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Differentiation of fish species in Taiwan Strait by PCR-RFLP and lab-on-a-chip system
Food Control 44 (2014) 26e34
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Differentiation of fish species in Taiwan Strait by PCR-RFLP and lab-on-a-chip system Shuangya Chen a, *, Yongxiang Zhang b, Hong Li c, Jiahe Wang a, Weiling Chen a, Yu Zhou a, Shan Zhou d a
Xiamen Entry-Exit Inspection and Quarantine Technology Center, No. 2165, Jiangang Road, Haicang District, Xiamen 361026, China Xinglin Entry-Exit Inspection and Quarantine Bureau, Xiamen 361022, China China National Accreditation Service for Conformity Assessment, Beijing 100088, China d Agilent Technologies Co. Ltd., Beijing 100102, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2014 Received in revised form 3 March 2014 Accepted 6 March 2014 Available online 27 March 2014
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-achip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-achip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the control of the adulteration of food with fish tissue content in Taiwan Strait. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Fish species identification Taiwan Strait PCR-RFLP Lab-on-a-chip
1. Introduction In recent years, more and more cases of adulteration including mislabeling, fraud and substitution with cheaper fish arose in the fish market. Hence, techniques that enable authentication of commercial fishery products are highly requested to guarantee accurate labeling and fraudulent substitution. Species identification of fish is typically based on morphological characteristics. However, most of the external features allowing morphological identification of whole fish are not apparent after processing. DNA and protein based methods are also used for fish identification. Protein method has less advantage on processed sample in which protein may be already denatured or degraded. DNA based methods tend to be more favorite and reliable due to their high specificity and sensitivity, strong stability and easy application. In particular, because of ease-of-use, low cost and repeatable, PCR-RFLP has become widely used for fish species identification, such as cod fish (Akasaki, Yanagimoto, Yamakami, Tomonaga, & Sato, 2006), mackerel (Aranishi, 2005), tuna (Lin & Hwang, 2007) and shark (Mendonca et al., 2009). The shortcomings of traditional electrophoresis include complicate preparation of the gel, long electrophoresis time and * Corresponding author. Tel.: þ86 592 3269929; fax: þ86 592 3269921. E-mail address: [email protected] (S. Chen). http://dx.doi.org/10.1016/j.foodcont.2014.03.019 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved.
large sampling volume. On the contrary, the new lab-on-a-chip system has advantages in simple and convenient manipulation and speed. Moreover, the lab-on-a-chip system is more secure due to eliminating the gel staining step and it has been used for differentiation of white fish (Dooley, Sage, Clarke, Brown, & Garrett, 2005). Taiwan Strait is a 180-km-wide, 360-km-long, and 60-m-deep channel separating Mainland China and Taiwan, and links the East China Sea with the South China Sea. As an important fishing resource, there are more than 700 ocean fish species. Among them over one hundred are important commercial species and most of them belong to Order Perciformes. Due to big variety in quality and price, differentiate fish with similar morphology but different in commercial value is an important issue. The purpose of the present work was to discriminate fish species in Taiwan Strait using PCR-RFLP approach and lab-on-a-chip system, and offer a rapid, ease-of-use, safe and effective analysis method for label certification. 2. Material and methods 2.1. Samples A total of 62 species (Table 1) were purchased from seafood markets of Xiamen, China, and taxonomically identified by an
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Table 1 Fish species collected for PCR-RFLP differentiation study. Order
Family
Species
Order
Family
Species
Perciformes
Serranidae
