METHOD FOR DIAGNOSING PORCINE ENZOOTIC PNEUMONIA BY POLYMERASE CHAIN REACTION Russian patent published in 2019 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. Invention is a method for diagnosis of porcine enzootic pneumonia by a polymerase chain reaction involving PCR reaction using a reaction mixture, primers to a Mycoplasma hyopneumoniae genome fragment using a thermocycler with preset temperature and time parameters of PCR, after amplification, the result is analyzed by electrophoresis in 2 % agarose gel using 50-fold TAE-buffer containing ethidium bromide in final concentration 1.2 mg/ml, and taking into account positive samples in ultraviolet light at wave length 254 nm, where the PCR reaction is carried out in one round, the target is a fragment of the gene of the specific surface protein p102 Mycoplasma hyopneumoniae, as a reaction mixture per one PCR reaction 15.25 mcl of a single reaction buffer containing 1.5 mmol MgCl₂; 1.0 mcl (10 pmol) of forward primer p102F; 1.0 mcl (10 pmol) of reverse primer p102R; 2.5 mql of dNTP (0.25 mmol of each dNTP); 0.25 mql (1.25 units) Taq-polymerase and 5.0 mcl of the analyzed DNA; as primers to fragment of gene of specific surface protein p102 Mycoplasma hyopneumoniae take one pair of primers – straight P102F 5'-AGACTCGCTTTTGATGCAACC-3', reverse P102R 5-AGGCTTTGGAAATACAGAACAAGC-3'; setting the following temperature and time parameters to the PCR amplification device: preliminary heating of the device ("pause") – 95 °C; primary denaturation of DNA – 95 °C – 5 minutes – 1 cycle; denaturation of DNA – 95 °C – 30 sec, hybridization of primers – 60 °C – 30 sec, elongation of complementary chains – 72 °C – 30 s, 40 cycles; final elongation 72 °C – 5 min - 1 cycle; positive samples are considered to be detected fragment with size of 201 base pairs.

EFFECT: invention enables to detect a Mycoplasma hyopneumoniae genome fragment in one round with a low probability of false-positive results.

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