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

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.

1. Introduction

The genus Chionoecetes (snow crabs) belongs to the phylum Arthropoda, subphylum Crustacea, order Decapoda, and family Oregoniidae, and is distributed across a wide bathymetric range from continental shelf to slope and bathyal zones in the North Pacific [1]. Seven species have been reported within this genus. Among them, Chionoecetes bairdi occurs primarily on the continental shelves of the eastern North Pacific, particularly in the Bering Sea and the Gulf of Alaska, where it supports important commercial fisheries [2,3]. C. opilio inhabits continental shelf and upper slope environments and is widely distributed throughout the North Pacific, including the Bering Sea and the Sea of Okhotsk [4,5]. In contrast, C. japonicus is typically associated with deeper continental slope habitats and is mainly reported to be in the East Sea and adjacent waters [6]. Other species, including C. tanneri, C. angulatus, C. pacificus, and C. elongatus, are generally reported from deeper slope environments of the North Pacific region [7,8]. These Chionoecetes species constitute valuable commercial resources and major commodities in the international seafood market, highlighting the need for effective management measures to ensure their sustainable use [5].

For C. opilio resources in Korea, systematic catch control began with the pilot introduction of the Total Allowable Catch (TAC) system in 1999 and its full implementation in 2002 for coastal gillnet and pot fisheries [9]. Despite TAC adoption, catches in Korea peaked at 4817 tons in 2007 but have steadily declined, resulting in unmet demand, averaging about 2184 tons annually between 2018 and 2023 [10,11]. This decline is mainly attributed to historical overfishing, illegal harvest, removal of immature and spawning individuals, and the long-term effects of ocean warming and changes in the food web. To mitigate these declines, recent amendments to Korean fisheries regulations prohibit the capture and sale of individuals with a carapace length below 9 cm or female snow crabs, as part of strengthened resource conservation measures [12].

As a result of strengthened domestic conservation regulations and continuing resource limitations, the supply of C. opilio in Korea remains constrained. To compensate for this shortfall, female C. bairdi products harvested in Japan—where the capture and commercial distribution of female snow crabs are legally permitted—have been imported into the Korean market following quarantine and safety inspections [13,14]. Although these imports comply with existing food safety and inspection regulations, female C. bairdi are morphologically very similar to C. opilio, making visual discrimination extremely difficult. Given that imported products are generally traded at lower prices, there is a considerable risk of unintentional mixing or fraudulent substitution during distribution, particularly in online and processed seafood markets.

Conventional species identification of snow crabs has relied largely on morphological characteristics. However, morphological identification becomes unreliable for closely related species or for specimens that have been processed, frozen, or partially damaged. These limitations highlight the need for more objective and reliable species identification methods.

Molecular techniques can identify target species quickly and accurately and therefore have great potential as countermeasures against species misidentification based on morphological classification [15]. Among various molecular approaches, multiplex polymerase chain reaction (PCR) based on single-nucleotide polymorphisms (SNPs) in mitochondrial DNA (mtDNA) has been widely developed as a powerful tool for the rapid and accurate identification of fish and other marine species [16]. Multiplex PCR assays are particularly advantageous in food science because they can distinguish species even after intensive processing, such as drying, canning, or fileting [17].

Mitochondrial DNA is especially suitable for species identification due to its high copy number, maternal inheritance, and relatively rapid evolutionary rate [18,19]. Compared with other molecular methods, Random Amplified Polymorphic DNA (RAPD) suffers from poor reproducibility, and PCR–restriction fragment length polymorphism (PCR-RFLP) analysis is limited by its dependence on restriction enzyme recognition sites [20,21,22]. In contrast, multiplex PCR using species-specific primers enables simultaneous detection of multiple target species with high specificity and accuracy [23,24].

Previous molecular studies on Chionoecetes have reported triplex PCR assays for C. opilio and C. japonicus, as well as ultrafast real-time PCR assays targeting C. japonicus, C. opilio, and Paralithodes camtschaticus [25,26]. However, no study has yet addressed the identification of Japanese female C. bairdi products imported into Korea.

In this study, the mitochondrial cytochrome c oxidase subunit I (COI) gene was analyzed to investigate phylogenetic relationships among three Chionoecetes species (C. bairdi, C. opilio, and C. japonicus) currently distributed in the Korean market. Based on species-specific sequence variation, diagnostic primers were designed, and optimal multiplex PCR conditions were established for the simultaneous identification of the three species. The results of this study are expected to provide a reliable molecular tool for species authentication in the snow crab trade and to contribute to consumer protection, fair seafood distribution, and effective fisheries resource management at both national and international levels.

