METHOD FOR DETECTING A GENOME OF A BRUCELLA INFECTION AGENT (BRUCELLA SPECIALES) IN FARM ANIMALS

摘要

FIELD: biotechnology.;SUBSTANCE: invention represents a method for detecting a genome of a brucellosis infection agent (Brucella spp.) in agricultural animals, involving recovering DNA from biological material from infected animals by a sorption method, staged one-stage polymerase chain reaction – with simultaneous amplification cycles with real-time detection using oligonucleotide primers specific for the genome region (Brucella spp.), probes, dyes and control samples in form of internal and positive, measurement of specific signal and control signal through channels of corresponding dyes and interpretation of results, where fluorescent detection is carried out, measuring a JOE(HEX)/Yellow accumulation of a fluorescent signal for a specific signal for testing the genome of the brucellosis infection agent (Brucella spp.), and FAM/Green channel – signal of internal control sample, if fluorescence signal accumulation curves are up to 35 cycle, result of reaction is considered positive, and if the curves do not cross the threshold line or cross it after 35 cycles, the result of the reaction is negative, and the interpretation of the results is carried out based on the presence/absence of the intersection of the fluorescence curve with the threshold line, wherein for internal control sample suspension of bacteriophage T4 with concentration of 5×10³ copies of nucleotide sequences at 1 mcl, and for a positive control sample a mixture of recombinant plasmid DNA containing a DNA fragment of Brucella spp. DNA is used and a fragment of bacteriophage T4 genome, taken in ratio 1:1, with the following nucleotide sequences: B7F TGAAGCTGCCTGCATCGGTC – forward primer, B7R CATAATGGCCGGGTGTTGGCT – reverse primer, B7P HEX-CAACAGCATGCAGCTTGGTCGTCAATC-BHQ1 – probe, T4F TACATATAAATCACGCAAAGC – forward primer, T4R TAGTATGGCTAATCTTATTGG – reverse primer, T4P FAM ACATTGGCACTGACCGAGTTC – probe.;EFFECT: invention enables reliable diagnosis of the Brucella speciales DNA exciter.;1 cl, 3 dwg, 4 tbl