Epinephelus akaara Epinephelus moara Epinephelus fuscoguttatus Epinephelus coioides Epinephelus awoara Epinephelus quoyanus Promicrops lanceolatus Plectropomus leopardus Plectropomus maculatus Cromileptes altivelis Lateolabrax japonicus Siniperci chuatsi Larimichthys crocea Nibea albiflora Otolithoides biauritus Chrysochir aureus Sciaenops ocelcatus Larimichthys polyactis Collichthys niveatus Collichthys lucidus Johnius grypotus Hapalogenys mucronatus Hapalogenys nigripinnis Plectorhinchus cinctus Parapristipoma trilineatum Acanthopagrus latus Acanthopagrus schlegel Parargyrops edita Pagrosomus major Rhabdosargus globiceps Oplegnathus fasciatus
Perciformes
Priacanthidae Lutjanidae Stromateidae Formionidae Carangidae
Priacanthus tayenus Lutjanus malabaricus Pampus argenteus Formio niger Trachinotus blochii Decapterus maruadsi Selaroides leptolepis Psenopsis anomala Nemipterus marginatus Scolopsis vosmeri Cheilinus undulatus Signus fuscescens Leiognathus equulus Echeneis naucrates Gerres filamentosus Kyphosus bigibbus Microcanthus strigatus Takifugu oblongus Takifugu xanthopterus Lagocephalus gloveri Lagocephalus wheeleri Lagocephalus lunaris Narke japonica Rhynchobatus djiddensis Raja porosa Dasyatis zugei Chiloscyllium plagiosum Mustelus griseus Scorpaenopsis neglecta Sebastiscus marmoratus Elentheronema tetradactylum
Sciaenidae
Coryphaenidae
Sparidae
Oplegnathidae
ichthyologist at Third Institute of Oceanography, State Oceanic Administration, P. R. China. Three individuals per species were tested. Fish muscle samples from live or frozen specimens of each species were obtained freshly and immediately processed by cutting small muscle portions (1e2 g) and subsequently put them at 20 C until use. 2.2. DNA extraction Total genomic DNA was extracted from fish muscle. Fresh and frozen pieces of samples (100e400 mg) were homogenized in liquid N2. DNA was extracted using Qiagen DNeasy Blood & Tissue Kit (Spin-Column Protocol) and eluted in ultrapure water. Concentrations (ng/mL) of DNA were assessed at 260 nm using a Thermo nanodrop 2000C spectrophotometer.
Centrolophidae Nemipteridae Labridae Siganidae Leiognathidae Echeneidae Gerreidae Kyphosidae Tetraodontiformes
Tetraodontidae
Torpediniformes Rajiformes Rajiformes Rajiformes Orectolobiformes Carcharhiniformes Scorpaeniformes Scorpaeniformes Mugiliformes
Narkidae Rhynchobatidae Rajidae Dasyatidae Orectolobidae Triakidae Scorpaenidae Scorpaenidae Polynemidae
2.4. RFLP analysis PCR products were digested with three restriction enzymes DdeI, HaeIII and NlaIII (New England Biolabs, Inc.). Restriction reactions were carried out in 5 mL final volume containing 2.5 mL of PCR product, 0.5 mL of restriction enzyme, and 0.5 mL of restriction enzyme buffer supplied by the manufactures and incubated at 37 C for 2 h. Reactions were terminated by incubation at 65 C for 20 min. PCR products (5 mL) were mixed with 1 mL 60 mmol/L EDTA to achieve a final concentration of 10 mmol/L EDTA. 1 mL of reaction mix was loaded on to Agilent DNA 1000 chips and analyzed on the 2100 Bioanalyzer. RFLP profile was detected and compared with the positions of size markers. 3. Results
2.3. PCR amplification
3.1. DNA extraction and PCR amplification
Cyt b gene primers (Russell et al., 2000) named L14735 (50 AAAAACCACCGTTGTTATTCAACTA-30 ) and H15149 (50 -GCICCTCARAATGAYATTTGTCCTCA-30 ) were synthesized by Takara Co., Ltd., and used to amplify a fragment of 464 bp. The amplification reactions were carried out in 25 mL final reaction volume containing 12.5 mL of 2 PCR mix (Guangzhou Dongsheng Biotech Co., Ltd.), 1 mL of each primer (10 mM), 1 mL of template DNA (2.5e25.0 ng/mL) and 9.5 mL sterile distilled water. The amplification conditions according to Agilent DNA Fish ID Ensemble Protocol (Agilent Technologies Inc.) on BIO-RAD PTC-200 DNA Engine Thermal Cycler were: initial denaturation at 95 C for 5 min, 40 cycles of amplification (denaturation at 95 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 30 s), and final extension at 72 C for 7 min. The PCR products were detected and compared with the positions of size markers using Agilent DNA 1000 Kit on the Agilent 2100 Bioanalyzer.