2. Materials and Methods

2.1. Sample Collection and DNA Extraction

Muscle tissue samples of three Chionoecetes species (C. bairdi, C. opilio, and C. japonicus) were obtained from the Marine Biological Resource Bank at the National Institute of Fisheries Science, Busan, Korea (

Table 1. Specimens of the three Chionoecetes species used in this study.

No.	Scientific Name	Sample Location	Collection Dates	Sample Size (n)		Specimen Accession Number
1	Chionoecetes bairdi	Japan	2024.12		276	NIFS-AR-25005257-25005532
2	Chionoecetes opilio	Korea	2025.01	98	213	NIFS-AR-25005539-25005636
		Russia	2025.04	39		NIFS-AR-25005680-25005718
		Japan	2025.05	76		NIFS-AR-25005719-25005794
3	Chionoecetes japonicus	Korea	2025.02		61	NIFS-AR-25005569-25005599

). Approximately 20 mg of each tissue sample was placed in a microcentrifuge tube, and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 180 μL of ATL buffer and 20 μL of Proteinase K were added, and the mixture was incubated at 56 °C for 8 h for complete lysis. After incubation, 200 μL of AL buffer and 200 μL of 99% ethanol were added and thoroughly mixed. The lysate was transferred to a DNeasy spin column and centrifuged at 6000× g for 1 min. Sequential washes were performed with 500 μL of Wash Buffer 1 (AW1) and Wash Buffer 2 (AW2), followed by centrifugation at 6000× g for 1 min and 14,000× g for 3 min, respectively. Columns were allowed to air-dry at room temperature to remove residual ethanol, and genomic DNA was eluted using 100 μL of AE buffer. DNA integrity was evaluated by 2.0% agarose gel electrophoresis (E-Graph Gel Documentation System, ATTO Corporation, Tokyo, Japan). DNA concentration was measured with a NanoPhotometer N60 Touch (Implen GmbH, Munich, Germany), and purity was assessed using the A260/A280 ratio. Only DNA samples with ratios between 1.8 and 2.0 were used. The extracted DNA was stored at −20 °C until subsequent analyses.

2.2. COI Amplification, Sequencing, and Genetic Analysis

The mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified with the universal primers LCO1490 and HCO2198 (

Table 2. Primer sequences and expected product sizes for PCR amplification of the three Chionoecetes species.

Primer	Sequence (5′→3′)	Product Size (bp)	Target Species
Universal COI primers (annealing temperature: 54 °C)
LCO1490	GGT CAA CAA ATC ATA AAG ATA TTG G	598	C. bairdi, C. opilio,
HCO2198	TAA ACT TCA GGG TGA CCA AAA AAT CA		and C. japonicus
Species-specific primers (annealing temperature: 60 °C)
CB_181F	GAT TTG GAA ATT GAC TAG TAC CT	598	C. bairdi
CO_377F	GTT GAT ATG GGG ATT TTT TCT C	401	C. opilio
CJ_583F	GAT CGA AAC TTG AAT ACA TCC	194	C. japonicus
Chi_756R	GCA TAA ATT ATT CCT AAT GTC CC	—

). Each PCR was carried out in a 20 μL reaction mixture containing 10 μL of Solg™ h-Taq PCR Smart Mix (SolGent Co., Ltd., Daejeon, Republic of Korea), 1 μL of each primer, 1 μL of template DNA, and nuclease-free water. Genomic DNA was normalized to 40 ng/µL prior to COI amplification to ensure consistent template input across samples. PCR amplification was performed on a Veriti™ 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) under the following conditions: an initial denaturation at 95 °C for 11 min; 35 cycles of denaturation at 95 °C for 50 s, annealing at 54 °C for 50 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 7 min. PCR products were verified on 2.0% agarose gels using a 1 kb Plus DNA ladder (Invitrogen, Carlsbad, CA, USA), purified with a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany), quantified using a NanoPhotometer N60 Touch (Implen GmbH), and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed on an ABI 3730XL DNA Analyzer (Applied Biosystems, Waltham, MA, USA).

The obtained sequences were assembled into consensus sequences using SeqMan Pro 17 (Lasergene 17, DNASTAR, Madison, WI, USA) and BioEdit v7.2 (Ibis Biosciences, Carlsbad, CA, USA). Species identity was confirmed by comparing consensus sequences with GenBank references via the NCBI BLAST web service, with ≥98% identity as the threshold. Polymorphism analyses, including haplotype number, polymorphic sites, haplotype diversity (Hd), and nucleotide diversity (Pi), were performed using DnaSP v5.10.01 (University of Barcelona, Spain).

2.3. Species-Specific Primer Design and Singleplex PCR

Consensus COI sequences of the three species were aligned with GenBank references (C. bairdi, OP429109.2; C. opilio, MT335860.1; C. japonicus, MT750295.1) to identify species-specific single-nucleotide polymorphisms (SNPs). Forward primers were designed based on 3′-end SNPs to generate distinct PCR products (Figure 1).

Singleplex PCR was performed to evaluate primer specificity and optimal annealing temperatures. Reactions (20 μL) contained 10 μL Solg™ h-Taq PCR Smart Mix, 1.5 μL of each species-specific forward primer, 1.5 μL universal reverse primer, 1 μL template DNA, and nuclease-free water. PCR cycling conditions were initial denaturation at 95 °C for 11 min, 35 cycles of 95 °C for 50 s, annealing at the optimized temperature (determined by a 10-step gradient PCR from 47 °C to 65 °C) for 50 s, and extension at 72 °C for 1 min; and a final extension at 72 °C for 7 min. Amplified products were visualized on 2.0% agarose gels using a 1 kb Plus DNA ladder for size estimation.

2.4. Multiplex PCR Optimization

Multiplex PCR was performed to simultaneously identify the three species using three species-specific forward primers (CB_181F, CO_377F, and CJ_583F) and one universal reverse primer (Chi_756R) (Table 2). Each 20 μL reaction contained 10 μL Solg™ h-Taq PCR Smart Mix, 0.5 μL of each forward primer (total 1.5 μL), 1.5 μL reverse primer, 1 μL template DNA, and nuclease-free water. Cycling conditions were initial denaturation at 95 °C for 11 min, 35 cycles of 95 °C for 50 s, optimized annealing temperature for 50 s (determined by a 4-step gradient PCR from 60 °C to 63 °C), and extension at 72 °C for 1 min; and a final extension at 72 °C for 7 min. PCR products were analyzed on 2.0% agarose gels, and fragment sizes were estimated using a 1 kb Plus DNA ladder. The slightly higher optimal annealing temperature required for multiplex PCR (62 °C), compared with that for singleplex reactions (60 °C), is likely attributable to primer competition and altered effective primer concentrations when multiple primer sets are combined in a single reaction.

2.5. Primer Sensitivity Testing

The sensitivity of the primers was evaluated by serially diluting genomic DNA from each species to final concentrations of 10, 1, 0.1, and 0.01 ng/µL under optimized multiplex PCR conditions. Each dilution was tested in triplicate, and PCR products were analyzed on 2.0% agarose gels to determine amplification performance.

The limit of detection (LoD) was defined as the lowest DNA concentration at which species-specific bands were consistently detected in at least two out of three independent replicates. This criterion was adopted to ensure analytical reproducibility while accounting for minor stochastic variation commonly observed at very low template concentrations.

In addition, to simulate potential market scenarios involving mixed-species products, equal amounts of genomic DNA from the three Chionoecetes species were combined and subjected to multiplex PCR analysis under the same conditions.

3. Results

3.1. Mitochondrial COI Gene Variation Among Chionoecetes Species

The mitochondrial COI gene region (598 bp) of three Chionoecetes species (C. bairdi, C. opilio, and C. japonicus) was amplified, and species identity was confirmed by comparison with reference sequences in NCBI GenBank. The obtained sequences showed over 99% sequence identity with reference COI sequences of C. bairdi, C. opilio, and C. japonicus. DNA sequence polymorphism analysis using DnaSP v5.10.01 revealed 43 haplotypes in 276 individuals of C. bairdi, 21 haplotypes in 213 individuals of C. opilio, and 7 haplotypes in 61 individuals of C. japonicus. Within each species, a single haplotype predominated, accounting for approximately 63% of C. bairdi (n = 173) individuals, and 80% of C. opilio (n = 166) and C. japonicus (n = 49). No haplotypes were shared among species, indicating clear interspecific genetic differentiation. Haplotype diversity (Hd) and nucleotide diversity (Pi) were 0.597 and 0.00146 for C. bairdi, 0.387 and 0.00100 for C. opilio, and 0.352 and 0.00064 for C. japonicus (

Table 3. Genetic diversity of the COI gene region in populations of three Chionoecetes species.

	Species
	C. bairdi	C.opilio	C. japonicus
Total Number of individuals (N)	276	213	61
Number of polymorphic sites	36	20	6
Number of haplotypes (H)	43	21	7
Haplotype diversity (Hd)	0.597	0.387	0.352
Nucleotide diversity (Pi)	0.00146	0.00100	0.00064

), suggesting higher intraspecific genetic diversity and a more complex population structure in C. bairdi.

