Purpose: The purpose of this work was to develop real-time PCR assays for the detection of peanut and for the detection of walnut, the primary tree nut allergen.
Methods: Primers and probes for real-time PCR were designed to target the internal transcribed spacer (ITS) regions in the rRNA genes of the target genomes. Primer and probe design used DNA sequence data from numerous types of plants likely to be found in food products, including nontarget tree nuts, legumes, grains, and spices, in order to specifically design the assay not to cross-react with these other plant materials. Real-time PCR was carried out using six to eight target levels corresponding to approximately 0.1 to 106 parts per million allergenic food.
Results: Assay performance was evaluated using reaction efficiency, statistical R2 value, and linear range. Results demonstrate that assay linearity spanned all six to eight orders of magnitude tested. Reaction efficiencies near the ideal of 100% were achieved. In general, there was low cross-reactivity with some of the non-targeted food products tested.
Significance: Food allergies affect approximately 4 to 6% of the U.S. population, and the Food Allergen Labeling and Consumer Protection Act (FALCPA) requires that foods containing any of eight major allergenic foods and food groups be labeled accordingly. This necessitates highly sensitive detection methods. This work was focused on the development of such highly sensitive detection methods for peanut and tree nuts, two of the major food allergens in the United States.