Abstract
The ability to rapidly and efficiently identify causative agents of dangerous human and animal
diseases is a prerequisite to diagnosis, prophylaxis and therapy. Such identification systems can
be developed based on DNA markers enabling differentiation between various bacterial strains.
One source of these markers is genetic polymorphism. An efficient method for detecting the most
stable polymorphisms without knowledge of genomic sequences is subtractive hybridization. In this
work we report an approach to typing of Burkholderia pseudomallei and B. mallei that cause
melioidosis and glanders, respectively. Typing is based on hybridization of bacterial genomes
with a DNA array of genomic markers obtained using subtractive hybridization. The array comprised
55 DNA fragments which distinguished the genomes of B. pseudomallei C-141 and B. mallei C-5
strains, and it was used to test 28 radioactively labeled B. pseudomallei strains and 8 B. mallei
strains. Each strain was characterized by a specific hybridization pattern, and the results were
analyzed using cluster analysis. 18 patterns specific to B. pseudomallei and 6 patterns specific
to B. mallei were found to be unique. The data allowed us to differentiate most studied
B. pseudomallei variants from one another and from B. mallei strains. It was concluded that DNA
markers obtained by subtractive hybridization can be potentially useful for molecular typing of
B. pseudomallei and B. mallei strains, as well as for their molecular diagnosis. The method
reported can be easily adapted for use both with DNA arrays and DNA microarrays with fluorescent
probes. |
