A Duplex Proofman-LMTIA Assay for Rapid Detection of Shrimp, Crab, and Fish Allergens in Food Products

Seafood allergy is a major global food safety concern, with fish, shrimp, and crab representing important allergenic sources. This study combined ladder-shape melting temperature isothermal amplification (LMTIA) with proofreading enzyme-mediated probe cleavage (Proofman) technology to establish a duplex assay for the rapid and simultaneous detection of crustacean (shrimp/crab) and fish allergens. Group-specific primers and Proofman probes were designed from conserved regions of shrimp and crab tropomyosin genes and the fish mitochondrial 16S rDNA gene. The optimized reaction temperature was 61 °C, and amplification was completed within 30 min. The assay showed high specificity, with no cross-reactivity with squid, shellfish, or common livestock and poultry meats. The limits of detection for pure genomic DNA were 5 pg/μL for shrimp and fish and 10 pg/μL for crab. In model meat mixtures, the limits were 4% (w/w) for shrimp and 8% (w/w) for fish and crab. Results from 12 commercial processed foods were consistent with label declarations. Compared with the reference LAMP method specified in the Chinese Entry–Exit Inspection and Quarantine Industry Standard, the assay retained high sensitivity while enabling duplex detection and reducing nonspecific amplification, supporting its potential for rapid food safety testing.