3.2. Species-Specific Primer Design and Validation

To design species-specific primers, consensus COI sequences of three Chionoecetes species (C. bairdi, C. opilio, and C. japonicus) were aligned with complete reference sequences from GenBank (C. bairdi, OP429109.2; C. opilio, MT335860.1; C. japonicus, MT750295.1) to identify diagnostic SNP sites. Species-specific SNPs were detected at positions 181 (C. bairdi), 377 (C. opilio), and 583 (C. japonicus), with no intraspecific variation observed across all analyzed individuals, indicating high diagnostic reliability (Figure 1). Using these diagnostic sites, three species-specific forward primers (CB_181F, CO_377F, and CJ_583F) and a universal reverse primer (Chi_756R) were designed (Table 2). When applied in PCR, these primer sets produced distinct amplicons of 598 bp (C. bairdi), 401 bp (C. opilio), and 194 bp (C. japonicus), both in single-template and mixed-template reactions (Figure 2). The optimal annealing temperature was determined to be 60 °C using gradient PCR (47–65 °C), which resulted in consistent, species-specific amplification. Amplicons were visualized on 2.0% agarose gels, and their sizes matched the expected lengths. No cross-reactivity was observed among the three species, confirming the high specificity of the primers. These results demonstrate that the designed primers enable accurate discrimination among the three Chionoecetes species and provide a rapid and reliable molecular tool for market sample authentication.

3.3. Specificity and Sensitivity of Multiplex PCR

The multiplex PCR simultaneously amplified distinct fragments of 598 bp (C. bairdi), 401 bp (C. opilio), and 194 bp (C. japonicus), with no evidence of nonspecific amplification or primer–dimer formation. The optimal annealing temperature was determined to be 62 °C, which yielded consistent, species-specific amplification. To evaluate assay sensitivity, genomic DNA from each species was serially diluted to 10, 1, 0.1, and 0.01 ng/µL, and PCR was performed in triplicate under identical conditions. Under optimized conditions, the multiplex PCR assay consistently detected each target species down to a DNA concentration of 0.01 ng/µL. Species-specific amplicons were observed in at least two out of three independent replicates at this concentration, which was therefore defined as the limit of detection (LoD) (Figure 3).

Moreover, when genomic DNA from the three species was combined in equal proportions, all expected fragments were simultaneously amplified, demonstrating that the assay is robust under mixed-template conditions representative of potential market adulteration.

4. Discussion

In this study, a multiplex PCR assay targeting species-specific variations was developed and applied to identify three Chionoecetes species (C. bairdi, C. opilio, and C. japonicus). This assay enabled the rapid and accurate simultaneous identification of these morphologically similar species, which are otherwise difficult to distinguish using conventional methods.

The higher genetic diversity observed in C. bairdi likely reflects its broader geographic origin and mixed market sources compared with the other two species. Although non-target crustacean species were not included in the present analysis, no nonspecific amplification or unexpected bands were observed among the three target Chionoecetes species under optimized conditions. These results indicate that the primer sets exhibit high analytical specificity within the tested species. Nevertheless, additional validation against non-target crab species, including other commercially distributed brachyuran crabs and closely related taxa, will be necessary to fully assess potential cross-reactivity and to confirm the robustness of the assay under broader market conditions.

Previous methods such as RAPD-PCR and PCR-RFLP have been reported for species identification; however, these approaches often involve multiple analytical steps and are less suitable for high-throughput simultaneous multi-species identification [20,21,22]. In contrast, the multiplex PCR approach used in this study can be performed with standard laboratory equipment, including a conventional PCR thermocycler and a gel documentation system, and it eliminates the need for restriction enzymes. It also allows multiple species to be detected in a single reaction, thereby offering greater cost- and time-efficiency [27,28].

In addition, earlier studies on Chionoecetes have included triplex PCR assays for C. opilio and C. japonicus, as well as ultrafast real-time PCR assays for C. japonicus, C. opilio, and Paralithodes camtschaticus [25,26]. However, no study has previously addressed the detection of C. bairdi, which is commonly imported into the Korean seafood market. The present study fills this gap by designing and validating species-specific primers that enable the simultaneous identification of all three commercially important Chionoecetes species.

Overall, the successful application of multiplex PCR in this study demonstrates its potential as a practical tool for the accurate labeling of Chionoecetes species, contributing to the establishment of sound distribution practices and ensuring seafood safety in Korea and global seafood markets.

5. Conclusions

The multiplex PCR assay developed in this study enables reliable discrimination of three Chionoecetes species with high specificity and sensitivity. This assay reduces the risk of species misidentification arising from morphological similarity and allows rapid and accurate species authentication in a single PCR. Overall, the proposed method provides a cost-effective and efficient tool for supporting sustainable snow crab resource management, preventing seafood fraud, and ensuring safe and transparent distribution in Korea and global markets. Further validation using processed seafood products and complex mixed-species samples will be required to fully confirm its applicability under commercial conditions.