Rapid and simultaneous identification and quantification of seven commonly consumed meat species in food products using two-tube TaqMan probe-based multiplex real-time PCR assays

Accurate enforcement of food labeling regulations requires sensitive methods to detect trace animal-derived ingredients. This study reports the development of a two-tube multiplex real-time PCR assay using TaqMan probes to simultaneously identify and quantify DNA from cattle, buffalo, goat, sheep, poultry, and porcine species in food products. An endogenous 18S rRNA control (141 bp) was included to minimize false negatives and normalize data, thereby improving quantification reliability. Specificity testing against 23 non-target species showed no cross-reactivity. The assay was validated across raw, processed, and admixed samples, detecting adulteration from 10% to as low as 1% (w/w) with recovery rates of 87.33-113.72%. Its detection limit reached 0.006 ng DNA per species. Application to 49 commercial meat products revealed buffalo adulteration in 90% of beef products, while no porcine DNA was detected. Overall, the system proved sensitive, reliable, and precise, making it suitable for regulatory monitoring of meat authenticity.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s10068-026-02275-w.