Among all the specimens, majorities (48 fish species) belong to Perciformes. Others belong to Tetraodontiformes, Rajiformes, Torpediniformes, Scorpaeniformes, Mugiliformes and Orectolobiformes. Most of Perciformes samples came from Serranidae, Sciaenidae, Sparidae and Coryphaenidae. All of the DNA extracts from 62 fish species produced 464 bp PCR fragment respectively, indicated that the DNA were successfully extracted and the template DNA could be used for PCR amplification. 3.2. RFLP analysis of fish species 3.2.1. RFLP analysis of groupers in family Serranidae Groupers are valuable fish species and in Taiwan Strait mostly point to fishes belongs to genera Epinephelus, Promicrops,
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Plectropomus and Cromileptes. Since there are huge price differences exist in different grouper species, meats of cheaper grouper are occasionally used to adulterate expensive ones, such as Cromileptes altivelis and Plectropomus leopardus. Sometimes, Lateolabrax japonicus and Siniperci chuatsi are also used to adulterate groupers. Previous publication showed that PCR approaches using 16S rRNA gene had been developed for grouper identification (Epinephelus and Mycteroperca species) from substitute species such as Nile perch (Lates niloticus) and wreck fish (Polyprion americanus) (Trotta et al., 2005). While some highly consumed expensive grouper species in China market, like P. leopardus and C. altivelis, were not belong to Epinephelus or Mycteroperca. Therefore this method was not appropriate in China market.
In present study, we collected 12 Serranidae species, including six Epinephelus species, two Plectropomus species, Promicrops lanceolatus, C. altivelis, L. japonicus and S. Chuatsi, and used a PCR-RFLP method to discriminate them. The restriction patterns of 12 fish species were shown in Fig. 1 and mean fragment sizes were shown in Table 2. Combined three restriction patterns, all the species except Epinephelus akaara and Epinephelus moara showed obvious differences in the patterns, suggesting the PCR-RFLP method is useful for differentiating L. japonicus and S. Chuatsi from grouper species, as well as differentiating C. altivelis and P. leopardus from other cheaper groupers. However, E. akaara and E. moara had same patterns, indicated these two grouper could not be distinguished by three enzymes. Furthermore, TaqI, MseI, EcoRI, BamHI and XhoI enzymes could not
Fig. 1. PCR-RFLP profiles of Serranidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, E. akaara; 2, E. moara; 3, E. fuscoguttatus; 4, E. coioides; 5, E. awoara; 6, E. quoyanus; 7, E. lanceolatus; 8, P. leopardus; 9, P. maculatus; 10, C. altivelis; 11, L. japonicus; 12, S. chuatsi.
S. Chen et al. / Food Control 44 (2014) 26e34 Table 2 PCR-RFLP fragment sizes of 12 Serranidae species. Sample
E. akaara E. moara E. fuscoguttatus E. coioides E. awoara E. quoyanus E. lanceolatus P. leopardus P. maculatus C. altivelis L. japonicus S. chuatsi
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Table 3 PCR-RFLP fragment sizes of 12 species belong to Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae.
