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

Abstract

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.

Health-Related Outcomes and Molecular Methods for the Characterization of A1 and A2 Cow’s Milk: Review and Update

“…The same research group developed another accurate and specific method based on real-time PCR for directly detecting A1 and A2 alleles in milk [107]. Two years later, Watanabe et al [108] developed a highly sensitive PCR-based method capable of detecting the A1 allele in A2 milk samples without necessitating DNA isolation. The method leverages the CycleavePCR technique to directly amplify the β-casein (β-CN) gene from raw milk samples.…”