engleski

Genotyping of Mycobacterium tuberculosis by ligation-mediated polymerase chain reaction (LMPCR)

Genotyping of Mycobacterium tuberculosis (M. tuberculosis) by means of molecular methods has become an important instrument for tuberculosis surveillance, control and prevention. The discovery of the insertion sequence IS6110, found in virtually all members of the M. tuberculosis resulted in great progress in M. tuberculosis strain differentiation. Thus, due to its mobility and its common presence in large number of copies, the IS6110 is widely used as genetic marker in genotyping of clinical M. tuberculosis isolates for epidemiological investigations. In this paper, our objective was to distinguish different strains of M. tuberculosis in clinical isolates by ligation-mediated polymerase chain reaction (LMPCR). This method enables to amplify large number of restriction fragments, which contain IS6110 sequences and variable, flanking sequences on the 5' side of IS6110. It is possible, due to the difference in number of copies of the IS6110 element and its position in the genome, to distinguish different M. tuberculosis strains. To conduct LMPCR, we constructed an asymmetrical, double-stranded DNA fragment (adapter - AD), which is covalently ligated by T4 DNA ligase to the cohesive ends obtained with digestion of M. tuberculosis genomic DNA by SalI restriction enzyme. For amplification of the products we used one primer specific for IS6110 element (T4) and a second specific for AD sequence (T1). These primers amplified only flanking sequences located on the 5' side of IS6110. The amplified DNA fragments obtained by LMPCR were well distinguished by electrophoresis on 2.5% agarose gels. The molecular size of amplicons ranged from 100 to 2, 000 pb and they number varied between 2 and 10, depending of the M. tuberculosis strain. Besides, LMPCR proved to be very sensitive, rapid, technically simple and reproducible for M. tuberculosis genotyping.

Mycobacterium tuberculosis; genotyping

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