A Multiplex PCR-Based Assay for Authentication of Six Commercially Important Cephalopod Species

Cephalopod products are widely distributed as frozen raw materials or cut portions, making morphology-based species identification difficult during commercial handling and inspection. In this study, we developed a conventional multiplex PCR assay for the simultaneous identification of six commercially important cephalopod species, Octopus vulgaris, O. ocellatus, O. minor, Enteroctopus dofleini, Dosidicus gigas, and Todarodes pacificus. Species-specific forward primers and a shared reverse primer were designed from the mitochondrial cytochrome c oxidase subunit I (COI) region to generate distinct diagnostic amplicons within a single reaction. The assay successfully produced species-resolved bands of 459, 365, 248, 194, 141, and 82 bp for O. vulgaris, E. dofleini, O. ocellatus, O. minor, D. gigas, and T. pacificus, respectively, with no ambiguous overlap among diagnostic fragments. Clear and reproducible amplification was obtained at annealing temperatures of 51–54 °C, with 52 °C selected as the standard condition, indicating useful operational tolerance for routine application. The assay also retained consistent diagnostic performance down to 1 ng of template DNA per reaction. These results demonstrate that the developed multiplex PCR assay provides a simple, rapid, and gel-based method for the preliminary identification of selected cephalopod species in frozen commercial materials and may be useful for seafood inspection and market surveillance.