METHOD OF IDENTIFYING DNA OF UREAPLASMA DIVERSUM BACTERIA IN BOVINE SEMEN BY REAL-TIME PCR Russian patent published in 2022 - IPC C12Q1/68 C12N15/00 

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, veterinary microbiology and genetic engineering and can be used to detect the microorganism Ureaplasma diversum in various biological material. Method involves sample preparation, DNA extraction, polymerase chain reaction with hybridization-fluorescent detection in real time using a set of oligonucleotides complementary to 16 S ribosomal RNA Ureaplasma diversum region containing forward (Udv_F5'-CATTTACTTGCATGAGTGAATG-3') and reverse (Udv_R5'-AGCTACGCGTCAATGAC-3') primers and fluorescent-labeled probe (Udv_Z5'-(ROX)-TGGATGAGGGTGCGACGTATCATCC-(BHQ)-3'), and a reaction mixture. Amplification is carried out according to the following program: 15 min at 95 °C; 10 s at 95 °C, 20 s at 60 °C, 10 s at 72 °C (5 cycles without fluorescent signal detection); 10 s at 95 °C, 20 s at 55 °C, 10 s at 72 °C (35 cycles with fluorescent signal detection on ROX/Orange channel). Amplification results are evaluated using the amplifier software, wherein the results are interpreted based on the presence (or absence) of the fluorescence curve intersection with the threshold line set at 0.05, and the presence of the genome of the Ureaplasma diversum bacterium is diagnosed.

EFFECT: invention enables to detect DNA of Ureaplasma diversum in sperm products intended for artificial insemination of cattle with high specificity and sensitivity.

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