Genotyping of Mycobacterium tuberculosis clinical isolates in Zhejiang province with spacer oligonucleotide typing and multiple loci variable number tandem repeat analysis

To analyze the genotypes of Mycobacterium tuberculosis clinical isolates in Zhejiang province with spacer oligonucleotide typing(spoligotyping) and multiple loci variable number tandem repeat(VNTR) analysis(MLVA),70 clinical strains of Mycobacterium tuberculosis isolated from Zhejiang province were selected randomly.After conventional cultivation,the bacterial DNA was extracted from these isolated strains and the PCR technology was used to amplify the total DR region with spoligotyping and to amplify simultaneously 15 loci of VNTR with MLVA.The clustering of genotypes was analyzed with BioNumberies(Version 5.0).Through spoligotyping,these clinical strains of T.tuberculosis could be categorized into two families:the Beijing family(70%) and non-Beijing family(30%).In the Beijing family,89.8%(44/49) belonged to the typical strains and these strains could be categorized into 4 genogroups with MLVA,i.e.I,II,III,IV genogroups,in which I genogroup(8.5$) consisted of 5 genotypes;II genogroup(18.3%) consisted of 13 genotypes' III genogroup(70.4%) consisted of 38 genotypes and IV genogroup(2.8%) consisted of 2 genotypes.The results using these two methods of genotyping matched quite well,from which all the Beijing family strains were MLVA-III and the non-Beijing family strains were distributed among MLVA-I,II,IV.All the 70 strains of M.tuberculosis could be divided into 58 genotypes including 53 unique genotypes by means of MLVA,while they could be divided into 18 genotypes including 12 unique genotypes by means of spoligotyping.Results of the susceptibility tests against the first-line drugs indicated that 71.4%(35/49) of the Beijing family strains were sensitive,while 28.6%(14/49) were resistant,but in the non-Beijing family strains,66.7% were sensitive and 33.3% were resistant.However,no significant difference existed between them as demonstrated by chi-square test(χ2=0.158,P0.05).From these observation,it is evident that significant genetic polymorphism exists among the clinical strains of M.tuberculosis isolated from Zhejiang province with the predominantly prevalent strains of Beijing family and without obvious association on the drug-resistance.It is also demonstrated that MLVA should be superior to spoligotyping to analyze the genotypes of M.tuberculosis,especially the Beijing family strains.