METHOD FOR SIMULTANEOUS GENE DIAGNOSIS OF FOUR ALLELES OF BETA-CASEIN IN CATTLE AND TEST SYSTEM FOR ITS IMPLEMENTATION Russian patent published in 2024 - IPC C12Q1/68 

FIELD: biotechnology.

SUBSTANCE: invention can be used in veterinary practice and zootechnics for diagnosis of 4 alleles of beta-casein (CSN2^A1, CSN2^B, CSN2^A2, CSN2^A3). Disclosed is a method for simultaneous gene diagnosis of 4 alleles of beta-casein: CSN2^A1, CSN2^B, CSN2^A2, CSN2^A3 in cattle and a test system for implementing the method. DNA is extracted from the biological material, a real-time polymerase chain reaction is carried out using three reaction mixtures containing a diluent, Taq DNA polymerase, primers having the following sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, *rev—reverse sequence is used, three positive and one negative control samples for each reaction mixture. When preparing for the reaction, the required volume of components is calculated based on the number of analyzed samples plus 4, the reagents are mixed, then 20 mcl of the prepared PCR mixture is added to the tubes prepared for PCR, 5 mcl of the control and analyzed samples are added to each PCR test tube, the tubes are placed in an amplifier, with the number of amplification cycles equal to 40. Analysis of the obtained data is carried out by comparing the amplified sections of genes, results are interpreted based on the presence or absence of intersection of the fluorescence curve with the threshold line set at the corresponding level; interpretation of the analysis results of the analyzed samples is carried out in accordance with Table 5.

EFFECT: invention enables simultaneous gene diagnosis of four mutant alleles of beta-casein: CSN2^A1, CSN2^B, CSN2^A2, CSN2^A3 in cattle; reduces time of PCR-RT.

2 cl, 5 tbl