Methodology and application of PCR‐RFLP for species identification in tuna sashimi

Abstract The Thunnini, or tuna, comprise many species with very different commercial values. The principal raw tuna product on the market is sashimi, for which the species used is difficult to identify through conventional morphological analysis. The present study amplified the cytochrome b gene (Cytb) of 4 major tuna species used for preparing sashimi—yellowfin tuna (Thunnus albacares), southern bluefin tuna (Thunnus maccoyii), bigeye tuna (Thunnus obesus), and Atlantic bluefin tuna (Thunnus thynnus)—and 4 species commonly mislabeled as components of tuna sashimi—albacore tuna (Thunnus alalunga), skipjack tuna (Katsuwonus pelamis), striped marlin (Tetrapturus audax), and swordfish (Xiphias gladius). Polymerase chain reaction (PCR) amplicons were digested with 5 restriction enzymes—Eco147 I, Hinf I, Mbo I, Xag I, and Hind II—to obtain characteristic restriction maps of the above‐mentioned raw tuna species and the commonly mislabeled species. An identification method using PCR restriction fragment length polymorphism (PCR‐RFLP) was established and validated using 39 commercial tuna sashimi samples, which verified that this method provides results consistent with those obtained by classical sequencing. PCR‐RFLP has several advantages over classical sequencing, such as simplicity, speed and accuracy. This technique could support species identification for raw tuna and sashimi.

domestic and international markets. For example, fresh striped marlin (Tetrapturus audax), swordfish (Xiphias gladius), and albacore tuna are often mislabeled, while yellowfin and bigeye tuna are mislabeled in sushi (Lowenstein, Amato, & Kolokotronis, 2009). In addition, between 60% and 94% of the fish sold as red snapper in the United States are mislabeled (Marko et al., 2004). Seafood mislabeling circumvents consumer choice, which poses health risks, impacts the normal business order of the market, and undermines conservation strategies. Therefore, it is necessary to establish a method to identify the species of raw tuna used to produce sashimi.
Methods for species identification primarily include conventional morphological identification, protein-based analysis, and molecular biology techniques based on PCR. Morphological identification relies on the specialist's knowledge of systematic taxonomy and longterm experience, but it is difficult to differentiate similar species with close genetic relationships using this approach. Protein-based analyses, exemplified by isozyme analysis and immunological analysis, still lack stability and specificity. Therefore, molecular biology techniques are currently more widely used for species identification in research (Aranishi, Okimoto, & Izumi, 2005;Calo-Mata et al., 2009). This includes random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and forensically informative nucleotide sequencing (FINS). However, these methods also exhibit some limitations, such as complex procedures, poor stability, and poor reproducibility (Chapela et al., 2007;Larraín, Díaz, Lamas, Uribe, & Araneda, 2014;Rasmussen & Morrissey, 2009). The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique, which was developed based on fingerprinting technology, is most widely used for species identification because of its simple procedure, low cost, and good reproducibility Wilwet, Jeyasekaran, Shakila, Sivaraman, & Padmavathy, 2018). Moreover, it just needs PCR thermocycler and electrophoresis apparatus, which means most laboratories could bear the cost, that is also a realistic factor in most developing countries.
The present study investigated 4 major tuna species for sashimi preparation (yellowfin, southern and Atlantic bluefin, and bigeye tuna) and 4 commonly mislabeled species (albacore and skipjack tuna, striped marlin, and swordfish). A method for identifying raw tuna species was established using PCR amplification of the cytochrome b gene (Cytb) and restriction enzyme digestion, and was validated in commercial samples. This method provides a novel approach for the rapid and simple identification of raw tuna species.

| Samples
Eight kinds of species samples including albacore tuna (cm), yellowfin tuna (hu), Atlantic bluefin tuna (lq), southern bluefin tuna (ms), bigeye tuna (dd), striped marlin (da), swordfish (ji), and skipjack tuna (jy) from main fishing ocean were collected by Shandong ZhongLu Oceanic Fisheries Co., Ltd. Each kind of species had 20 muscle samples from 20 individual fish, which were morphologically identified by expert. The 39 commercial sashimi products labeled as yellowfin tuna (Shu), southern bluefin tuna (Sms), bigeye tuna (Sdd), and Atlantic bluefin tuna (Slq) were purchased randomly from 4 major supermarkets and 4 sushi restaurants with different suppliers and batches in Qingdao, China.

