Multiplex PCR Assay for the Rapid and Accurate Identification of Three Chionoecetes Species

In this study, a multiplex PCR assay was developed for the rapid and accurate identification of three Chionoecetes species (Chionoecetes bairdi, C. opilio, and C. japonicus) available in global seafood markets. The morphological similarity between imported female C. bairdi from Japan and native C. opilio in Korea complicates visual discrimination and raises concerns over potential mislabeling. To address this issue, mitochondrial cytochrome c oxidase subunit I (COI) gene sequences were analyzed to identify species-specific SNPs, and primers were designed accordingly. Singleplex PCR confirmed species-specific amplification among the three target species, and the optimal annealing temperature was determined. The multiplex PCR simultaneously amplified distinct fragments of 598 bp (C. bairdi), 401 bp (C. opilio), and 194 bp (C. japonicus), with no nonspecific amplification or primer–dimer formation. Sensitivity testing revealed a detection limit of 0.01 ng/µL for all three species, defined as the lowest DNA concentration at which species-specific bands were consistently observed in at least two out of three replicates. These results demonstrate that the developed multiplex PCR is a reliable, rapid, and cost-effective tool for accurate species identification, supporting sustainable resource management, preventing seafood fraud, and ensuring safe distribution in both Korea and global seafood markets.