FIELD: biotechnology, veterinary microbiology and genetic engineering.
SUBSTANCE: invention relates to the field of biotechnology, veterinary microbiology and genetic engineering and can be used for the detection of toxigenic Pasteurella multocida in various biological material. The method includes sample preparation, DNA isolation, real-time polymerase chain reaction with hybridization-fluorescence detection using a set of oligonucleotides for joint amplification of Pasteurella multocida kmt1 and toxA Pasteurella multocida genes, containing direct - Pm_F 5'-TGCCACTTGAAATGGGAAATG-3'; toxA_F 5'-CAGTGAAAGCACGGCTGA-3' and reverse -Pm_R 5'-AATAACGTCCAATCAGTTGCG-3'; toxA_R 5'-CATAAGCAGGAAGTTCCCAGT-3' primers and fluorescently labeled probes Pm_Z 5'-R6G-TGTGAGTGGGCTTGTCGGTAGTCT-BHQ-3'; toxA_Z 5'-Cy5-TCAGCGCCTTTTCGCGGATATGCTG-BHQ-3', and the reaction mixture. The amplification results are evaluated using the cycler software, and the results are interpreted based on the presence or absence of the intersection of the fluorescence curve with a cut-off line set at 0.05, and the presence of the genome of the bacterium Pasteurella multocida and the toxA gene is diagnosed.
EFFECT: method allows for rapid confirmation of the presence of toxigenic Pasteurella multocida variants in cultures and samples of biological material: respiratory swabs and internal organs from different types of animals: pigs, cattle, small cattle, rabbits, poultry, with high sensitivity and specificity.
1 cl, 2 dwg, 1 tbl, 2 ex
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