Development of a multi-DNA barcode screening method for plant-derived allergen species discrimination

Plant-derived allergens, such as peanuts, sesame seeds, nuts, and cereals, are frequent triggers of food allergies. DNA barcoding enables rapid and accurate multi-species identification. This study systematically evaluated four DNA barcodes —ITS2, matK , rbcL , and psbA-trnH —to identify the most effective one for allergen detection. Validation with commercial food products confirmed the practical applicability of the selected barcode. Among the evaluated barcodes, ITS2 performed best, with 100 % success in both amplification and sequencing. In contrast, psbA-trnH had the lowest sequencing success (80.77 %). ITS2 also showed a distinct barcoding gap in genetic distance analysis, unlike the overlapping ranges of other barcodes, confirming its superior reliability for identifying plant-derived allergenic species. When applied to commercial foods, the ITS2-based method achieved 98.44 % identification accuracy (63/64). It is noteworthy that 53.13 % (34/64) of samples lacked allergen labeling. ITS2 offers a robust and applicable approach for detecting plant-derived allergens in commercial products. • Plant-derived allergenic species encompass a broad spectrum of 26 allergens. • Systematic screening identified optimal DNA barcodes for target allergens detection. • ITS2 barcodes enable precise differentiation among allergenic species. • The method can be used to detect commercially available foodstuffs.