| DNA extraction
Thirty milligrams of fish flesh was cut into pieces and placed in a centrifuge tube. DNA extraction was performed according to the manufacturer's protocol. The DNA concentration and purity were measured using the NanoPhotometer Pearl. The samples were stored at −20°C.
Conditions for the PCR were as follows: pre-denaturation at 95°C for 10 min; 35 cycles of 94°C for 1 min, 53°C for 1 min, 72°C for 1 min; and extension at 72°C for 10 min.
The PCR amplicons were subjected to electrophoresis on a 1.0% agarose gel at 120 V for 40 min. The experimental results were observed and recorded using a gel imaging system. The PCR amplicons were sequenced by Sangon Biotech Co., Ltd. were selected from the NCBI Web site as reference genes. A phylogenetic tree was constructed using the MEGA 7 software (https:// www.megas oftwa re.net) with 1,000 bootstrap replicates. The

TA B L E 1 PCR system of Cyt b gene
Kimura 2-parameter was used for modeling, and neighbor-joining was used to construct the phylogenetic tree.

| Restriction enzyme analysis
The cleavage sites of the Cytb PCR amplicons were analyzed using DNAMAN (https://www.lynnon.com). Five species-specific restriction enzymes were selected and used to digest the PCR amplicons of the Cytb gene: Eco147 I, Hinf I, Mbo I, Xag I, and Hind II (for identification of samples dd and jy only).
The PCR amplicons (10.0 μl) were mixed with 1.0 μl of the respective restriction enzymes, 2.0 μl of 10× FastDigest ® Buffer, and 17.0 μl of deionized water. After enzyme digestion at 37°C for 60 min, the results were analyzed using agarose gel electrophoresis on a 3.0% gel.

| PCR amplification
A single fragment was amplified from each experimental sample. No fragment length polymorphism was detected. All fragments had a length of 357 bp (Figure 1), as expected.

| Sequence alignment and phylogenetic tree analysis
The sequencing products were aligned against the GenBank database to determine the Cytb region in the mitochondrial DNA (mtDNA). The phylogenetic tree constructed based on the Cytb gene of the 8 fish species is shown in Figure 2.  Figure 3 and are consistent with the predictions made using the DNAMAN software.

| Atlantic bluefin tuna (Thunnus thynnus)
The Cytb PCR amplicon of Atlantic bluefin tuna was digested by Hinf

| Southern bluefin tuna (Thunnus maccoyii)
The Cytb PCR amplicon of southern bluefin tuna was digested by

| Striped marlin (Tetrapturus audax)
The Cytb PCR amplicon of striped marlin was digested by Eco147 I   into 3 bands of 124-,

| Bigeye tuna (Thunnus obesus)
The hence, a single band of 357-bp length was obtained upon digestion by these enzymes. The enzyme digestion results for bigeye tuna are shown in Figure 9 and are consistent with the predictions made by the DNAMAN software.

| Results of commercial sample analysis
After the method of identifying raw tuna by PCR-RFLP was es- Detailed results of the identification of the commercial samples are shown in Table 3.

| D ISCUSS I ON
The rapid development of modern molecular biotechnology has led to an evolution in research approaches for species identification ranging from morphological, physiological, and biochemical levels, to the molecular level. The basis for identification has also evolved from the shape, size, color, bone, and structure of the fish, to macrobiomolecules, such as DNA, RNA, and proteins (Liu, Xu, Wu, Xie, & Feng, 2016). PCR-RFLP has been successfully applied for the identification of a variety of fish species and their products due to its simplicity and low cost. Wolf, Hübner, and Lüthy (1999) identified 8 species of carp using PCR-RFLP. Chakraborty, Aranishi, and Iwatsuki (2005) developed a PCR-RFLP approach that could successfully identify 3 closely related hairtail species (genus Trichiurus). Akasaki, Yanagimoto, Yamakami, Tomonaga, and Sato (2006) found that PCR-RFLP could rapidly identify 9 species of cod. Espiñeira, González-Lavín, Vieites, and Santaclara (2008) used PCR-RFLP to distinguish 7 species of anglerfish. Chen et al. (2012) successfully identified 5 species of pufferfish using PCR-RFLP analysis and a chip bioanalysis system. (2007)

ACK N OWLED G M ENTS
This work was supported by National Key Research and Development Program of China (2016YFF0201805).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.