Development of a Touchdown—Duplex PCR Assay for Authentication of Sheep and Goat Meat

Authenticity in foods of animal origin like meat is an arduous task, since there are various cases of adulteration where morphological identification is not applicable. Several incidences of meat adulteration and mislabeling are being reported all over the world; hence, robust analytical techniques are required to investigate such food frauds. To provide a valid test method for simultaneous detection of closely related species like sheep and goat meat, a touchdown approach in a duplex PCR assay was planned and developed. The developed assay utilized a set of common forward primer and species-specific reverse primers targeting the mitochondrial cytochrome B (cytb) gene of both sheep and goat. The PCR assay produced amplicons of 254 bp and 462 bp respectively for sheep and goat, and no cross reactivity reported with related species such as cattle, buffalo, pig, and chicken. Using DNA mixtures of both the species, a sensitivity of 10 ng and 5 ng were reported for goat and sheep respectively. The limit of detection performed using meat mixtures of sheep and goat were reported with a relative limit of 5% for sheep meat and 10 % for goat meat. The applicability of the assay was evaluated on real-world samples procured from local retailers, which revealed a 50% mislabeling among the two species. The optimized assay can provide a valid test method for detection of sheep and goat adulteration/mislabeling/substitution.