Evaluation Of Typing Approach In Molecular Epidemiology Of Mycobacterium Tuberculosis Complex

Objective1.To develop a new,rapid and easy method for species identification of Mycobacterium tuberculosis Complex(MTBC).2.To evaluate the feasibility of two genotyping methods,Spoligotyping and VNTR typing,for Mycobacterium tuberculosis Complex.Methods1.Seven sets of primers(Mtbcl-7) for MTBC were used to amplify the genomic DNA from the reference strains and 96 clinical isolates.2.Mycobacterium tuberculosis strains were genotyped by Spoligotyping and VNTR.Results1.The specificities of seven sets of primers were identified by PCR with 3 MTBC reference strains,9 non-mycobacterium reference strains and 27 non-tuberculosis mycobacterium(NTM) reference strains.The results showed that mycobacterium and non-mycobacterium were identified by Mtbc1;NTM and MTBC were identified by Mtbc2;M. microti was identified by Mtbc3;M.bovis and M.bovis BCG were identified by Mtbc4;M. africanum subtype I and M.tuberculosis were identified by Mtbc5;M.bovis BCG was identified by Mtbc4 and Mtbc6 together;M.M.canettii was identified by Mtbc5 and Mtbc7 together.Of 96 clinical isolates,20 NTM,3 M.microti,1 M.bovis,1 M.bovis BCG,3 M. africanum subtypeâ… ,1 M.M.caprae,3 M.M.canettii,64 M.africanum subtypeâ…¡and M. tuberculosis were identified.2.By Spoligotyping method,59 strains were typed to 11 genotypes,in which 83.1%(49 /59)strains belonged to Beijing genotype.By VNTR method,these strains were divided into 53 genotypes.The VNTR typing showed high discriminatory power,especially for the Beijing genotype strains.Forty-nine Beijing genotype strains could be subdivided into 43 different VNTR genotypes.In these samples collected,10 VNTR loci showed different discrimination according to their allelic diversity.Eight Locus showed high discrimination,while two locus showed moderate discrimination.Conclusions1.It showed that PCR was a rapid and effective method for the species identification of MTBC.It could be used for molecular epidemiology.2.VNTR genotyping is a simple and fast method.Its numerical result facilitates the comparison among strains of Mycobacterium tuberculosis from different labs.The discriminatory power of VNTR is higher than that of Spoligotyping.There was significantly different discrimination between VNTR and Spoligotyping in the clustered rate of M. tuberculosis from the different regions and the proportion of Beijing genotype.3.The results indicated that either Spoligotyping or VNTR technique is very useful for the epidemiological investigation of tuberculosis in China.The strains in different regions have different character in China.Especially VNTR typing that is easy to popularize will play an important role in the epidemiological investigation of tuberculosis.