Detection and quantification of the canine DNA in meat DNA mixes and meat mixtures by real time PCR and digital droplet PCR

Accurate detection of animal species in meat products is essential for food safety, regulatory compliance, and the prevention of food fraud. This study presents the development and validation of a modified quadruplex quantitative PCR (qPCR) assay and singleplex droplet digital PCR (ddPCR) for the detection and quantification of canine DNA in meat and DNA mixtures. Two- and three-species samples containing canine, pork, and chicken components (0.1 %–100 %) were prepared, including heat-treated variants. A simplified quantification approach using a single 100 % DNA reference diluted to 10 ng/µL was applied in both methods. Results from qPCR and ddPCR were statistically evaluated using ANOVA, RMSD, and coefficient of variation (CV). Both methods yielded accurate results across most concentration ranges, with no significant differences in calculated percentages. However, qPCR exhibited significantly higher variability than ddPCR, especially at extreme concentrations (<1 % and >99 %). ddPCR showed superior performance in three-species mixtures and yielded lower RMSD values overall. Despite higher variability at detection limits inherent to PCR technology, both methods are suitable for routine quantification of canine DNA in meat products. The study confirms that a single-reference standard is a cost-effective and reliable alternative to full calibration curves for species quantification.