METHOD FOR DETECTING AND GENOTYPING RNA OF PORCINE REPRODUCTIVE-RESPIRATORY SYNDROME VIRUS Russian patent published in 2019 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for detecting and genotyping RNA of porcine reproductive and respiratory syndrome virus, involving RNA extraction from biological material of infected pigs by a sorption method, synthesis of cDNA on the RNA matrix by staging a single-step multiplex reverse transcription reaction and polymerase chain reaction with the addition of an internal control sample based on bacteriophage MS2 and a positive control sample, consisting of an internal control sample based on bacteriophage MS2, European and American genotypes of porcine reproductive-respiratory syndrome virus, conducting 40 amplification cycles with real-time detection using oligonucleotide primer reproductive-respiratory syndrome virus virus-specific viruses, fluorescent-labeled probes and control samples, measuring accumulation of a fluorescent signal through channels of corresponding fluorescent dyes and interpreting results based on presence or absence of intersection of the fluorescence curve with the threshold line, 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 containing nucleic acid fragments of European and American genotypes of porcine reproductive and respiratory syndrome virus and a native bacteriophage MS2 genome fragment taken in ratio of 1:1:1, wherein accumulation of fluorescent signal is measured in channels: FAM/Green for a specific signal of the European genotype of porcine reproductive and respiratory syndrome virus; JOE/Yellow for a specific signal of the American genotype of the same virus and Cy5/Red for the internal control signal if the fluorescent signal accumulation curves are up to 35 cycles, then 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 reaction result is negative.

EFFECT: invention provides reliable diagnosis of porcine reproductive and respiratory syndrome and detecting various serotypes of the virus.

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