Fragment size/bp DdeI
HaeIII
NlaIII
447 446 194, 269 447 462 457 451 469 115, 340 122, 330 89, 117, 259 447
119, 169, 177 119, 170, 178 178, 288 180, 295 117, 174 38, 182, 251 48, 133, 160 39, 103, 127, 189 38, 102, 128, 187 59, 75, 103, 233 114, 321 138, 147, 188
180, 285 181, 287 182, 284 182, 286 183, 280 495 181, 286 90, 101, 152 88, 98, 244 479 446 108, 373
Sample
H. mucronatus H. nigripinnis P. cinctus P. trilineatum A. latus A. schlegel P. edita P. major R. globiceps O. fasciatus P. tayenus L. malabaricus
Fragment size/bp DdeI
HaeIII
NlaIII
95, 123, 255, 261 122, 327 89, 120, 215 456 65, 392 462 169, 298 166, 256 396 200, 245 150, 289 458
182, 287 129, 342 129, 333 47, 134, 293 463 463 130, 149, 191 129, 148, 191 128, 332 131, 335 119, 130, 151 126, 150, 176
92, 106, 127, 168 95, 105, 297 184, 279 485 184, 285 88, 110, 270 94, 107, 291 91, 105, 287 82, 89, 285 102, 146, 186 92, 105, 256 364
Fig. 2. PCR-RFLP profiles of 12 fish species belong to Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, H. mucronatus; 2, H. nigripinnis; 3, P. cinctus; 4, P. trilineatum; 5, A. latus; 6, A. schlegel; 7, P. edita; 8, P. major; 9, R. globiceps; 10, O. fasciatus; 11, P. tayenus; 12, L. malabaricus.
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differentiate E. akaara and E. Moara either (data not shown), implied that other gene or other method such as DNA sequence analysis may be chosen to discriminate E. akaara and E. moara. 3.2.2. RFLP analysis of 12 morphologically similar species in family Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae Some fish species from the family Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae have a joint name “bream” in China and have similar morphological characteristics, which result in false identification, especially when the fish were
processed into filets. 12 common fish species (Hapalogenys mucronatus, Hapalogenys nigripinnis, Plectorhinchus cinctus, Parapristipoma trilineatum, Acanthopagrus latus, Acanthopagrus schlegel, Parargyrops edita, Pagrosomus major, Rhabdosargus globiceps, Oplegnathus fasciatus, Priacanthus tayenus and Lutjanus malabaricus) in the Xiamen fish market coming from five different fish families were chosen to compare with the PCR-RFLP files. PCR-RFLP patterns generated with enzymes DdeI, HaeIII and NlaIII were shown in Fig. 2 and mean fragment sizes were shown in Table 3. The great difference of the patterns showed that these morphologically similar species could be discriminated with three
Fig. 3. PCR-RFLP profiles of Sciaenidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, L. crocea; 2, N. albiflora; 3, O. biauritus; 4, C. aureus; 5, S. ocelcatus; 6, L. polyactis; 7, C. niveatus; 8, C. lucidus; 9, J. grypotus.)
S. Chen et al. / Food Control 44 (2014) 26e34 Table 4 PCR-RFLP fragment sizes of 9 Sciaenidae species. Sample
L. crocea N. albiflora O. biauritus C. aureus S. ocelcatus L. polyactis C. niveatus C. lucidus J. grypotus
Fragment size/bp DdeI
HaeIII
NlaIII
121, 332 125, 318 116,317 468 445 440 441 446 166, 302
131, 342 127, 159, 174 73,111,125,143 124, 133, 180 75, 117, 130, 146 128, 338 126, 336 130, 335 476
80, 92, 107, 219 102, 365 90,96,119,164 105, 370 91, 105, 285 90, 100, 287 88, 98, 286 91, 103, 285 187, 283
enzymes. Among them, the patterns of P. edita and P. major were very similar, the only difference between them was that PCR product of P. edita was digested to two fragments of around 169 bp and 298 bp by DdeI, while PCR product of P. major was digested to two fragments of around 166 bp and 256 bp. 3.2.3. RFLP analysis of 9 Sciaenidae species Large yellow croaker (Larimichthys crocea) is an economically important marine fish in China. Due to its high price, other cheaper fish species such as Nibea albiflora were found to be dyed with stain and labeled as yellow croaker by some vendors, especially in those frozen products. Thus, it is necessary to identify L. crocea from morphologically