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Article

Development of a Multiplex Polymerase Chain Reaction Method for the Simultaneous Identification of Four Species of Genus Lagocephalus (Chordata: Vertebrata)

by
Hye Min Lee
1,†,
Chun Mae Dong
2,†,
Mi Nan Lee
2,
Eun Soo Noh
2,
Jung-Ha Kang
2,
Jong-Myoung Kim
3,
Gun-Do Kim
4,*,‡ and
Eun-Mi Kim
5,*,‡
1
CellQua, Inc., Seongnam 13595, Republic of Korea
2
Biotechnology Research Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea
3
Department of Fisheries Biology, Pukyong National University, Busan 48513, Republic of Korea
4
Department of Microbiology, Pukyong National University, Busan 48513, Republic of Korea
5
Research Planning Division, National Institute of Fisheries Science, Busan 46083, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors also contributed equally to this work.
Fishes 2025, 10(10), 501; https://doi.org/10.3390/fishes10100501
Submission received: 21 August 2025 / Revised: 17 September 2025 / Accepted: 19 September 2025 / Published: 7 October 2025
(This article belongs to the Special Issue Molecular Genetics and Genomics of Marine Fishes)
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Abstract

Pufferfish are an economically important food in Asia despite the potential risk of tetrodotoxin (TTX) poisoning. To promote food safety by ensuring the correct identification of pufferfish species, we developed common and species-specific primer sets for four Lagocephalus species (Lagocephalus spadiceus, Lagocephalus cheesemanii, Lagocephalus wheeleri, and Lagocephalus inermis) based on analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) in various pufferfish species commonly distributed and/or legally sold in Korea. The common primers were developed based on complete sequence data acquired from GenBank. The total length of fragments amplified by the common primer set was 1280 bp. Then, species-specific multiplex polymerase chain reaction (PCR) amplification was conducted for the four target species, obtaining 980 bp for L. spadiceus, 859 bp for L. cheesemanii, 672 bp for L. wheeleri, and 563 bp for L. inermis. Multiplex PCR is an important tool for the simple, rapid, accurate, and simultaneous identification of target species. The newly developed primer sets will contribute to reducing the occurrence of TTX poisoning and protect consumer rights by eradicating the mislabeling or fraudulent use of pufferfish products.
Keywords:
COI; mitochondrial DNA; multiplex species-specific PCR; pufferfish; species identification
Key Contribution: Development of species-specific genetic markers for four species of genus Lagocephalus.

1. Introduction

Fish represent a significant food resource and contain high contents of nutrient and proteins, including collagen [1]. Pufferfish are consumed for their unique flavor, low fat content, and high nutritional value in many Asian countries, including Korea, Japan, China, Taiwan, and Malaysia [2,3]. A limited number of pufferfish species are also consumed in the United States [4]. However, pufferfish can be fatally toxic to humans because the heat-stable tetrodotoxin (TTX) is present in various tissues including the skin, muscle, liver, intestine, and gonads. TTX content varies among pufferfish according to the species, habitat, sex, season, and even physical treatment such as freezing or thawing [5,6,7]. TTX is detected mainly in the liver and ovaries of marine pufferfish and in the skin of brackish and freshwater species. TTX accumulates throughout the food chain following its production by bacteria, leading to toxic levels in pufferfish [8,9,10]. For example, the ingestion of toxic pufferfish eggs by a different pufferfish species was found to efficiently increase TTX levels in the latter species [11,12]. Some marine and freshwater pufferfish contain saxitoxins (STXs), which can cause paralytic shellfish poisoning, in addition to or instead of TTX [13]. Because TTX rivals STXs for the highest toxicity levels among natural poisons found in fisheries products, and has a severe mortality rate, its detection is critically important in the field of seafood hygiene and safety.
TTX poisoning occurs globally through the consumption of home-made dishes containing pufferfish; these events may be caused by a lack of pufferfish food preparation skills, species misidentification by consumers, or species mislabeling by producers [14]. During the past 5 years, 430 poisoning cases and 52 deaths related to the consumption of TTX from pufferfish have been reported worldwide [14]. Many countries have acted to prevent TTX poisoning by establishing regulations related to pufferfish consumption, or prohibiting it outright. The Ministry of Food and Drug Safety of Korea allows the trade of only 21 pufferfish species in domestic and imported products and the National Fishery Products Quality Management Service published methods for distinguishing pufferfish species and recommendations for their safe handling. Information on the 21 pufferfish species that are commercially available in Korea is presented in Table 1. Most of these species are poisonous; however, reported poisoning cases have mainly been caused by species of the family Tetraodontidae [15].
The Tetraodontidae family contains the most species within order Tetraodontiformes, comprising 27 genera and 184 species worldwide [17]; among these, 10 genera and 33 species have been reported from the seas around Korea [18]. Tetraodontidae remains taxonomically controversial due to morphological variation within species and other difficulties associated with classification [17]. Most edible pufferfish in Korea belong to the Tetraodontidae, and most of these belong to the genera Takifugu and Lagocephalus. The major species causing toxicity in European and Asian countries are within genus Lagocephalus Swainson, 1839. These species are abundant in temperate and tropical waters, have adapted well to the open sea, being distributed in the East Sea, the southern West Sea, the South Sea, and off the coast of Jeju Island in Korea [19]. In Korea, the most popular pufferfish species used in cooking are within genus Takifugu, followed by Lagocephalus, with the latter species considered more accessible due to their lower price. Free trade agreements in Asia have led to the expansion of fishing areas from the coast to the open sea, such that about 28,050 tons of pufferfish have been imported to Korea from China, Japan, Myanmar, India, and Indonesia within the past 5 years, of which 13,320 tons of Lagocephalus accounted for about 47% of the total pufferfish import. However, almost all pufferfish are imported frozen, which impedes accurate morphological species classification due to the factors such as body and fin discoloration [18]. For example, the nearly inedible diamondback puffer (Lagocephalus guentheri) has been disguised and imported as the edible cheeseman’s puffer (Lagocephalus cheesemanii), and L. guentheri and starry blowfish (Arothron stellatus) have been mixed and packed with L. cheesemanii and marketed as edible pufferfish. In the light of these cases, food poisoning related to the consumption of falsely labeled pufferfish is a growing concern both domestically and globally.
Molecular techniques can identify target species quickly and accurately, and therefore have potential as countermeasures against fish species misidentification based on morphological classification [10]. Among various molecular techniques, multiplex polymerase chain reaction (PCR) based on single-nucleotide polymorphisms (SNPs) of mitochondrial DNA (mtDNA) has been widely developed as a useful tool for the rapid classification of fish and other marine species [20]. Multiplex PCR assays are widely applied in food science to distinguish species that have undergone intense processing, such as drying, canning, or fileting [21]. It is also effective for species that are difficult to distinguish from closely related species. Thus, multiplex PCR is well suited for identifying taxonomically controversial pufferfish species that have few useful external characters for taxonomic differentiation.
Within genus Lagocephalus, only L. cheesemanii, L. inermis, and L. wheeleri were initially consumed as food; however, some countries now additionally consume L. spadiceus based on a recent report that it is non-toxic [22]. Matsuura (2010) [23] described L. wheeleri as a junior synonym of L. spadiceus, raising concern that the latter species may become more widely consumed despite the potential risk of TTX toxicity. Because many countries have used L. wheeleri and L. spadiceus without distinction, the Ministry of Food and Drug Safety of Korea revised its recommendations in 2019 to clarify the common and scientific names of edible pufferfish species, to prevent confusion (MFDS notification no. 2019-57). Because the toxicity of L. spadiceus has not been clearly verified, it is not harvested in Korea; however, it may be imported along with other seafood. It is necessary to find a method for distinguishing the controversial species L. spadiceus from the three edible Lagocephalus species and L. wheeleri. Therefore, this study aimed to develop a rapid and reliable MSS-PCR method to distinguish the potentially toxic L. spadiceus from the edible species L. cheesemanii, L. wheeleri, and L. inermis.
In this study, we analyzed SNPs and developed multiplex species-specific multiplex PCR (MSS-PCR) primer sets from the cytochrome c oxidase subunit I (COI) gene of mtDNA sampled from the commonly consumed L. cheesemanii, L. wheeleri, and L. inermis and the controversial species L. spadiceus, based on specimens collected from Korea, India, China, and Japan, respectively. This method provides a practical tool that can be directly applied to seafood safety monitoring, including import inspection, prevention of mislabeling during distribution, reinforcement of routine surveillance by regulatory authorities, and assurance of safety certification in international trade to protect consumers.

