Triplex Proofman-LMTIA: A Rapid, Specific, and Sensitive Assay for Detecting Wheat, Peanut, and Soybean Allergens in Foods

Wheat, soybean, and peanut are recognized as major food allergens, with their prevalence rising globally, necessitating rapid and reliable detection methods. A new detection approach was developed in this research, which integrates Ladder-shape Melting Temperature Isothermal Amplification (LMTIA) with Proofreading Enzyme-Mediated Probe Cleavage (Proofman) technology to enable the concurrent identification of wheat, soybean, and peanut allergens. Compared with the loop-mediated isothermal amplification (LAMP) method under the experimental conditions set in this study, this approach can reduce the false-positive results associated with LAMP, and it does not rely on sophisticated instrumentation required by technologies like mass spectrometry. The GAG56D (wheat), Ara h 2.01 (peanut), and Lectin (soybean) genes were selected as target genes for the three allergens. Specific primers and probes were designed according to these target genes, and the reaction system was optimized. A systematic evaluation of the triplex Proofman-LMTIA method was then conducted regarding its specificity, sensitivity, limit of detection, and repeatability. Finally, the method’s practical applicability was validated using commercial products. The optimized system achieved simultaneous detection within 40 min at 61 °C, showing no cross-reactivity with common foods. The method demonstrated good sensitivity, with a sensitivity of 5 pg/μL for genomic DNA and a detection limit of 5% (w/w) in a powder matrix, along with excellent repeatability. In practical sample testing, the results were fully consistent with product label declarations, accurately identifying single and multiple allergen contaminations. The Proofman-LMTIA detection method, with its rapid, simple, sensitive, and specific characteristics, demonstrates significant potential for applications in food safety supervision.