similar species. Some researches on genetic analysis of L. crocea have been reported (Cui, Liu, Li, You, & Chu, 2009; Wu, Liu, Cai, Ye, & Wang, 2011). However, the study on identification method of this species is very limited so far. Here, we collected 9 Sciaenidae species (L. crocea, N. albiflora, Otolithoides biauritus, Chrysochir aureus, Sciaenops ocelcatus, Larimichthys polyactis, Collichthys niveatus, Collichthys lucidus and Johnius grypotus) from Xiamen market and intended to use PCR-RFLP method to discriminate large yellow croaker from other croakers. PCR-RFLP patterns generated with enzymes were shown in Fig. 3 and mean fragment sizes were shown in Table 4. The result indicated that L. crocea could be easily discriminated from other eight fish species with three enzymes. While L. polyactis, C. niveatus and C. lucidus had the same RFLP patterns, indicating that they could not be discriminated by this method. Liu, Chen, Dai, and Zhuang (2010) compared 75 COI gene sequences (650 bp) of 30 species of Sciaenidae and found that the genetic differentiation (0.004) between C. niveatus and C. lucidus
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has not yet reached the level of species variation, which is different from the morphological conclusion. The small difference in COI gene sequences between two species also suggested that DNA method using COI gene would be insufficient for discriminate them. With the present result, other fast-evolving genes such as NADH dehydrogenase (ND) gene or internal transcribed spacer (ITS) sequence may be better for the discrimination of L. polyactis, C. niveatus and C. lucidus. 3.2.4. RFLP analysis of 5 puffer species The meat of puffer fish is delicious, nutritious and has high economic value. So that puffer fish are known as “the king of fish”. However, deaths by puffer fish toxin which has high toxicity, had been reported all over the world every year. There are more than forty puffer fish species in China, only seven or eight species among them are safe to eat. In order to confirm the puffer species of the food and protect the health of consumers, simple and effective identification method is needed to differentiate puffer fish species. Due to geography and temperature reasons, the distribution of puffer fish species is limited. In Taiwan Strait the most common puffer species are low toxic Takifugu oblongus, Takifugu xanthopterus, Lagocephalus gloveri, Lagocephalus wheeleri and toxic Lagocephalus lunaris. In present study, PCR-RFLP method and lab-on-achip system were applied to discriminate these five puffer species. Isoelectric focusing electrophoresis (IEF), sodium dodecyl sulfate e polyacrylamide gel electrophoresis (SDS-PAGE) and twodimensional electrophoresis (2DE) techniques using protein molecules were applied to identify puffer fish species (Chen & Hwang, 2002; Chen, Shiau, Noguchi, Wei, & Hwang, 2003; Chen, Shiau, Wei, & Hwang, 2004). PCR-RFLP was also reported using mitochondrial 16S rRNA gene to differentiate nine puffer fish species collected from the coastal area of Okinawa Islands in Japan (Ishizaki et al., 2006). A PCR method was also established for detecting puffer fish, while this method can not differentiate puffer fish species (Chen et al., 2010). PCR-RFLP patterns generated with enzymes DdeI, HaeIII and NlaIII were shown in Fig. 4 and mean fragment sizes were shown in Table 5. Results showed that patterns generated with enzymes DdeI were different between Takifugu and Lagocephalus. T. oblongus and T. xanthopterus both generated only one band using DdeI enzyme, suggesting that no DdeI site existed in the PCR amplicon. While all three Lagocephalus species generated two bands bigger than 100 bp.
Fig. 4. PCR-RFLP profiles of puffer species obtained on microchips. PCR products were digested by restriction enzymes (M, Molecular mass marker; 1, T. oblongus; 2, T. xanthopterus; 3, L. gloveri; 4, L. wheeleri; 5, L. lunaris.).
