OBJECTIVETo develop a standardized IS6110-restriction fragment length polymorphism (RFLP) method, used for evaluating the capacity of genotyping.

METHODSIS6110-RFLP of 78 Mycobacterium (M.) tuberculosis strains were studied by bio-molecular techniques including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis, together with data analysis by software Gel-Pro analyzer 3.1 and BioNumerics (Version 5.0).

RESULTSIS6110-RFLP method was established and standardized successfully, including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis and usage of the analysis software with standard parameters. By this method, 78 M. tuberculosis isolates were classified into 75 genotypes which belonged to 11 different clusters. Of all the isolates, 66.7% (52/78) belonged to a main cluster.

CONCLUSIONStandard IS6110-RFLP method was established successfully. This method had powerful capacity for genotyping and strain level identification and could be used for the surveillance on pathogens of M. tuberculosis in China.

Bacterial Typing Techniques ; methods ; DNA, Bacterial ; Genome, Bacterial ; Genotype ; Molecular Sequence Data ; Mycobacterium tuberculosis ; classification ; genetics ; isolation & purification ; Polymorphism, Restriction Fragment Length

Objective To establish and evaluate the standardized protocol of multiple loci variable numbers tandem repeats (VNTR) analysis (MLVA) for genotyping Mycobacterium tuberculosis (M.tuberculosis).Methods 15 VNTR loci were chosen for genotyping 54 Chinese M.tuberculosis strains by PCR-electmphoresis-based VNTR analysis and the results were analyzed by software BioNumerics (Version 5.0).Results MLVA method was successfully established and standardized,including the standard protocol for bacterial culture,DNA isolation,PCR and agarose gel electrophoresis and the software analysis. 15 VNTR loci were confirmed,including ETRA,ETRB,ETRC,ETRD,ETRE,MIRU10,MIRU16,MIRU23,MIRU26,MIRU27,MIRU39,MIRU40,Mtub21,Mtub30 and Mtub39,to be suitable for MLVA analysis of M.tuberculosis.Conclusion The standardized MLVA method has been established successfully.This method is simple and has powerful capacity for genotyping and strain differentiation,can be used for the network surveillance on pathogens of M.tuberculosis,and the data are comparable between laboratories.It is valuable for tracing the source and studying the trend of prevalence during investigation of M.tuberculosis infections.

Investigations on the genetic diversity of Mycobacterium tuberculosis in China have shown that Beijing genotype strains play a dominant role. To study the association between the M. tuberculosis Beijing genotype and the drug-resistance phenotype, 1286 M. tuberculosis clinical isolates together with epidemiological and clinical information of patients were collected from the center for tuberculosis (TB) prevention and control or TB hospitals in Beijing municipality and nine provinces or autonomous regions in China. Drug resistance testing was conducted on all the isolates to the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, and ethambutol). A total of 585 strains were found to be resistant to at least one of the four anti-TB drugs. The Beijing family strains consisted of 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains, whereas the non-Beijing family strains comprised 202 (58.05%) drug-sensitive strains and 146 (41.95%) drug-resistant strains. No significant difference was observed in prevalence (χ= 2.41, P > 0.05) between the drug-resistant and drugsensitive strains among the Beijing family strains. Analysis of monoresistance, multidrug-resistant TB, and geographic distribution of drug resistance did not find any relationships between the M. tuberculosis Beijing genotype and drug-resistance phenotype in China. Results confirmed that the Beijing genotype, the predominant M. tuberculosis genotype in China, was not associated with drug resistance.
Antitubercular Agents ; therapeutic use ; China ; epidemiology ; Drug Resistance, Multiple, Bacterial ; Genetic Variation ; Genotype ; Humans ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Phenotype ; Tuberculosis, Multidrug-Resistant ; drug therapy ; epidemiology