METHOD FOR DETECTING RNA OF SCHMALLENBERG DISEASE VIRUS IN FARM ANIMALS Russian patent published in 2019 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for extracting RNA from biological material by choice: whole blood, blood serum, tissue and organ fragments by sorption method, synthesis of cDNA on RNA matrix by single-step staging with addition of internal and positive control samples of multiplex reverse transcription reaction and polymerase chain reaction – with 45 amplification cycles with real-time detection using oligonucleotide primers of the Schmallenberg virus specific for the region of the genome of the fluorescent-labeled probe and control samples, measuring accumulation of a fluorescent signal through channels of corresponding fluorescent dyes and interpreting results based on the presence/absence of intersection of the fluorescence curve with the threshold line Threshold, according to the invention for internal control sample suspension of bacteriophage MS2 with concentration of 5 × 10³ copies of nucleotide sequences per 1 mcl, and for a positive control sample, a mixture of recombinant plasmid DNA containing a fragment of the genome of the Schmallenberg virus exciter and a fragment of the bacteriophage MS2 genome taken in ratio 1:1, with the following nucleotide sequences: Shm-F 5 'TCAGATTGTCATGCCCCTTGC 3' – forward primer; Shm-R 5' TCGGCCCCAGGTGCAAATC 3' – reverse primer; Shm-Z JOE-ACCATCGGCCCAGGTGCATCCCTTA-BHQ1 – probe; MS2F, 5'-TGGCACTACCCCTCTCCGTATTCAC-3 – forward primer; MS2R, 5'-GTACGGGCGACCCCACGATGAC-3' – reverse primer; MS2P, 5'-Cy5-CACATCGATAGATCAAGGTGCCTACAAGC-3' BHQ2 – probe, wherein accumulation of fluorescent signal is measured in channels: JOE/Yellow for specific signal of Schmallenberg virus agent; Cy5/Red for the internal control signal, if the fluorescence signal accumulation curves are up to 35 cycles, the result of the 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.

EFFECT: invention enables higher diagnostic accuracy in the method for detecting RNA of the Schmallenberg disease virus in farm animals.

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