2. Materials and Methods

2.1. Sampling and DNA Extraction

We sampled muscle tissues from four Lagocephalus species that were stored by the National Institute of Fisheries Science (Busan, Republic of Korea). All specimens were morphologically classified before analysis. A total of 90 specimens of L. spadiceus (n = 3), L. cheesemanii (n = 31), L. wheeleri (n = 45), and L. inermis (n = 11) were used to design primers for species identification. To determine the specificity of the MSS-PCR primer set, template DNA was extracted from 13 non-target species (Takifugu obscurus, Takifugu xanthopterus, Takifugu chinensis, Takifugu poecilonotus, Takifugu rubripes, Takifugu porphyreus, Takifugu vermicularis, Takifugu stictonotus, Takifugu niphobles, Takifugu pardalis, Sphoeroides pachygaster, Ephippion guttifer, and Diodon holocanthus). These 13 non-target pufferfish species were all authenticated specimens that were available in the National Institute of Fisheries Science (NIFS) repository at the time of testing. All specimens were preserved in 99.9% ethanol before genomic DNA extraction. The specimens are described in Table 2.
For DNA extraction, muscle or fin tissue was collected from each sample. Genomic DNA was extracted using a magnetic bead-based MagExtractor DNA Multi-Prep Kit (TNT Research, Gimhae, Republic of Korea) with the automated KingFisher Flex DNA extraction system (Thermo Fisher Scientific, Waltham, MA, USA) or a DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). About 20 mg of muscle or tissue was combined with 140 μL of proteinase K buffer (5 M NaCl, 1 M tris, 0.5 M ethylenediaminetetraacetic acid, 10% sodium dodecyl sulfate) and 10 μL proteinase K (20 mg/mL) and incubated at 56 °C overnight. Lysate was combined with 650 μL lysis buffer and 40 μL beads to extract DNA using the automated DNA extraction system. Commercial kits were used following the manufacturers’ protocols. The concentration and purity of genomic DNA were quantified using a spectrophotometer (Nanodrop ND-1000, Thermo Fisher Scientific) and the samples were stored at −20 °C until use.

2.2. Sequencing and Species Identification

To develop the common primers and classify Lagocephalus species, all complete and partial sequences of Lagocephalus species were acquired from the National Center for Biotechnology Information (NCBI) GenBank database (available online: https://www.ncbi.nlm.nih.gov/nucleotide (accessed on 15 September 2025)). Because genus Lagocephalus was revised relatively recently [16,23], all species described as valid in the World Register of Marine Species (WoRMS) were searched for sequence retrieval. The mtDNA COI regions of L. spadiceus (KM667972.2), L. gloveri (=L. cheesemanii; MT903226.1), L. wheeleri (AP009538.1 and FJ434551.1), L. inermis (KT933000.1), other Lagocephalus species and non-target species of the families Tetraodontidae, Diodontidae, and Ostraciidae obtained from NCBI (listed in Table 3) were aligned using BioEdit v7.2.5 software (available online: https://bioedit.software.informer.com/ (accessed on 15 September 2025)) to investigate inter- and intra-specific variation and conserved regions and design a common primer set for the genus Lagocephalus (Figure 1). The non-target species in Table 3 were chosen with reference to the National Fishery Products Quality Management Service publications that enumerate major pufferfish taxa; from the list of publications, we included only species for which cytochrome c oxidase subunit I (COI) sequences are available in NCBI. To extend coverage beyond this availability-constrained specimens, we additionally conducted in silico specificity assessments against COI sequences from 43 pufferfish species (Table 3). All sequences used for primer design are listed in Table 3; information on the common primers for genus Lagocephalus is provided in Table 4.
To identify the four target species, we conducted PCR amplification using an ABI Veriti Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) with 2 µL of genomic DNA (10 ng), 0.4 µL each of 10 pmol forward (Lag_COI_F) and reverse (Lag_COI_R) primers, 0.4 µL of 10 mM dNTP mixture, 0.2 µL of DNA Taq polymerase (Anti-HS Taq, TNT Research), 2 µL of 10× PCR buffer, and distilled water to a total volume of 20 µL. The PCR cycle consisted of 11 min of denaturation at 95 °C; 35 cycles of 50 s at 95 °C, 50 s at 56 °C, 50 s at 72 °C; and a 7 min final extension at 72 °C. The mitochondrial COI genes of 13 non-target species were amplified using the universal primers VF2_t1 (5′-TGT AAA ACG ACG GCC AGT CAA CCA ACC ACA AAG ACA TTG GCA C-3′) and FishR2_t1 (5′- CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA A -3′) [16], under the same conditions described above, and sequenced prior to designing species-specific primers. PCR products were visualized using a gel documentation system (AE9000 E-Graph, ATTO, Tokyo, Japan) and purified using the QIAquick PCR purification kit (Qiagen).
Sequencing was performed at our laboratory using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and an ABI 3730xl DNA Analyzer (Applied Biosystems). Purified PCR products were sequenced bidirectionally using dye-termination reaction. The sequencing PCR condition was 96 °C for 2 min; 25 cycles of 96 °C for 15 s, 50 °C for 5 s, and 60 °C for 2 min. Following sequencing PCR, 1 μL of 50 mM EDTA (pH 8.0), 1 μL of 600 mM sodium acetate (pH 5.2), and 25 μL of 99.9% ethanol were added to each reaction. Samples were centrifuged at 3000 rpm (2272× g) for 45 min at 4 °C, washed once with 70% ethanol, and air-dried at room temperature for 10 min. DNA pellets were resuspended in 10 μL of Hi-Di Formamide (Applied Biosystems) and analyzed on the ABI 3730xl DNA Analyzer.

