A highly sensitive PCR method for A1 allele detection in A2 milk samples without DNA isolation.

We previously developed a genotyping method to detect the A1 and A2 alleles of the bovine β-casein gene. This method required DNA extraction from hair samples. Recently, demand for A2 milk (milk from cows homozygous for the A2 allele) has increased, and dairy farms are required to have certification to produce A2 milk. Here, we describe the development of a new, simple, and sensitive genotyping method for the β-casein gene that does not require DNA extraction. This method uses the CycleavePCR technique and can amplify the β-casein gene directly from raw milk samples. Genotypes obtained from the milk samples (n = 27) were completely coincident with those obtained from genomic DNA. In addition, this method could quantify the A1 allele in the milk samples. The limit of detection for the A1 allele in A2 milk was 2%. The copy numbers of the A1 allele corresponding to the 2% detection limit were estimated to be 30.5 ± 24.3 molecules/μL. These findings indicate that this new genotyping method is simple and fast for detecting the A1 allele in milk samples and can therefore be potentially used to certify A2 milk.