Effect of thermal processing on the performance of the novel single-tube nested real-time PCR for the detection of walnut allergens in sponge cakes

•Single-tube nested real-time PCR as a high sensitive assay to trace walnut DNA•Detection of Juglans regia, nonspecific LTP mRNA, encoding for the Jug r 3 allergen•Comparison of conventional real-time PCR with single-tube nested real-time PCR•Assessment of assay performance to detect walnut in batter and cake.

Walnut is commonly included as an ingredient in bakery products, being widely appreciated by many consumers. However, walnut is also classified as an allergenic food, representing a potential risk for the health of sensitised individuals when present in processed foods as a hidden allergen. In the present work, we developed a single-tube nested real-time PCR system to trace walnut in bakery products and assessed the effect of heat treatment on the performance of the technique. The proposed approach, targeting the sequence encoding for the Jug r 3 allergen, enabled lowering the relative limit of detection (LOD) of the conventional real-time PCR assay from 0.005% to 0.001% of walnut in both batter and sponge cakes. The absolute LOD was 1 pg of walnut DNA (1.6 DNA copies) in both mixtures, evidencing that the performance of the method was not affected by the heat processing. To our knowledge we proposed for the first time a single-tube nested real-time PCR system as a cost-effective, robust and powerful tool for high-throughput DNA-based detection and quantification of walnut allergens in raw and processed foods.