t-value raw data of ddPCR for Quantification of Meat Species in Rendered Animal Fats

Rendered fats are highly susceptible to species mislabeling due to processing-induced loss of visual markers, threatening religious dietary compliance (Halal/Kosher) and food safety management systems. Current quantitative methods require scarce matrix-matched certified reference materials (CRMs), limiting routine surveillance in resource-limited settings. This study develops a fit-for-purpose screening method using pre-calibrated duplex ddPCR with empirical correction coefficients (K-factors) to enable rapid, cost-effective species authentication without routine CRM dependency. Species-specific biological coefficients (t) were predetermined from pure fats (n=23), and matrix correction factors (CF) were established through recovery experiments. The method was validated across five laboratories using four independent ddPCR platforms. For adulteration levels ≥10% (w/w), mean recoveries ranged 96.8–107.8% with inter-laboratory RSD <7.5%. Mass fraction limits of quantification (LOQ) were 5% (bovine) and 10% (equine/ovine/porcine), suitable for regulatory screening. Analysis time was 2.5 hours per sample. In blind testing of commercial samples (n=8), the method correctly identified all declared and undeclared species without false positives. This screening-level approach supports HACCP-based CCP monitoring and enables rapid corrective actions.