A Novel Normalized Quantitative Real-Time PCR Approach for Ensuring Roe Deer (Capreolus capreolus) Meat Authenticity in Game Meat Foods

Roe deer meat is a prized game product in many European countries. However, concerns exist regarding the accuracy of the amount of declared roe deer in processed game meat foods. This study aimed to develop a reliable method for the detection and quantification of roe deer in commercialized game meat products. A TaqMan probe-based quantitative real-time PCR (qPCR) assay was designed, targeting a single-copy 120-bp region of the roe deer agouti signaling protein (ASIP) encoding gene. The method employed the normalized ∆Cq approach to establish a calibration curve for roe deer detection and quantification within 0.05-50% (<i>w</i>/<i>w</i>) in complex raw and processed matrices. The method proved to be specific for roe deer identification, achieving limits of detection and quantification of 0.04 ng of roe deer DNA and 0.05% (<i>w</i>/<i>w</i>) of roe deer in simulated pâté. Following validation with blind samples, highlighting the precision and trueness of the approach, the assay was applied to 46 market samples from four European origins (Poland, Portugal, France, and Spain). The analysis revealed significant discrepancies between declared roe deer content and actual levels in all roe deer labeled products. The global analysis of results, combining the previous survey on red deer species with present roe deer data, identified 61% of mislabeled/adulterated samples due to the absence of deer species, substitution of roe deer with red deer, substitution of fallow deer with other deer species and red deer with pork, and undeclared addition of roe deer. This study demonstrates the effectiveness of the developed qPCR method for accurate roe deer meat authentication in foods, showing its usefulness as a tool for routine food inspection to ensure labeling compliance.