2.3. Sequencing and Phylogenetic Analyses

The obtained bidirectional sequences were assembled using SeqMan Pro v7.1.0 (DNASTAR, Madison, WI, USA) and haplotype sequences were analyzed with DnaSP v5.1 (available online: https://www.ub.es/dnasp (accessed on 15 September 2025)). Haplotype sequences were compared to other nucleotide sequences registered in GenBank using the NCBI BLAST web service (National Center for Biotechnology Information, Bethesda, MD, USA; available online: https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 15 September 2025)) to verify species identity and remove potential sequence errors. Haplotype data from the amplified mtDNA COI region were used for phylogenetic analysis.
To identify genetic distances and relationships within Lagocephalus, haplotype sequences were aligned with the complete and partial sequences of each Lagocephalus species, reference sequences of Matsuura and Satoh (2017) [24] obtained from GenBank, and Sphoeroides pachygaster as an outgroup (Table 5) using the ClustalX multiple alignment tool in BioEdit v7.2.5. Phylogeny was inferred under a Maximum Likelihood framework in MEGA 12 using the Tamura–Nei (TN93) model with a discrete Gamma distribution to accommodate among-site rate variation (+G; 5 categories; shape ≈ 0.41). Branch support was evaluated with 1000 bootstrap replicates [25].

2.4. Species-Specific Multiplex Primer Design

Gene sequences of Lagocephalus species and non-target species obtained from GenBank (Table 3) were aligned with sequenced haplotype data. Species-specific primers were designed to target the COI region within the aligned sequences, excluding intra-species mutational genes caused by point mutations, to detect SNPs with inter-species specificity. Forward primers for each target species were selected based on the presence of an SNP at the 3′ end of the primer sequence and a difference of ≥100 bp between species (Figure 2). Primer mismatches within the 3′-terminal region are highly detrimental to priming efficiency and primers deliberately incorporating a single nucleotide polymorphism (SNP) at the 3′ end are widely used to discriminate common polymorphisms, pathogenic variants, and other rare mutations [26,27]. Accordingly, this principle was applied to the design of species-specific primers in the present study. The developed species-specific primers had similar annealing temperatures, GC content < 40%, and limited potential for self-dimerization.

2.5. Single and Multiplex PCR

Single and multiplex PCR amplification were conducted to determine the suitable annealing temperatures, species-specific reactions, and cross-reactivity of the newly developed primers. Single PCR was performed with a reaction mixture comprising 1 µL of template DNA (10 ng) for each species, 0.2 µL of Lagocephalus universal reverse primer (Lag_COI_R) (10 pmol), 0.2 µL of species-specific forward primer (LS_COI_F, LC_COI_F, LW_COI_F, or LI_COI_F) (10 pmol), 0.2 µL of 10 mM dNTP mixture, 0.1 µL of DNA Taq polymerase (Anti-HS Taq, TNT Research), 1 µL 10× PCR buffer, and distilled water to a total volume of 10 µL. PCR amplification was performed with 11 min of initial denaturation at 95 °C; 35 cycles of 50 s at 94 °C, 50 s at 56–62 °C, 50 s at 72 °C; and a 7 min final extension at 72 °C. To determine the optimum annealing conditions, gradient PCR was conducted using a temperature ramp from 56 °C to 62 °C.
For multiplex PCR, the reverse primer for Lagocephalus and all forward primers for the four target species were combined. PCR amplification was performed with a total volume of 10 µL, containing 1 µL of template DNA (10 ng), 0.2 µL of 10 mM dNTP mixture, 0.1 µL of DNA Taq polymerase (Anti-HS Taq, TNT Research), 1 µL of 10× PCR buffer, distilled water, 0.5 µL of Lagocephalus universal reverse primer (Lag_COI_R) (10 pmol), and 10 pmol of each species-specific forward primer (0.05 µL LS_COI_F, 0.1 µL LC_COI_F, 0.05 µL LW_COI_F, and 0.5 µL LI_COI_F). Gradient PCR was performed with an initial denaturation at 95 °C for 11 min; 32 cycles of 94 °C for 50 s, 56–62 °C for 50 s, 72 °C for 1 min; and a final extension at 72 °C for 7 min. Both single and multiplex PCRs were conducted using the ABI Veriti Fast Thermal Cycler (Applied Biosystems) and amplified products were electrophoresed on 1.5% agarose gel (140 V, 40 min) and visualized using the AE9000 E-Graph gel documentation system (ATTO). PCR amplicons were sequenced for comparison with approved nucleotides from the NCBI BLAST web service.

2.6. Specificity and Sensitivity of Multiplex PCR

To determine the specificity of the novel MSS-PCR primer set, we tested template DNA from the 13 non-target species of families Tetraodontidae and Diodontidae. The DNA template concentrations of the four target species (L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis) were 10, 1, 0.1, and 0.01 ng/µL, respectively. MSS-PCR amplification was conducted using an ABI Veriti Fast Thermal Cycler (Applied Biosystems) with the reaction mixture comprising 1 µL of template DNA, 1 µL of 10× PCR buffer, 0.2 µL of 10 mM dNTP mixture, 0.1 µL of DNA Taq polymerase (Anti-HS Taq, TNT Research), 0.5 µL of Lagocephalus universal reverse primer (Lag_COI_R) (10 pmol), and 10 pmol of each species-specific forward primer (0.05 µL LS_COI_F, 0.1 µL LC_COI_F, 0.05 µL LW_COI_F, and 0.5 µL LI_COI_F), and distilled water for a total volume of 10 µL. The PCR amplification conditions were as follows: preheating at 95 °C for 11 min; 32 cycles of 94 °C for 50 s, 59 °C for 50 s, 72 °C for 1 min; and final extension at 72 °C for 7 min. The amplified products were stained with 6× Loading STAR solution (Dyne Bio, Seongnam, Republic of Korea) and analyzed using 1.5% agarose gel electrophoresis at 140 V for 40 min.

3. Results

3.1. Common Primers for Genus Lagocephalus

We analyzed the mitochondrial COI gene to develop molecular markers for the simple and rapid identification of four pufferfish species: L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis. The optimal annealing temperature for the developed common primer set was 56 °C and a total fragment of 1280 bp was amplified from the mtDNA COI region of the four target species (Figure 3). The final length of the amplified COI gene fragment was 1194 bp after trimming poor-quality sequences.

