Laboratory-Specific Comparison of KASP and a Locally Implemented Gel-Based T-ARMS PCR Workflow for Targeted CSN2 Codon-67 Genotyping in a Holstein–Friesian Panel

Targeted CSN2 codon-67 genotyping supports A2-oriented breeding and herd management. We compared Kompetitive Allele-Specific PCR (KASP) with a locally implemented gel-based tetra-primer amplification refractory mutation system PCR (T-ARMS PCR) workflow in a balanced, non-population-representative Holstein–Friesian panel using re-audited Sanger-supported reference classifications. Both assays distinguish His67-associated (A1-type) from Pro67-associated (A2-type) β-casein classes rather than complete CSN2 alleles. The paired analysis included 96 independent samples. KASP produced 90 reference-concordant, four reference-discordant, and two no-call outcomes (93.8% all-record reference-concordant yield), whereas T-ARMS PCR produced 77 reference-concordant, six reference-discordant, and 13 ambiguous outcomes (80.2%). Concordance among callable/evaluable results was 95.7% and 92.8%, respectively. In the primary comparison, KASP showed a 13.5 percentage point higher yield (95% CI, 4.2–22.9 percentage points; p = 0.015), mainly because it generated fewer non-interpretable outcomes. All callable KASP discordances involved Pro67/Pro67 reference samples called His67/Pro67. Because no independent DNA dilution, new DNA extraction, repeat KASP run, or new Sanger sequencing was performed, their cause and reproducibility remain unresolved. Formal repeatability and between-run reproducibility were not systematically evaluated; therefore, this comparison is not a formal assay validation and does not define T-ARMS PCR performance beyond the tested local configuration. Discordant, ambiguous, or high-impact classifications require independent repeat testing or sequencing confirmation.