Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR)

•Mitochondrial DNA content per cell varies at least fivefold between tissues.•Targeting mitochondrial DNA for species quantification is unsuitable.•Droplet digital PCR is advantageous over qPCR in food quality assurance and control.•DdPCR shows limits of quantification and detection of 0.01% and 0.001%, respectively.•No cross-reactivity was detected in 14 different species using the nuclear F2 gene. Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (−70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems.