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Volume 11
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10.3390/fishes11030129
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Article

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

by
Chun Mae Dong
1,
Hee Jeong Park
1,
Eun Soo Noh
1,
Seung-Hwan Lee
2,
In Joon Hwang
1,
Jung-Ha Kang
1 and
Hyo Sun Jung
1,*
1
Biotechnology Research Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea
2
Dokdo Fisheries Research Center, East Sea Fisheries Research Institute, National Institute of Fisheries Science, Samho-ro 229, Buk-gu, Pohang 37709, Republic of Korea
*
Author to whom correspondence should be addressed.
Fishes 2026, 11(3), 129; https://doi.org/10.3390/fishes11030129
Submission received: 28 January 2026 / Revised: 19 February 2026 / Accepted: 21 February 2026 / Published: 24 February 2026
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Abstract

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.
Keywords: multiplex PCR; mitochondrial DNA; species identification; Chionoecetes bairdi; Chionoecetes opilio; Chionoecetes japonicus multiplex PCR; mitochondrial DNA; species identification; Chionoecetes bairdi; Chionoecetes opilio; Chionoecetes japonicus

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MDPI and ACS Style

Dong, C.M.; Park, H.J.; Noh, E.S.; Lee, S.-H.; Hwang, I.J.; Kang, J.-H.; Jung, H.S. Multiplex PCR Assay for the Rapid and Accurate Identification of Three Chionoecetes Species. Fishes 2026, 11, 129. https://doi.org/10.3390/fishes11030129

AMA Style

Dong CM, Park HJ, Noh ES, Lee S-H, Hwang IJ, Kang J-H, Jung HS. Multiplex PCR Assay for the Rapid and Accurate Identification of Three Chionoecetes Species. Fishes. 2026; 11(3):129. https://doi.org/10.3390/fishes11030129

Chicago/Turabian Style

Dong, Chun Mae, Hee Jeong Park, Eun Soo Noh, Seung-Hwan Lee, In Joon Hwang, Jung-Ha Kang, and Hyo Sun Jung. 2026. "Multiplex PCR Assay for the Rapid and Accurate Identification of Three Chionoecetes Species" Fishes 11, no. 3: 129. https://doi.org/10.3390/fishes11030129

APA Style

Dong, C. M., Park, H. J., Noh, E. S., Lee, S.-H., Hwang, I. J., Kang, J.-H., & Jung, H. S. (2026). Multiplex PCR Assay for the Rapid and Accurate Identification of Three Chionoecetes Species. Fishes, 11(3), 129. https://doi.org/10.3390/fishes11030129

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