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Intralaboratory testing of the PCR protocol for molecular genetic identification of bacteria of the genus Staphylococcus spp

The results of optimization of the Staphylococcus spp. identification protocol by polymerase chain reaction with agarose gel detection and approbation of the protocol with wild strains selected from dogs are presented. Determination of the parameters of specificity and sensitivity of the method was performed on museum strains of cocci S. epidermidis ATCC 14990, S. aureus ATCC 25923, S. aureus subsp. aureus UKM B-918, S. pneumoniae ATCC 49619 and E. faecalis ATCC 194433. DNA extraction was performed using the IndiSpin Pathogen Kit. The ready PCR mix NEB OneTaq® 2X Master Mix with Standard Buffer was used to prepare the reaction mixture. Primers targeted to the tuf gene region using an amplification product of 370 bp were used for the study. The reaction results were recorded in a 2% agronomic gel with the addition of ethidium bromide at a concentration of 0.5%. The optimal annealing temperature was determined by the temperature gradient method. In a study of the specificity of the method, three museum strains of staphylococci were identified as positive, while strains of other cocci did not give reaction products. The sensitivity study of the method was to detect the amplification product in seven dilutions of bacterial suspension that meet McFarland turbidity standards, the lowest concentration was further diluted 10, 100 and 1,000 times. The last dilution, which showed the presence of the amplification product corresponds to 2×106 CFU in 200 μl of saline used for DNA isolation. PCR protocol was tested on wild staphylococcal strains. Ear and nasal swabs of dogs, as well as washes from the transfer cage were selected for the study. The primary inoculation of the material was carried out on mannitol salt agar, on this medium only the growth of halophilic microorganisms is possible. Growth was found on 17 Petri dishes. The PCR washings of these cups indicated the presence of staphylococci in the test materials. The results of in-laboratory PCR testing indicate that the primer we used gives high indicators of specificity and sensitivity. Our tested technique can be used to confirm the presence of Staphylococcus spp. bacteria in the primary culture of smears taken from dogs.

Key words: PCR, tuf gene, approbation of primers, optimization of primers, dog microflora, Staphylococcus spp.

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