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Seafood substitution poses a significant threat to the global seafood market, driven by increasing consumer demand, and fraudulent practices like species mislabeling. Among seafoods, crustaceans are valuable commodities and are particularly vulnerable to substitutionary activities due to their morphological similarities. This study focuses on developing rapid and accurate identification methods to combat seafood fraud. To address this, we developed species specific C-LAMP and mPCR-LFD assays targeting the COI gene of three mussel species (Perna canaliculus, Mytilus galloprovincialis, and Perna viridis) and three crab species (Portunus pelagicus, Portunus sanguinolentus and Charybdis feriata). The C-LAMP and mPCR - LFD assays conditions were optimized by varying the temperature and time. The C-LAMP assay demonstrated high accuracy at 63 °C for 30 mins. The C-LAMP exhibited high sensitivity for P. viridis and P. canaliculus, with limits of detection of 0.003 and 0.01 ng/ reaction, respectively. Similarly, the mPCR-LFD assay achieved high sensitivity, detecting DNA concentrations as low as 0.1 ng, 0.006 ng, and 0.01 ng for blue swimming crab, three spotted crab and crucifix crab, respectively. Further, in-house validation using various processing methods (boiling, steaming, frying, and canning) using the developed assay demonstrated high sensitivity and robustness. Out-house validation of crab species revealed 28 % of mislabeling rate. Therefore, the C-LAMP and mPCR-LFD assay developed in this study is simple, rapid, and cost-effective for authenticating seafood products. Hence, these novel molecular techniques will offer a promising solution for routine fishery examinations to stakeholders, industry people, and regulatory authorities, which contributes to seafood safety and authenticity.
