METHOD OF DETECTING DNA OF AFRICAN SWINE FEVER VIRUS BY REAL-TIME POLYMERASE CHAIN REACTION Russian patent published in 2018 - IPC C12Q1/68 

FIELD: veterinary medicine.

SUBSTANCE: invention relates to veterinary virology, specifically to means of diagnosing African swine fever (ASF). Method for detecting DNA of the African swine fever virus by real-time polymerase chain reaction comprises isolating DNA using control samples (quality control of virus isolation and control of the reaction parameters); amplification of DNA using synthetic primers and a fluorescent probe, including denaturation at 95 °C, cycling: denaturation – annealing – elongation and estimation of the result from the fluorescent signal accumulation curves for each of the given channels. Recombinant plasmid with a fragment of the vp72 gene of the African swine fever virus is used as the control of the quality of virus isolation. Recombinant plasmid with a fragment of the healthy pig tissue gene is used as the control of reaction parameters. Synthetic oligonucleotide primers and a fluorescent probe, complementary to the preserved genome region of the African swine fever virus gene vp72 region, are taken in the ratio 1:1:0.5 and have the following nucleotide composition: SEQ NO 1:5'-CTG-CTC-ATG-GTA-TCA-ATC-3' – forward primer, SEQ NO 2:5'-GAT-ACC-ACA-AGA-TCG-CCG-3' – reverse primer, SEQ NO 3:5'-FAM-CCA-CGG-GAG-GAA-TAC-CAA-CCC-AGT-G-BHQ1-3' – fluorescent probe, where BHQ1 denotes a dark fluorescence quenching agent attached to 3'-terminal nucleotide, and FAM is a fluorescent dye attached to nucleotide C. Amplification is carried out under the following conditions: denaturation at 95 °C for 90 s, then cycling with detection, comprising denaturation at 95 °C for 15 s, annealing – 60 °C – 30 s and elongation – 72 °C for 4 s. Cycle: denaturation – annealing – elongation is repeated 40 times. Then, accumulation of the fluorescent signal is measured on channels: FAM for specific signal; HEX for internal control signal; CY5 for the signal of exogenous internal control. If the accumulation curves of the fluorescent signal reaches up to 36 cycles, then the result of the reaction is considered positive, and if the curves do not cross the threshold line or cross it after 36 cycles, the result of the reaction is negative.

EFFECT: invention makes improves accuracy of detecting DNA of the African swine fever virus by real-time polymerase chain reaction.

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