansu Province.Though Spoligotyping has lower discriminatory power than MLVA, it can use to classify M.tuberculosis complex isolates in taxon or subspecies(it can detect Beijing genotype strains),has consummate international spoligotyping database and can compare genotyping in different regions.MLVA is available for time- save, labor-saving,easiness to compare with others,and having better discriminatory power to those isolates with a low IS6110(less than 5 copies)copy number.Generally,in these two PCR- based methods,the discriminative ability of MLVA is better than the Spoligotyping.Using spoligotyping and MLVA together can provide adequate discrimination for molecular epidemiological analysis in comparison to IS6110-RFLP, when applied to regional areas,and can get a perfect genotyping result.These two PCR-based methods has the merit of easy and fast(get the result in 24h),repeated in different laboratories,needing few DNA.Especially,the resulting data can be easily coded and exchanged between different laboratories.It inaugurates a new approach to study source of infection,molecular evolution of epidemigenic strain and establishment of a global database.