3.2. Species Identification and Phylogenetic Analysis

Sequences amplified from the common primer set for Lagocephalus were compared to other COI nucleotide sequences registered in GenBank. The sequences generated for four Lagocephalus species were classified into three haplotypes for L. spadiceus (Hap_1-Hap_3), six haplotypes for L. wheeleri (Hap_4-Hap_9), three haplotypes for L. cheesemanii (Hap_10-Hap_12), and five haplotypes for L. inermis (Hap_13-Hap_17) (Figure 4). Haplotypes differed among L. spadiceus individuals; the major haplotypes were Hap 4_4 for L. wheeleri (51%), Hap_10 for L. cheesemanii (77%), and Hap_13 for L. inermis (45%). BLASTn comparison of common-primer COI amplicons to GenBank showed 100% query coverage and the following percent-identity ranges (best-hit accessions in parentheses): L. spadiceus 99.25~99.66% (KM667972.2); L. cheesemanii 99.83~100.00% (MT903226.1; deposited under L. gloveri); L. wheeleri 99.58~100.00% (FJ434551.1); and L. inermis 99.41~99.50% (KT933000.1) (see Table S1).
To verify recent taxonomical revisions, we performed a phylogenetic analysis by based on a neighbor-joining phylogenetic tree, substituting sequences obtained in this study into the reference sequences to confirm the relationships among Lagocephalus species (Figure 4). The results confirmed that all 11 species within Lagocephalus were well differentiated. A comparison of haplotype sequences from L. cheesemanii obtained in this study with reference sequences from Matsuura and Satoh (2017) [24] and all GenBank L. gloveri and L. cheesemanii nucleotides showed genetic similarities of 99–100%, excluding accession nos. KU945235.1 and FJ434548.1, which were clustered as L. wheeleri. Our findings also clearly showed that L. gloveri and L. cheesemanii sequences were merged into a single clade.
The distribution of overall mean inter- and intraspecific genetic distances of the four Lagocephalus species were compared using genetic divergence analysis (Table 6). The intraspecific genetic divergence showed a minimum of 0.1% (L. cheesemanii—L. cheesemanii) and maximum of 0.4% (L. spadiceus—L. spadiceus), and all values were lower than the interspecific genetic distances. Interspecific genetic divergence analysis showed that L. spadiceus and L. wheeleri differed by 6.3% and were closest among the four species; the greatest genetic distance was between L. wheeleri and L. inermis, at 18.3%. Based on the 544 bp alignment, the results showed that L. spadiceus has about 96% homology with L. guentheri, which was the most closely related species in the phylogenetic tree, approximately 93% homology with L. wheeleri, which has similar morphological characters, and >10% genetic distance with the remaining Lagocephalus species. In particular, L. spadiceus differed genetically from L. inermis, L. lunaris, and L. suezensis by >20% (Table S2).

3.3. Species-Specific Primer Design and Single PCR

Single PCR amplification using the species-specific primers developed in this study showed high specificity for each of the four target species. The appropriate annealing temperatures were 59–61 °C for LS_COI_F/Lag_COI_R and LC_COI_F/Lag_COI_R, 60–61 °C for LW_COI_F/Lag_COI_R, and 57–58 °C for LI_COI_F/Lag_COI_R, at which specific fragments were amplified for L. spadiceus (980 bp), L. cheesemanii (859 bp), L. wheeleri (672 bp), and L. inermis (563 bp). The amplified fragments of each species were easily distinguished by gel electrophoresis.

3.4. MSS-PCR

To overcome difficulties in developing a multiplex PCR methodology such as selecting an appropriate annealing point and preventing dimer formation, we tested the ratio of PCR components for each species-specific primer, PCR cycling conditions, and gradient PCR to determine the optimal amplification temperatures. Consequently, we confirmed that the optimal annealing temperature was 59 °C for all four target species in multiplex PCR, and that the conditions of the developed multiplex PCR method were adequate for product amplification using single DNA samples. As shown in Figure 5, the newly developed species-specific primers rapidly and accurately detected amplicons of L. spadiceus (980 bp), L. cheesemanii (859 bp), L. wheeleri (672 bp), and L. inermis (563 bp). To verify the PCR amplification according to individual variation, all 90 samples used in the present study were amplified with the newly developed MSS-PCR primer set and all samples were correctly classified as one of the four target species.

3.5. Specificity and Sensitivity of MSS-PCR

The specificity of the MSS-PCR primers was verified using genomic DNA from the 13 non-target species (Table 2), with no cross-reaction (Figure 6). To confirm the PCR amplification threshold and verify the reliability of the MSS-PCR method, we conducted a sensitivity test by diluting the DNA of the four target species 10-fold, from 10 to 0.01 ng. The results showed that L. spadiceus demonstrated a sensitivity of 0.01 ng and that the detection limit for L. cheesemanii, L. wheeleri, and L. inermis was 0.1 ng (Figure 7).

