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In the past few years, DNA microarrays have become a fundamental tool for the detection and analysis of nucleic acid sequences because they enable massively parallel molecular analysis to be carried out in a miniaturized format with a very high throughput. The technology of DNA microarray has been applied in fundamental research, human genetics, infectious disease diagnosis, genotyping,genetic expression monitoring, and for large-scale analysis of mutations in genes, which could increase the probability for certain diseases or influence the metabolism of drugs. DNA microarray technique based on the hybridization of a target DNA with oligonucleotide probes on the microchips allows rapid and accurate genotyping of microbial samples at the molecular level in a highly parallel format. The identification of a pathogen and its antibiotic resistance at the molecular level can be performed in a much shorter time and offers greater information content. We have developed several types of oligonucleotide microarrays with horseradish peroxidase - based detection for the identification of genes and determination of single nucleotide polymorphisms (SNPs). The technique was applied for the identification of genes encoding betalactamases – bacterial enzymes responsible for the development of bacterial resistance to betalactam antibiotics. The method of oligonucleotide microarray consisted of several steps involving DNA extraction, amplification and labeling of target DNA with biotin multiplex PCR, and the subsequent hybridization of a PCR product with specific oligonucleotide probes immobilized on microchip surface of different origin. After hybridization biotin in DNA duplexes was developed with the streptavidin–HRP conjugate followed by colorimetric detection of the enzyme. Special algorithms were developed for molecular design of sets of oligonucleotide probes which ensured specific identification of a number of genes and SNPs in a single assay. Two different hybridization formats (direct and sandwich) were studied in order to enhance the sensitivity and specificity of the multiassays. We succeeded to reduce the procedure time to 4 hours from DNA isolation. The sensitivity and dynamic range of colorimetric detection for biotin-labeled oligonucleotide was demonstrated to be rather similar to those for Cy3-based fluorescent detection widely used in DNA microarrays. Potential of the developed colorimetric detecting system was demonstrated in the microarray for simultaneous identification of SNPs in genes of TEM-, SHV, and CTX-M types of beta-lactamases. The limit of detection for the SNP detection by colorimetric and fluorescent microarrays was comparable, and the discrimination power for the selected SNPs was higher for the colorimetric microarray. The results of all diagnostic arrays were validated for the detection of extended spectrum beta-lactamase (ESBL) and carbapenemase genes using clinical isolates and standard DNA sequencing. The microarray platform developed appears to be well suited for the clinical microbiology routine. The work was supported by the RSF (Project No. 15-14-00014). - 99