[Identification and differentiation of mycobacteria through the normal and multiplex polymerase chain reaction (PCR)]
2006
Bonovska, M.G.(National Diagnostic Research Veterinary Inst., Sofia (Bulgaria))
Three different mycobacterial genomic fragments were used for the identification and differentiation of Mycobacterium species. The oligonucleotide primers ТВ 15 and ТВ 19 were chosen from the groEL gene presented in the Mycobacterium sp.; the primers IS41 and IS43 were chosen from insertion sequence IS6110 presented in Mycobacterium tuberculosis complex; primers PT1 and PT2 were chosen from the mtp40 gene identified as a specific-species of M. tuberculosis genomic fragment. Normal and multiplex PCR was carried out with the observed primers. Genome DNAs isolated from Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M, avium, M. phlei and M. chelonae were used for the PCR amplification. ТВ-primers were generated into normal PCR by synthesis of PCR products in size of 600 bp from M. tuberculosis, M. bovis и M. avium and IS-primers of 300 bp from M. tuberculosis, M. bovis and M. bovis BCG. PCR amplification products with size of 400 bp were obtained from the PT-primers only with M. tuberculosis. Multiplex PCR was realized with two and three pairs of primers in different quantity proportion. The primers mixture TB+PT (1:1) was synthesized by one fragment PCR with size of 600 bp with M. bovis, M. avium, M. phlei и M. chelonae and by two fragment PCR from 400 и 600 bp with M. tuberculosis. Amplification products were not observed in M. flavescens. One fragment of 600 bp from M. bovis и M. bovis BCG and two fragments of 300 и 600 bp from M. tuberculosis were amplificated by primers mixture of TB+IS (2:1). Two fragments with size of 300 and 600 bp from M. bovis and M. bovis BCG and three fragments of 300, 400 and 600 bp from M. tuberculosis were obtained from primers mixture TB15+PT+IS (2:1,5:1). Research results proved that in the conditions of the appropriate combination on the used primers the multiplex PCR could be successfully applied for the differentiation of the different mycobacterium species that was impossible with normal PCR technique
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