New high-sensitive rhAmp method for A1 allele detection in A2 milk samples.

Cows' milk may contain two types of β-casein: A1 and A2. A1 digestion is associated with the release of β-casomorphine-7 peptide, which can cause adverse gastrointestinal effects. Two methods - high-resolution melting (HRM) and rhAmp® SNP genotyping - were developed to identify the β-casein gene (CSN2) A1 and A2 alleles directly in milk. DNA milk samples from 45 animals were examined and 10 samples were also sequenced to confirm the accuracy of the assays. The analytical sensitivities of both strategies for A1 allele identification were evaluated by testing decreasing dilutions of A1 allele DNA copies (500 - 5 copies) in the A2 sample. The limits of detection for A1 in A2 samples were 10% (100 copies) and 2% (10 copies) for HRM and rhAmp, respectively. Both techniques were specific, differentiating between A1 and A2 alleles. However, we recommend rhAmp genotyping testing over HRM because of its enhanced sensitivity for A1.