Rapid Screening of Species Adulteration in Rendered Fats by Precalibrated Droplet Digital PCR

Rendered fats are highly susceptible to species mislabeling because processing removes visual markers, threatening Halal/Kosher compliance and food safety management. We developed a precalibrated droplet digital PCR (ddPCR) screening method using operational quantification coefficients K, eliminating routine dependence on scarce matrix-matched certified reference materials for fresh or once-frozen-thawed fats. Species-specific biological coefficients t_i were established from pure fats (n = 23), and interspecies signal conversion factors CF from aqueous-phase recovery experiments. Validated across five laboratories and four ddPCR platforms, the method achieved interlaboratory RSD_R of 2.88-8.58% at 5-50% (w/w) mass fractions and mean recoveries of 99.45-101.71% at validated levels (H2-H5). Near the limit of quantification (LOQ), porcine at 5% and equine at 10% showed systematic positive bias (recoveries 140.64% and 121.22%), requiring semi-quantitative reporting; ovine at 10% showed negative bias (85.02%) and requires confirmatory testing in interlaboratory application. In-house LOQs were 5% (porcine) and 10% (bovine, equine, ovine); interlaboratory reporting limits were 5% (porcine), 10% (equine), and 20% (bovine, ovine). Analysis takes 2.5 h per sample. In blind testing of six samples, species were correctly detected and quantified in all five commercial fats; a plant-based control yielded no signal, falling outside the validated scope. This screening-level approach supports HACCP (Hazard Analysis and Critical Control Points)-based monitoring at critical control points (CCPs) in rendering facilities and customs inspection stations.