4. Discussion

The global rise in seafood production has increased the risk of fraud or accidental mislabeling, leading to growing concern about food safety [28]. Accidental poisoning through the consumption of pufferfish species that are strictly prohibited in many countries, but illegally or accidentally caught and consumed as seafood, is becoming a critical concern. For example, since the opening of the Suez Canal in 1869, Lessepsian species, which invaded from the Red Sea and rapidly spread throughout the Mediterranean Sea, have caused serious zoogeographical and ecological problems [29]. These Lessepsian species included Lagocephalus pufferfish, which are among the worst threats to public health in Europe. Many countries have already reported TTX poisoning through the deliberate or accidental consumption of Lagocephalus species [30]. Both suppliers and consumers commonly confuse toxic and non-toxic pufferfish species due to their morphological similarity, particularly after processing; for example, identifying the species included in fish filets or fish balls is highly challenging for food inspectors [31].
In the past, morphological and component analyses were widely conducted to identify the species included in various seafood types; however, the recent development of molecular analysis methods has the potential to transform food inspection processes. Several protein-based methods for fish species identification have been developed, but these analytical methods have limitations that can impede classification, as proteins lose their biological activity after animal death and proteins tend to become denatured under heating and high-pressure treatments [32]. In contrast, DNA is ubiquitous, relatively stable under food processing, and easy to examine [33]. Notably, even small mtDNA samples can be analyzed due to their higher copy numbers per cell compared with nuclear DNA. Furthermore, mtDNA is heritable and strongly conserved, with little duplication, no introns, and very short intergenic regions, making it a useful molecular marker for species identification and evolutionary studies of marine organisms [34]. In particular, multiplex PCR methods that can rapidly detect various species at high sensitivity within a single step are becoming widely adopted for food authentication [35].
Although many studies have analyzed the taxonomy of genus Lagocephalus, the classification and systematics of this genus remain unclear. Matsuura et al. (2011) [36] described confusion in the distinction of Lagocephalus species, particularly among L. cheesemanii, L. gloveri, L. guentheri, L. wheeleri, and L. spadiceus. Matsuura & Satoh (2017) [24] concluded that L. gloveri is a junior synonym of L. cheesemanii based on the morphological characteristics and DNA analysis of specimens collected from Australia, New Zealand, and the western North Pacific.
Therefore, these two species are regarded as the same species (L. cheesemanii) in the present study. Additionally, L. spadiceus was suggested to be a senior synonym of L. wheeleri by Matsuura (2010) [23], based solely on morphological classification, without molecular analysis. Giusti et al. (2019) [37] also treated L. wheeleri as L. spadiceus, following Matsuura (2010) [23], who performed taxonomical revisions based solely on the morphological classification of both species. However, the genetic distance between the L. spadiceus and L. wheeleri specimens used in this study was 6.7%, supporting their consideration as separate species. Han et al. (2017) [18] considered L. wheeleri and L. spadiceus as independent species due to their morphological differences, based on morphological analyses, meristics, and a genetic difference of about 7%. Giusti et al. (2019) [37] also evaluated the reliability of L. spadiceus and L. guentheri data in GenBank and the Barcode of Life Data (BOLD, available online: https://www.boldsystems.org/ (accessed on 15 September 2025)), and detected some invalid taxonomic descriptions. Giusti et al. (2019) [37] reported that most L. guentheri specimens collected in the Mediterranean were misclassified as L. spadiceus; they suggested that 32.5% of COI sequences and 43.7% of cytochrome b sequences in GenBank and 30% of COI nucleotides in BOLD were incorrectly registered. They also treated L. wheeleri as L. spadiceus, following Matsuura (2010) [23], who performed taxonomical revisions based solely on the morphological classification of both species.
However, it remains difficult to treat these as the same species because no direct genetic comparisons of L. wheeleri and L. spadiceus have been conducted using verified specimens. Giusti et al. (2019) [37] proposed that most L. spadiceus sequences registered in GenBank and BOLD could actually be L. guentheri, and predicted that L. wheeleri and L. spadiceus sequences would be identical because L. spadiceus was revised as a senior synonym of L. wheeleri by Matsuura (2010) [23]. They finally recognized the practical difficulties inherent in taking a firm stand because the morphology of all reported specimens can no longer be observed, and current studies that do not consider morphological differences in the shape and color of the caudal fins of L. spadiceus and L. guentheri are considered unreliable. In the present study, we considered L. spadiceus and L. wheeleri as independent species based on their genetic distance, a complete sequence of L. guentheri obtained from a specimen caught recently in Bohai Bay, China [38], and a sequence from an L. guentheri specimen found in Nigeria [39]; species classification was performed with reference to sequences registered in GenBank (accession nos. MT903227.1 and KY442712.1).
Although some studies have argued that L. spadiceus is non-toxic or contains little toxin [2,40], others have detected TTX in the brain, liver, and skin of this species [41,42]. Monaliza and Samsur (2011) [3] performed toxicity assessments of TTX from L. spadiceus via mouse bioassays; the injected mice showed neurotoxin infection, with symptoms including suffocation and cramp. Amano et al. (2022) [43] detected TTX in the brain and liver of L. spadiceus specimens collected from Enoshima, Kanagawa Prefecture, Japan. In Turkey, L. spadiceus may not be landed or sold due to its potential toxicity, according to Turkish Commercial Fisheries Notification 3/1 (RG17/5/2013-28650) [29]. Therefore, the food safety of L. spadiceus remains unclear, and in Korea there is no consensus on whether it is safe for consumption.
Previous studies that examined genus Lagocephalus have conducted taxonomic and phylogenetic studies based on morphological characteristics, as well as nucleotide sequence analysis [17,24,37]. Family Tetraodontidae is the most speciose taxon among teleosts, with diverse morphological variation and many controversies at the species and higher taxonomic levels; it remains difficult to clearly identify these species, and food poisoning due to misclassification is a global concern. Various DNA-based methods have been applied to detect commercial seafood fraud, including multiplex PCR, restriction fragment length polymorphism PCR, random amplified polymorphic DNA PCR, amplified fragment length polymorphism PCR, and forensically informative nucleotide sequencing, all of which are based on polymorphisms within the DNA sequences of different species [44]. DNA barcoding is also commonly used for species classification, based on a short fragment of the mitochondrial COI gene [20]. Kim and Kang (2023) [45] developed a systematic method that included nucleotide sequencing of mtDNA COI and cytochrome b gene and simple-sequence repeat amplification to identify pufferfish species, particularly those in genus Lagocephalus; their method examined the nucleotide sequences of seafood products, which allowed the reliable identification of unknown fish species, but at greater time and sequencing costs than those of other detection methods. Simpler DNA-based methods such as restriction fragment length polymorphism PCR, species-specific PCR and real-time PCR have been suggested for rapid pufferfish species identification [31,42,46,47]. However, no multiplex PCR method has been developed for simultaneous detection of the three edible Lagocephalus species (L. cheesemanii, L. wheeleri, and L. inermis). Accordingly, we developed species-specific primers for four Lagocephalus species, including these three edible species and one ambiguous species (L. spadiceus) whose toxicity remains uncertain.
To date, most cases of TTX poisoning through mislabeling have been reported from Asia, due to the fraudulent substitution of pufferfish for other fish products such as cod, mullet roe, or angelfish [4]. TTX poisoning has occurred in the United States following the consumption of frozen fish products labeled as monkfish, but identified as a Lagocephalus species based on combined molecular and morphological methods [48]. To prevent TTX poisoning through accidental consumption, the Japanese Ministry of Health and Welfare (presently the Ministry of Health, Labour, and Welfare) published guidelines for the consumption of edible pufferfish in 1983, followed by updates in 1993 and 1995 [8]. The United States Food and Drug Administration allowed the importation of tiger pufferfish (Takifugu rubripes) only from Japan and only under certain conditions; domestic harvest was also allowed, but only for non-toxic species [48]. In contrast, the European Union prohibited the sale of all species within family Tetraodontidae (regulation no. 854/2004). In other countries, the fishing of pufferfish is prohibited by the Egyptian government [49]; only L. gloveri and L. wheeleri can be sold in markets or processed for consumption in Taiwan [46]; and China prohibits the sale of fresh pufferfish but allows the culture of Takifugu rubripes and Takifugu obscurus in certified facilities and their sale after processing, with codes required on packaging to track their origin [4].
Despite the potential risk of TTX poisoning through misidentification or mislabeling, some pufferfish species are economically significant and marketed in raw, whole-body form or in processed products such as dried fish filets or fish balls. Where the consumption of pufferfish is unavoidable, government organizations should ensure the rapid, accurate detection and management of toxic species during the importation and distribution processes. The MSS-PCR method developed in the present study is anticipated to be useful for this purpose.

5. Conclusions

Among Tetraodontidae family members, Lagocephalus species have been frequently reported or caught in Asia, the Mediterranean Sea, and the Pacific Ocean. TTX poisoning through the consumption of these species represents a potential public health concern. Although many countries have long regulated or completely banned the import and distribution of pufferfish, TTX poisoning due to species substitution or seafood adulteration is inevitable as international trade and worldwide seafood consumption increase. To prevent poisoning via the accidental consumption of toxic pufferfish, we developed species-specific primers for the rapid, simultaneous detection of three edible species (L. cheesemanii, L. wheeleri, and L. inermis) and one ambiguous species (L. spadiceus) based on an MSS-PCR assay. The mitochondrial COI gene region was analyzed to design common and species-specific primers for these four target species. A total fragment of 1280 bp was amplified from all specimens and amplicons of L. spadiceus (980 bp), L. cheesemanii (859 bp), L. wheeleri (672 bp), and L. inermis (563 bp) were successfully detected. The MSS-PCR method developed in this study showed reliable accuracy and sensitivity, and represents an effective method for potential application in establishing the safety of seafood products and eradicating illegal distribution activities such as fraud and mislabeling. However, the specimens used in this study originated from Korea, Japan, China, and India; therefore, the primer set developed in this study may not cover the full breadth of global diversity. The MSS-PCR primers target species-diagnostic interspecific SNPs and are intended to function irrespective of sample processing. Nevertheless, independent validation across additional populations and regions is warranted and future work should extend testing to broader Indo-Pacific and Mediterranean specimens.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fishes10100501/s1, Table S1: BLASTn summary for common-primer COI amplicons of four Lagocephalus species. For each haplotype (Hap 1–17; see Figure 4), the table reports Query Coverage (alignment length ÷ query length × 100), Percent Identity, and the best-hit GenBank accessions. Species names follow the accepted taxonomy (e.g., L. cheesemanii; records deposited under the junior synonym L. gloveri are indicated as such).; Table S2: Overall mean distances of inter-species and intra-species of the specimens used in the present study. Values are the average number of base substitutions per site for the mitochondrial COI gene, estimated under the Tamura–Nei (TN93) model from 1194 bp alignment.

