Development of multiplex RAA coupled with lateral flow detection (mRAA-LFD) for rapid identification of three swimming crab species of Portunidae family

Abstract

Commercially valuable portunid crab meat products are vulnerable to economically motivated adulteration and species substitution, as the removal of exoskeletal features and subsequent processing preclude reliable morphological discrimination. This study developed a rapid multiplex recombinase-aided amplification coupled with lateral flow detection (mRAA-LFD) assay for the simultaneous authentication of Portunus pelagicus (PP), P. sanguinolentus (PS), and Charybdis feriata (CF). Species-specific primer sets were designed from polymorphic regions of the mitochondrial cytochrome c oxidase subunit I (COI) gene, producing characteristic amplicons of 147 bp (PP), 117 bp (PS), and 176 bp (CF), with integrated biotin-, TAMRA-, and Cy5-labeled primers enabling multiplex lateral flow detection. Optimal multiplex amplification was achieved at 37 °C for 30 min, followed by LFD visual detection within 5–10 min. The developed assay exhibited analytical specificity, producing positive signals only for the targeted crab species and no cross-reactivity was observed with non-target shellfish species. The detection limits were 0.4 ng/reaction by RAA and 0.2 ng/reaction by LFD for PP, 0.4 ng/reaction for PS, and 0.1 ng/reaction for CF by both RAA and LFD techniques. All thermally processed crab samples were successfully authenticated regardless of various cooking treatments. Analysis of commercial products revealed that 13 of 15 samples (86.7%) were correctly labeled as P. pelagicus, demonstrating its suitability for species authentication in complex food matrices and practical seafood traceability applications. Overall, the developed mRAA-LFD assay provides a rapid, sensitive, and equipment-light platform for on-site authentication of portunid crab species and offers a practical tool for seafood traceability, labeling verification, and food fraud prevention.