2024-10-232024-10-232023-01-010372-5480https://doi.org/10.24099/vet.arhiv.1821https://hdl.handle.net/11452/46933A2 milk popularity is increasing across the world and novel molecular techniques have been evaluated to develop reliable methods. This study aimed to genotype Holstein-Friesian cows concerning their A1/A2 status using Real-time PCR assay with the specifically designed FRET hybridization probes. In this context, DNA samples were obtained from 310 Holstein-Friesian milk samples. Concerning the Real-time PCR assay, the melting temperature of each amplicon was analyzed and the melting data was converted to a derivative plot using the LightCycler 480 System. The sensor probe was designed to match the wild-type sequence in the target DNA. In the Real-time PCR assay, the melting peaks obtained in the Real-time PCR assay were highly decisive and consistent for each genotype regarding CCT?CAT alteration. The results indicated a remarkably high frequency of the A2 allele (68%) and a considerable frequency of heterozygous animals (0.41). Population genetic analysis showed intermediate levels of genetic variability and biodiversity. The A2-herd conversion process is a complex process consisting of genetic testing of both cows and calves, evaluating replacement rates, and the conversion of heterozygotes by using A2-genotyped bull semen. In this sense, the key point is a reliable and rapid genotyping method to produce A1-free milk. This study suggests that Real-time PCR assay with the specifically designed FRET hybridization probes is a preferable method for A2 genotyping, and may be useful for further studies and instructive for companies or breeders who aim to produce A2 milk.eninfo:eu-repo/semantics/closedAccessQuantitative trait lociMilk-production traitsDairy-cattleVariantCsn2ProteinsGeneCattleA2 milk& beta;-caseinGenetic testingFret hybridization probesScience & technologyLife sciences & biomedicineVeterinary sciencesArticle00104417490000127928693310.24099/vet.arhiv.1821