An ARMS-integrated high-resolution melting assay for reliable discrimination of the A1/A2 polymorphism in the bovine CSN2 gene

Practical, rapid, and cost-efficient screening tools are needed to verify A2 milk purity and support selective breeding programs. Although several genotyping methods are available, their routine implementation is often limited by equipment costs and labor-intensive post-PCR processing. We hypothesized that combining allele-specific primer design with High-Resolution Melting (HRM) analysis would enhance the sensitivity of CSN2 A1/A2 discrimination by generating allele-specific amplicons with more clearly separated melting profiles than conventional HRM, which relies on the subtle melting difference associated with a single nucleotide polymorphism. To test this, we developed an ARMS-integrated HRM assay targeting the CSN2 c.245C > A variant. The assay produced distinct melting patterns that consistently discriminated A1A1, A1A2, and A2A2 genotypes across blood, hair follicle, and milk DNA samples without post-PCR electrophoresis. Application to commercial milk products further demonstrated its potential as a product-level screening tool. In complementary mixture experiments, qPCR showed high linearity (R 2 = 0.9930) in predefined DNA mixtures and detected minor A1-derived signal at 5% in milk mixtures, although low-level bias indicated semi-quantitative rather than absolute performance. Overall, the results support the usefulness of this ARMS-integrated HRM approach for practical discrimination of the CSN2 A1/A2 polymorphism and for rapid closed-tube A2 milk screening and breeding-oriented genotyping, although broader analytical validation is still needed for routine quantitative authentication. • A1 admixture was detected at 5% in controlled milk-mixture experiments. • Turnaround time was reduced from 6.0 h to 2.5 h versus AS-PCR. • Genotype calls were concordant across blood, hair follicles, and milk. • Per-sample reagent costs were reduced by omitting gel electrophoresis. • The assay enabled routine screening of A2-labelled milk products.