An optimized rapid assay for dual detection of peanut and soybean allergens in food

Rapid and accurate detection of allergenic components, such as peanuts and soybeans, in food products is crucial for ensuring the dietary safety of individuals with allergies. In this study, a rapid dual detection method for peanut and soybean allergens was established based on ladder-shape melting temperature isothermal amplification (LMTIA) combined with proofreading enzyme-mediated probe cleavage (Proofman). Specific primers and Proofman probes targeting the peanut Ara h 2.01 gene and the soybean lectin gene sequences were designed. This method facilitates simultaneous detection of peanut and soybean allergens in foods within 30 minutes. The detection sensitivity of the method for genomic DNA (gDNA) of peanuts and soybeans reached 5 pg µL^-1, and the minimum detectable limit for allergenic peanut (or soybean) components in non-allergenic matrices was 1%. The designed Proofman-LMTIA method has excellent specificity, and the detection results for the 9 categories of commercial food samples were completely consistent with the allergen labels on the products. Furthermore, this study verified the detection sensitivity of the method by comparing it with the standard singleplex LAMP method, and further confirmed its practical application feasibility through the detection of actual samples. The dual Proofman-LMTIA method established in this study enables rapid and accurate identification of peanut and soybean allergens in food products, which will facilitate market supervision of food allergens.