Author Contributions

Conceptualization, E.-M.K. and G.-D.K.; methodology, E.-M.K., C.M.D. and H.M.L.; software, C.M.D. and H.M.L.; validation, M.N.L., C.M.D. and E.S.N.; formal analysis, E.-M.K. and H.M.L.; investigation, E.-M.K., C.M.D. and H.M.L.; writing—original draft preparation, E.-M.K. and H.M.L.; writing—review and editing, E.-M.K., C.M.D. and H.M.L.; supervision, J.-H.K., G.-D.K. and J.-M.K.; project administration, E.-M.K.; funding acquisition, E.-M.K. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by a grant from the National Institute of Fisheries Science (R2025018), Republic of Korea.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding authors on reasonable request.

Conflicts of Interest

Author Hye Min Lee was employed by the CellQua company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Information of the common primers for the genus Lagocephalus designed from mitochondrial COI sequences of L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis available in GenBank of NCBI. Boxes indicate developed primer sets.
Figure 1. Information of the common primers for the genus Lagocephalus designed from mitochondrial COI sequences of L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis available in GenBank of NCBI. Boxes indicate developed primer sets.
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Figure 2. Nucleotide alignment and information of species-specific forward primers designed from the mtDNA COI gene of the Lagocephalus species L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis for species identification. Sequences of boxes indicate newly developed species-specific primers.
Figure 2. Nucleotide alignment and information of species-specific forward primers designed from the mtDNA COI gene of the Lagocephalus species L. spadiceus, L. cheesemanii, L. wheeleri, and L. inermis for species identification. Sequences of boxes indicate newly developed species-specific primers.
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Figure 3. PCR products of COI gene fragments amplified by the newly developed common primer set Lag_COI_F and Lag_COI_R from Lagocephalus species. A total length of the amplicons of all four target species was 1280 bp. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane 1–3: L. spadiceus; lane 4–6: L. cheesemanii; lane 7–9: L. wheeleri; lane 10–12: L. inermis.
Figure 3. PCR products of COI gene fragments amplified by the newly developed common primer set Lag_COI_F and Lag_COI_R from Lagocephalus species. A total length of the amplicons of all four target species was 1280 bp. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane 1–3: L. spadiceus; lane 4–6: L. cheesemanii; lane 7–9: L. wheeleri; lane 10–12: L. inermis.
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Figure 4. Maximum-likelihood tree inferred from ~544 bp fragment of the mitochondrial COI gene from L. spadiceus (Green; 3 haplotypes), L. wheeleri (Yellow; 6 haplotypes), L. cheesemanii (Blue; 3 haplotypes), and L. inermis (5 haplotypes). LS-IN and LC-IN: Indian L. spadiceus and L. cheesemanii population; LW-KR, LC-KR, and LI-KR: Korean L. wheeleri, L. cheesemanii, and L. inermis population; LW-CN, LC-CN, and LI-CN: Chinese L. wheeleri, L. cheesemanii, L. inermis population; LW-JP: Japanese L. wheeleri population. Accession number of each reference sequence is indicated.
Figure 4. Maximum-likelihood tree inferred from ~544 bp fragment of the mitochondrial COI gene from L. spadiceus (Green; 3 haplotypes), L. wheeleri (Yellow; 6 haplotypes), L. cheesemanii (Blue; 3 haplotypes), and L. inermis (5 haplotypes). LS-IN and LC-IN: Indian L. spadiceus and L. cheesemanii population; LW-KR, LC-KR, and LI-KR: Korean L. wheeleri, L. cheesemanii, and L. inermis population; LW-CN, LC-CN, and LI-CN: Chinese L. wheeleri, L. cheesemanii, L. inermis population; LW-JP: Japanese L. wheeleri population. Accession number of each reference sequence is indicated.
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Figure 5. Identification of the four Lagocephalus species by multiplex PCR. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane S: size marker for four Lagocephalus species; lane 1–3: L. spadiceus (980 bp); lane 4–6: L. cheesemanii (859 bp); lane 7–9: L. wheeleri (672 bp); lane 10–12: L. inermis (563 bp).
Figure 5. Identification of the four Lagocephalus species by multiplex PCR. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane S: size marker for four Lagocephalus species; lane 1–3: L. spadiceus (980 bp); lane 4–6: L. cheesemanii (859 bp); lane 7–9: L. wheeleri (672 bp); lane 10–12: L. inermis (563 bp).
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Figure 6. The result of specificity test of multiplex PCR with four target species of Lagocephalus and thirteen nontarget species. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane S: size marker for four Lagocephalus species; lane 1–4: L. spadiceus (980 bp), L. cheesemanii (859 bp), L. wheeleri (672 bp), L. inermis (563 bp); lane 5–17: Takifugu obscurus, T. xanthopterus, T. chinensis, T. poecilonotus, T. rubripes, T. porphyreus, T. vermicularis, T. stictonotus, T. niphobles, T. pardalis, Sphoeroides pachygaster, Ephippion guttifer, Diodon holocanthus.
Figure 6. The result of specificity test of multiplex PCR with four target species of Lagocephalus and thirteen nontarget species. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane S: size marker for four Lagocephalus species; lane 1–4: L. spadiceus (980 bp), L. cheesemanii (859 bp), L. wheeleri (672 bp), L. inermis (563 bp); lane 5–17: Takifugu obscurus, T. xanthopterus, T. chinensis, T. poecilonotus, T. rubripes, T. porphyreus, T. vermicularis, T. stictonotus, T. niphobles, T. pardalis, Sphoeroides pachygaster, Ephippion guttifer, Diodon holocanthus.
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Figure 7. Sensitivity test of four Lagocephalus species by multiplex PCR with newly developed primers. PCRs were performed using genomic DNA of four species of the genus Lagocephalus with a ten-fold serial diluted from 10 ng/µL, 1 ng/µL, 0.1 ng/µL, 0.01 ng/µL. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane 1–4: 10 ng, 1 ng, 0.1 ng, 0.01 ng.
Figure 7. Sensitivity test of four Lagocephalus species by multiplex PCR with newly developed primers. PCRs were performed using genomic DNA of four species of the genus Lagocephalus with a ten-fold serial diluted from 10 ng/µL, 1 ng/µL, 0.1 ng/µL, 0.01 ng/µL. Lane M: 100 bp DNA ladder (Dyne, Republic of Korea); lane 1–4: 10 ng, 1 ng, 0.1 ng, 0.01 ng.
Fishes 10 00501 g007
Table 1. The list of twenty-one edible pufferfish species permitted by the Ministry of Food and Drug Safety of Korea. It has been updated in 2019 reflecting additional scientific names of the five pufferfish (underlined) that cause confusion by using the different scientific names in other countries from those in Korea. Edible parts of the allowed pufferfish are presented by the National Fishery Products Quality Management Service of Korea.
Table 1. The list of twenty-one edible pufferfish species permitted by the Ministry of Food and Drug Safety of Korea. It has been updated in 2019 reflecting additional scientific names of the five pufferfish (underlined) that cause confusion by using the different scientific names in other countries from those in Korea. Edible parts of the allowed pufferfish are presented by the National Fishery Products Quality Management Service of Korea.
FamilyNameScientific NameEdible Portion
MuscleSkinTestis
1TetraodontidaeGrass pufferTakifugu niphobles,
Takifugu alboplumbeus		◯
2Fine patterned pufferTakifugu poecilonotus,
Takifugu flavipterus		◯
3Panther pufferTakifugu pardalis◯
4Vermiculated pufferTakifugu snyderi◯ ◯
5Genuine pufferTakifugu porphyreus◯ ◯
6Yellow pufferTakifugu obscurus◯ ◯
7Red-eyed pufferTakifugu chrysops◯ ◯
8Tiger pufferTakifugu rubripes◯◯◯
9Eyespot pufferTakifugu chinensis◯◯◯
10Striped pufferTakifugu xanthopterus◯◯◯
11Spotty back pufferTakifugu stictonotus◯ ◯
12Smooth-backed blowfishLagocephalus inermis◯◯◯
13Rough-backed pufferLagocephalus wheeleri,
Lagocephalus spadiceus		◯◯◯
14Cheeseman’s pufferLagocephalus gloveri 1,
Lagocephalus cheesemanii		◯◯◯
15Slackskinned pufferSphoeroides pachygaster◯◯◯
