DNA-based authentication for insect-based feedstuffs: The case study of Tenebrio molitor and Hermetia illucens

The rising demand for new protein sources increases the risk of fraud, particularly through misleading labelling, that can pose biosecurity hazards, such as allergic reactions in sensitised individuals. Ensuring authenticity of insect-based feed and food items is a top priority within the European Union. Addressing this need, this study developed two qPCR assays for insect species-specific detection/quantification in commercial meals, hydrolysates, and various animal feed products. These methods differentiated and quantified Tenebrio molitor (yellow mealworm) and Hermetia illucens (black soldier fly), enabling the authentication of various commercial feed and food matrices. qPCR targeting cytochrome b gene of T. molitor and NADH dehydrogenase gene of H. illucens detected DNA concentrations as low as 2 pg/mu L for T. molitor and 0.2 pg/mu L for H. illucens, identifying these insects in mixtures down to 0.24 % of inclusion in aquaculture feed. Additionally, both qPCR assays share the same thermal cycling conditions, enabling to detect T. molitor and H. illucens in a single run. In conclusion, this study demonstrates that these methods have strong analytical performance, confirming their suitability across complex matrices in food and feed applications. The qPCR offers a rapid, cost-effective tool for verifying EFSAauthorized insects in EU supply chains, from production to consumption.