Multiplex PCR Combined With Lateral Flow Dipstick Assay for the Species Authentication of Three Swimming Crabs

Accurate identification of commercially important crab species is crucial for avoiding seafood fraud and ensuring food safety for consumers. A novel approach for fast and effective authentication of three economically important crabs of the Portunidae family was employed in the present study. A multiplex PCR in conjunction with a lateral flow dipstick assay (mPCR-LFD) targeting the mitochondrial cytochrome C oxidase subunit I (COI) gene was developed to differentiate three common portunid crabs, including blue swimming crab, three-spotted crab, and crucifix crab. Three pairs of species-specific primers were designed with an estimated amplicon size of 183, 431, and 598 bp for Portunus pelagicus, Portunus sanguinolentus, and Charybdis feriata, respectively. The developed primers demonstrated high specificity, with no cross-reactivity between the target and closely related species. The assay showed high sensitivity by detecting DNA concentrations as low as 0.1, 0.006, and 0.01 ng for blue swimming crab, three-spotted crab, and crucifix crab, respectively. Several cooking procedures, including boiling, steaming, frying, and pressure canning, had no significant impact on the efficiency of the developed mPCR-LFD. Field assessment of samples revealed that 4 of the15 samples (26.7%) were mislabeled. The developed mPCR-LFD assay offers a rapid, sensitive, and specific tool for identifying portunid crabs, thereby enhancing food safety and combatting seafood fraud in the commercial fisheries sector.