Development of a real-time PCR system for the detection of the potential allergen fish in food

Abstract

Fish is one of the most important triggers of food-allergic reactions. Regulation (EU) No 1169/2011 governs the labelling of allergenic ingredients including fish. A real-time PCR assay, using TaqMan® probes, was applied to detect fish in food. For generating primer and probes, the Hoxc13 gene was selected. Based on the alignment of available DNA sequences from this gene from different fish species in public nucleic acid database, specific oligonucleotides were generated. To cover all relevant species of the phylogenetic class fish, the CTfish-system consists of two forward primers, three reverse primers and one TaqMan® probe. The real-time PCR method is able to detect specifically fish species belonging to the subclass Teleostei. The sensitivity is in an animal food product 100 mg/kg and in a vegetable food matrix 10 mg/kg relating to the fresh weight. The calculated limit of detection (LOD95 %) of fish DNA in background DNA is 2.5 copies. The method developed is robust against small, deliberate changes in the reaction conditions. The CTfish-system can be used to sensitively and selectively identify the presence of fish DNA in food.