Rendered fats are vulnerable to species adulteration and also to low-level accidental species cross-contamination which would exclude them from some religious diets. Species verification testing is difficult, particularly techniques that can be used in an in-process setting. They lack obvious morphological markers (e.g., adipocyte size and distribution, connective tissue architecture, and species-specific fat coloration). Conventional quantitative PCR (qPCR) suffers from matrix inhibition and amplification bias in lipid-rich matrices unless expensive matrix-matched Certified Reference Materials are available for each run.
This paper (open access) reports the inter-laboratory validation of a precalibrated droplet digital PCR (ddPCR) screening method which avoids these problems. Species-specific biological coefficients were established from pure fats and interspecies signal conversion factors from aqueous-phase recovery experiments.
Validation was across five laboratories and four ddPCR platforms.
The authors report that the method achieved acceptable reproducibility and recoveries at adulteration 5–50% (w/w) but was semi-quantitative at lower concentrations corresponding to accidental contamination. Near the limit of quantification, porcine at 5% and equine at 10% showed systematic positive bias, requiring semi-quantitative reporting; ovine at 10% showed negative bias 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.
The authors conclude that this screening-level approach supports monitoring of critical control points (CCPs) in rendering facilities, and also regulatory screening testing at customs inspection stations.
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