Development of Rapid Alkaline Lysis–Polymerase Chain Reaction Technique for Authentication of Mithun (Bos frontalis) and Yak (Bos grunniens) Species

<i>Bos frontalis</i> (Mithun) and <i>Bos grunniens</i> (yak) are crucial to the culture, food security, and economy of Southeast Asia, especially in India and China, respectively. Their genetic closeness to <i>Bos indicus</i> (indicine cattle) and <i>Bos taurus</i> (taurine cattle) necessitates precise methods for meat origin authentication. This study introduces a DNA-based technique to distinguish Mithun and yak species using the alkaline lysis (AL) protocol for DNA extraction, followed by species-specific polymerase chain reaction (PCR) to amplify unique mitochondrial D-loop regions, yielding 489 bp and 422 bp amplicons, respectively. The AL-PCR method showed high specificity for both species, with no cross-amplification with other related species. The method's effectiveness was validated across various sample preparations, including raw, cooked, autoclaved, microwaved, and fried samples. The AL-PCR assay is highly sensitive, detecting as little as 1 pg of Mithun DNA and 100 pg of yak DNA, and can identify down to 0.1% of these species in binary mixtures. This approach is rapid and cost-effective, offering significant benefits for consumer protection, promoting Mithun and yak farming, and addressing food safety and traceability issues.