16Towny pufferTakifugu flavidus◯
17DiodontidaePacific burrfishChilomycterus affinis, Chilomycterus reticulatus◯◯◯
18Spiny pufferDiodon holocanthus◯◯◯
19Bleeker’s porcupinefishDiodon liturosus◯◯◯
20Spotted porcupinefishDiodon hystrix◯◯◯
21OstraciidaeTrunkfishOstracion immaculatus◯ ◯
1 Following [16], we treat Lagocephalus gloveri as a junior synonym of L. cheesemanii and use L. cheesemanii as the accepted name throughout. Where necessary, legacy database entries labeled as L. gloveri are indicated but interpreted as L. cheesemanii in this study. Notes. Symbols: ◯ = edible portion permitted; blank = not specified.
Table 2. Details of the Lagocephalus specimen used in the development of MSS-PCR primer set and thirteen non-target species for specificity test.
Table 2. Details of the Lagocephalus specimen used in the development of MSS-PCR primer set and thirteen non-target species for specificity test.
No.Country of
Origin		Sampling
Date		Sample
Size		LocalitySpeciesRegistration
Number (NIFS a)
1IndiaApril 20183-Lagocephalus
spadiceus		NFRDI-FI-TS-0049323
NFRDI-FI-TS-0049327
NFRDI-FI-TS-0049329
2KoreaMay 20182Seogwipo-si,
Jeju		Lagocephalus
cheesemanii		NFRDI-FI-IS-0025360
NFRDI-FI-IS-0025364
3KoreaMay 20181JejuNFRDI-FI-IS-0025361
4KoreaMay 20181Jeju-si,
Jeju		NFRDI-FI-IS-0025366
5KoreaMay 20181BusanNFRDI-FI-IS-0025368
6KoreaJune 20181Pohang,
Gyeongbuk		NFRDI-FI-TS-0049457
7ChinaApril 201821-NFRDI-FI-IS-0025473~0025496
8IndiaApril 20184-NFRDI-FI-TS-0049324~0049326
NFRDI-FI-TS-0049328
9KoreaMay 20184BusanLagocephalus
wheeleri		NFRDI-FI-IS-0025288~0025291
10KoreaMay 20186Geoje,
Gyeongnam		NFRDI-FI-IS-0025292~0025297
11KoreaMay 20181Hupo-myeon, UljinNFRDI-FI-IS-0025367
12ChinaApril 201820-NFRDI-FI-IS-0025377~0025396
13JapanMay 201814NagasakiNFRDI-FI-TS-0049330~0049343
14ChinaApril 20189-Lagocephalus
inermis		NFRDI-FI-IS-0025452~0025456
NFRDI-FI-IS-0025459
NFRDI-FI-IS-0025460
NFRDI-FI-IS-0025462
NFRDI-FI-IS-0025465
15KoreaJuly 20181Jeju-si,
Jeju		NFRDI-FI-TS-0049458
16KoreaOctober 20181Jeju-si,
Jeju		NFRDI-FI-TS-0068234
17KoreaJuly 20111Chagwido,
Jeju		Diodon holocanthusNFRDI-FI-TS-0068232
18SpainApril 20141Las PalmasEphippion guttiferNFRDI-FI-IS-0010276
19KoreaApril 20141Tongyeong, GyeongnamTakifugu chinensisNFRDI-FI-TS-0049417
20KoreaMarch 20181Boryeong,
Chungnam		Takifugu obscurusNFRDI-FI-TS-0049421
21KoreaMay 20181Tongyeong, GyeongnamTakifugu stictonotusNFRDI-FI-IS-0025284
22KoreaMay 20181Sinan,
Jeonnam		Sphoeroides
pachygaster		NFRDI-FI-IS-0025369
23KoreaMay 20181Mokpo,
Jeonnam		Takifugu snyderiNFRDI-FI-IS-0025316
24KoreaApril 20181Tongyeong, GyeongnamTakifugu pardalisNFRDI-FI-TS-0049407
25ChinaApril 20181-Takifugu porphyreusNFRDI-FI-IS-0025397
26KoreaMarch 20181Boryeong,
Chungnam		Takifugu niphoblesNFRDI-FI-TS-0049389
27KoreaMay 20181JejuTakifugu poecilonotusNFRDI-FI-IS-0025340
28ChinaMay 20181Weihai,
Shandong		Takifugu rubripesNFRDI-FI-IS-0025555
29ChinaApril 20181FujianTakifugu xanthopterusNFRDI-FI-IS-0025596
a NIFS represent the National Institute of Fisheries Science. Notes. Registration Number (NIFS): Institutional specimen catalog number assigned by the National Institute of Fisheries Science (formerly NFRDI), Republic of Korea; not a public database accession (e.g., GenBank).
Table 3. The information of sequence data obtained from NCBI GenBank for primer design.
Table 3. The information of sequence data obtained from NCBI GenBank for primer design.
FamilyGenusSpeciesAccession no.
TetraodontidaeLagocephalus  L. spadiceusKM667972.2
  L. gloveri
  (=L. cheesemanii)		MT903226.1
  L. wheeleriAP009538.1 and FJ434551.1
  L. inermisKT933000.1
  L. guentheriMT903227.1
  L. laevigatusAP011934.1
  L. lagocephalusAP011933.1
  L. lunarisFJ434550.1
  L. sceleratusMH550879.1 and AP011932.1
  L. suezensisKP013619.1
Arothron  A. firmamentumAP006742.1
  A. stellatusAP019615.1
  A. mappaAP011931.1 and AP019602.1
  A. hispidusFJ434547.1
  A. nigropunctatusAP019605.1
Canthigaster  C. valentineAP011912.1
Chelonodon  C. patocaAP009541.1
Ephippion  E. guttiferKY498013.1 and KP641370.1
Feroxodon  F. multistriatusGU673338.1
Sphoeroides  S. pachygasterFJ434553.1 and AP006745.1
Takifugu  T. alboplumbeusKY514070.1 and MN965579.1
  T. bimaculatusKP973944.1
  T. chinensisAP009534.1
  T. chrysopsAP009525.1
  T. exascurusAP009540.1
  T. flavidusKJ562276.1
  T. niphoblesAP009526.1 and FJ434554.1
  T. oblongusFJ434555.1
  T. obscurusAP009527.1
  T. pardalisAP009528.1
  T. poecilonotusAP009539.1 and FJ434557.1
  T. porphyreusAP009529.1
  T. pseudommusKY514075.1 and KP641558.1
  T. rubripesAJ421455.1 and AP006045.1
  T. stictonotusAP009530.1 and FJ434558.1
  T. vermicularisAP009532.1
  T. xanthopterusFJ434560.1
Torquigener  T. hypselogeneionAP011927.1
DiodontidaeDiodon  D. liturosusKY682080.1
  D. holocanthusAP009177.1
  D. hystrixKY677758.1
Chilomycterus  C. reticulatusLC659947.1 and AP009188.1
OstraciidaeOstracion  O. immaculatusAP009176.1
Table 4. Sequences of common primers for Lagocephalus and species-specific primers for four target species.
Table 4. Sequences of common primers for Lagocephalus and species-specific primers for four target species.
No.Primer NameSequence (5′→3′)Product SizeTarget Species
Common primer set (COI gene region)
1Lag_COI_ FAGC CTY CTT ATT CGV GCA1280 bp  Common
  primer for
  genus
  Lagocephalus
2Lag_COI_RATB ARR GAG CCR ATY GAG
Species-specific forward primers
3LS_COI_FGGC AAT CTC GCC CAT GCC980 bp  L. spadiceus
4LC_COI_FCCG CCA TTT CCC AAT ACC AAA CT859 bp  L. cheesemanii
5LW_COI_FATT CTT TGG CCA CCC CGA G672 bp  L. wheeleri
6LI_COI_FATG GTG TGG GCA ATA ATG GCT563 bp  L. inermis
Table 5. List of the nucleotide sequences used for phylogenetic analysis. Reference sequences were obtained from NCBI GenBank and in this study.
Table 5. List of the nucleotide sequences used for phylogenetic analysis. Reference sequences were obtained from NCBI GenBank and in this study.
SpeciesAccession NumberReference
Lagocephalus spadiceusKM667972.2NCBI GenBank
KR861535.1–KR861536.1NCBI GenBank
KY130423.1NCBI GenBank
PX218734–PX218736Data generated in this study
Lagocephalus gloveri 1MT903226.1NCBI GenBank
KY984986.1NCBI GenBank
LC155436.1–LC155437.1NCBI GenBank
JQ681795.1NCBI GenBank
Lagocephalus cheesemaniiLC155433.1–LC155435.1NCBI GenBank
PX218737–PX218767Data generated in this study
Lagocephalus wheeleriAP009538.1NCBI GenBank
FJ434551.1NCBI GenBank
PX218768–PX218812Data generated in this study
Lagocephalus inermisKT933000.1NCBI GenBank
GU674214.1NCBI GenBank
KP266776.1NCBI GenBank
PX218813–PX218823Data generated in this study
Lagocephalus guentheriMT903227.1NCBI GenBank
KY442712.1NCBI GenBank
Lagocephalus laevigatusAP011934.1NCBI GenBank
Lagocephalus lagocephalusAP011933.1NCBI GenBank
Lagocephalus lunarisGQ461750.1NCBI GenBank
Lagocephalus sceleratusMH550879.1NCBI GenBank
Lagocephalus suezensisKP013619.1NCBI GenBank
Sphoeroides pachygaster
(Outgroup)		AP006745.1NCBI GenBank
1 Several GenBank accessions are registered as L. gloveri (e.g., LC155436.1–LC155437.1, KY984986.1, MT903226.1, JQ681795.1); consistent with our phylogeny, these are treated as L. cheesemanii herein.
Table 6. Overall mean distances of inter-species and intra-species of the specimens used in the present study. Values are the average number of base substitutions per site for the mitochondrial COI gene, estimated under the Tamura–Nei (TN93) model from 1194 bp alignment.
Table 6. Overall mean distances of inter-species and intra-species of the specimens used in the present study. Values are the average number of base substitutions per site for the mitochondrial COI gene, estimated under the Tamura–Nei (TN93) model from 1194 bp alignment.
Species1234
1  L. spadiceus0.004
2  L. cheesemanii0.1110.001
3  L. wheeleri0.0630.1080.003
4  L. inermis0.1730.1770.1830.002
Notes. Diagonal values in bold indicate mean intraspecific distances; off-diagonal entries are mean interspecific distances.
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Lee, H.M.; Dong, C.M.; Lee, M.N.; Noh, E.S.; Kang, J.-H.; Kim, J.-M.; Kim, G.-D.; Kim, E.-M. Development of a Multiplex Polymerase Chain Reaction Method for the Simultaneous Identification of Four Species of Genus Lagocephalus (Chordata: Vertebrata). Fishes 2025, 10, 501. https://doi.org/10.3390/fishes10100501

AMA Style

Lee HM, Dong CM, Lee MN, Noh ES, Kang J-H, Kim J-M, Kim G-D, Kim E-M. Development of a Multiplex Polymerase Chain Reaction Method for the Simultaneous Identification of Four Species of Genus Lagocephalus (Chordata: Vertebrata). Fishes. 2025; 10(10):501. https://doi.org/10.3390/fishes10100501

Chicago/Turabian Style

Lee, Hye Min, Chun Mae Dong, Mi Nan Lee, Eun Soo Noh, Jung-Ha Kang, Jong-Myoung Kim, Gun-Do Kim, and Eun-Mi Kim. 2025. "Development of a Multiplex Polymerase Chain Reaction Method for the Simultaneous Identification of Four Species of Genus Lagocephalus (Chordata: Vertebrata)" Fishes 10, no. 10: 501. https://doi.org/10.3390/fishes10100501

APA Style

Lee, H. M., Dong, C. M., Lee, M. N., Noh, E. S., Kang, J.-H., Kim, J.-M., Kim, G.-D., & Kim, E.-M. (2025). Development of a Multiplex Polymerase Chain Reaction Method for the Simultaneous Identification of Four Species of Genus Lagocephalus (Chordata: Vertebrata). Fishes, 10(10), 501. https://doi.org/10.3390/fishes10100501

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