METHOD FOR DETECTING DNA OF PASTEURELLA MULTOCIDA AGENT IN BIOLOGICAL MATERIAL OF ANIMALS AND FODDERS USING POLYMERASE CHAIN REACTION IN REAL TIME Russian patent published in 2024 - IPC C12Q1/68 

FIELD: veterinary microbiology.

SUBSTANCE: method is developed for detecting DNA of pasteurellosis agent (Pasteurella multocida) in animals by polymerase chain reaction. Method is implemented by DNA amplification with fluorescent detection in "real time" mode. DNA of the infectious agent is recovered by sorption from animal materials: blood, blood serum, fragments and suspensions of parenchymal organs, lymph nodes, milk from the affected lobes of the udder, washings from the nasal mucosa, as well as fodders, cultures of microorganisms, washings from surfaces. Further, a single-step polymerase chain reaction with fluorescent detection is carried out with 40 real-time amplification cycles. Oligonucleotide primers, probes, fluorescent dyes are used specific for the DNA genome of the infectious disease agent. Internal control sample in the form of a suspension of T4 bacteriophage with concentration of 5×10³ phage particles per 1 mcl and a positive control sample in the form of a mixture containing in the same volume ratio fragments of genomes of an infectious agent of an animal and bacteriophage T4 with a nucleotide sequence: T4F: 5´-TACATATAAATCACGCAAAGC-3´, T4R: 5´-TAGTATGGCTAATCTTATTGG-3´, T4P: CY5-5´-ACATTGGCACTGACCGAGTTC-3´-BHQ1, for a positive control sample, a fragment of the genome of the Pasteurella multocida agent with the following nucleotide sequence 5´–3´ is used: Pm-F: 5´-AACGTCCAATCAGTTGCGCC-3´, Pm-R: 5´-AGTGGGCTTGTCGGTAGTC-3´, Pm-P R6G-5´-CTCAACACACCAAACTCTGCCCAAC-3´-BHQ1.

EFFECT: broader functional capabilities and high accuracy of detecting pasteurellosis agent (Pasteurella multgbiSa).

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