Acta NaturaeActa NaturaeActa Naturae2075-8251Acta Naturae Ltd1078210.32607/20758251-2010-2-3-101-109ArticlesСтатьиResearch ArticleOligonucleotide Microarray for the Identification of Carbapenemase Genes of Molecular Classes A, B, and DOligonucleotide Microarray for the Identification of Carbapenemase Genes of Molecular Classes A, B, and DUlyashovaM M

Chemistry Faculty

KhalilovaYu I

Chemistry Faculty

RubtsovaM Yu

Chemistry Faculty

mrubtsova@gmail.comEdelsteinM VAlexandrovaI AEgorovA M

Chemistry Faculty

Lomonosov Moscow State UniversityZAO 'NPP IMMUNOTEK', (NPP IMMUNOTECH, JSC)Institute of Antimicrobial Chemotherapy, Smolensk State Medical AcademyBurdenko Institute of Neurosurgery, Russian Academy of Medical SciencesLomonosov Moscow State University1509201023VOL 2, NO3 (2010)ТОМ 2, №3 (2010)10110917012020Copyright ©; 2010, Ulyashova M.M., Khalilova Y.I., Rubtsova M.Y., Edelstein M.V., Alexandrova I.A., Egorov A.M.Copyright ©; 2010, Ulyashova M.M., Khalilova Y.I., Rubtsova M.Y., Edelstein M.V., Alexandrova I.A., Egorov A.M.2010Ulyashova M.M., Khalilova Y.I., Rubtsova M.Y., Edelstein M.V., Alexandrova I.A., Egorov A.M.Ulyashova M.M., Khalilova Y.I., Rubtsova M.Y., Edelstein M.V., Alexandrova I.A., Egorov A.M.https://creativecommons.org/licenses/by/4.0https://actanaturae.ru/2075-8251/article/view/10782

This work is a report on the development of a method of hybridization analysis on DNA microarrays for the simultaneous identification and typing of carbapenemase-encoding genes. These enzymes are produced by the microorganisms that are responsible for causing infectious diseases. The method involves several steps, including DNA extraction from clinical samples and amplification of carbapenemase genes by multiplex PCR with simultaneous labelling by biotin. Following that, hybridization of the labeled PCR products with oligonucleotide probes immobilized on the surface of a nitrocellulose-based DNA microarray occurs. The biotin molecules attached to the DNA duplexes are detected by using conjugates of streptavidin-horseradish peroxidase, which is then quantified by colorimetric detection of the enzyme. We have designed the required oligonucleotide probes and optimized the conditions of the membrane microarray-based hybridization analysis. Our method allows to identify 7 types of carbapenemase genes belonging to the molecular classes A, B, and D, and it also allows additional typing into genetic subgroups. The microarrays have been tested with the control strains producing the carbapenemase genes which have been characterized by sequencing. The developed method of hybridization analysis was employed to investigate clinical strains of Pseudomonas spp. and Acinetobacter spp., which produce carbapenemases of different classes based on phenotypic testing. All strains of Acinetobacter baumanii resistant to carbapenems were producers of two carbapenemase OXA-type genes (OXA-51, in combination with OXA-23 (1 strain), OXA-40 (5 strains), or OXA-58 (4 strains)). The metallo-β-lactamase VIM-2 type gene was detected in all Pseudomonas aeruginosa strains resistant to carbapenems. Testing of carbapenem-sensitive strains did not detect any carbapenemase genes. The microarray method for the identification of carbapenemase genes is very accurate and highly productive. It can be employed in clinical microbiological laboratories for the identification and study of carbapenemase epidemiology.

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