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Genotyping of resistant and sensitive strains of Mycobacterium tuberculosis by ligation-matiated polymerase chain reaction (LMPCR)

Tuberculosis is the leading infectious cause of death worldwide. The World Health Organisation (WHO) estimates more than 8 million of new cases of tuberculosis per year and about 3 million deaths caused by this disease every year. Croatia takes place among the countries with still high incidence of tuberculosis (35/100, 000). Recently, 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 (genotyping) and enabled epidemiological investigations of clinical M. tuberculosis isolates. Herein, our objective was to distinguish between resistant and sensitive strains of M. tuberculosis in clinical isolates by means of ligation-mediated polymerase chain reaction (LMPCR). This method enables differentiation between M. tuberculosis strains by the copy number of IS6110 element and its position in the genome. We examined 62 of M. tuberculosis isolates, of which 48 were resistant and 14 sensitive to five antituberculosis drugs. High molecular weight genomic DNA was extracted from M. tuberculosis colonies by cells treatement with lysozyme, proteinase K, NaCl and ethanol precipitation. 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 the second primer 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 their number varied between 2 and 10, depending of M. tuberculosis strain. We obtained several different profiles of M. tuberculosis in both resistant and sensitive strains, of which many were identical. Those profiles will be processed by image master analyser (Pharmacia Biotech) and the results will be statistically evaluated to indicate the eventual significant difference between the resistant and sensitive strains of M. tuberculosis. Since DNA fragments were well distinguished without further analyses, we can conclude that LMPCR is a very reproducible method for M. tuberculosis genotyping. Besides, LMPCR proved to be very sensitive, rapid and technically simple to perform.

Mycobacterium tuberculosis; genotyping

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