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Table 5 PCR-RFLP fragment sizes of 5 puffer species. Sample
T. oblongus T. xanthopterus L. gloveri L. wheeleri L. lunaris
Fragment size/bp DdeI
HaeIII
NlaIII
375 372 215, 236 178, 238 123, 325
63, 75, 305 138, 296 130, 299 119, 132, 183 45, 128, 255
467 460 476 71, 83, 93, 106, 164 126, 343
NlaIII profiles had three types, one band for T. oblongus, T. xanthopterus and L. gloveri, several small bands for L. wheeleri, and two bands of around 126 bp and 343 bp for L. lunaris. For HaeIII profiles, T. xanthopterus and L. gloveri produced similar patterns different from other samples. According to the above data, none of the puffer species could be identified only by one enzyme. Combined three enzymes patterns, these puffer species could be differentiated from each other.
3.2.5. RFLP analysis of 24 other commercial ocean fish species in Xiamen market It is significant to expand the range of fish species to verify the practicality of this PCR-RFLP method. Twenty four other commercial ocean fish species in Xiamen market were collected. Among the species, 15 samples belong to Perciformes. PCR-RFLP patterns were shown in Fig. 5. Other 9 samples belong to other orders and PCRRFLP patterns of these fishes were shown in Fig. 6. Mean fragment sizes of all 24 species were shown in Table 6. The results showed that profiles of each species had specificity and these 24 species could be differentiated via three enzymes. 4. Discussion The cytochrome b (cyt b) gene contains both slowly and rapidly evolving codon positions, as well as more conservation and more reliable regions. Therefore, this gene has been considered one of the most frequently used gene for phylogenetic studies at the species or genus level (Rasmussen & Morrissey, 2008). In the present study,
Fig. 5. PCR-RFLP profiles of 15 Perciformes species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, P. minor; 2, F. niger; 3, T. blochii; 4, D. maruadsi; 5, S. leptolepis; 6, P. anomala; 7, N. marginatus; 8, C. undulatus; 9, S. fuscescens; 10, L. equulus; 11, E. naucrates; 12, S. vosmeri; 13, G. filamentosus; 14, K. bigibbus; 15, M. strigatus.)
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Fig. 6. PCR-RFLP profiles of 9 fish species in other order obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, N. japonica; 2, R. djiddensis; 3, R. porosa; 4, D. zugei; 5, C. Plagiosum; 6, M. griseus; 7, S. neglecta; 8, S. marmoratus; 9, E. tetradactylum.)
we investigated the PCR-RFLP profiles of fish species in Taiwan Strait using cyt b gene to discriminate them. The result shows that the RFLP profile is specific, effective, rapid and suitable for food inspection. Although the method described above is useful for identification purposes, it is not suited for samples containing a mixture of different species as profiles become too complex to interpret. The method is not ideally appropriate for heavily processed products either, such as canned samples. A smaller (<200 bp) PCR target would be better suited for canned food.
Compare with conventional gel electrophoresis, lab-on-a-chip system made RFLP profiles more accurate, sensitive, rapid and easier to perform. DNA fragments greater than 25 bp of chip 1000 were expected detected using lab-on-a-chip system. However, some small fragments lower than 50 bp were inconsistently observed. This was probably because they were too close to the sizing limit (25 bp) or did not fluoresce sufficiently to be detected due to low concentration. These small fragments displayed low peak height on the electropherogram and need to be manually adjusted.
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Quarantine of the People’s Republic of China (AQSIQ) and a grant (No. 3502Z20134055) from Science and Technology Program of Xiamen City.
Table 6 PCR-RFLP fragment sizes of 24 fish species. Sample
P. minor F. niger T. blochii D. maruadsi S. leptolepis P. anomala N. marginatus C. undulatus S. fuscescens L. equulus E. naucrates S. vosmeri G. filamentosus K. bigibbus M. strigatus N. japonica R. djiddensis R. porosa D. zugei C. plagiosum M. griseus S. neglecta S. marmoratus E. tetradactylum
Fragment size/bp DdeI
HaeIII
NlaIII
465 454 161, 281 120, 341 161, 318 469 448 467 114, 122, 214 120, 296 510 482 479 481 121, 332 142, 337 479 490 478 488 236 444 92, 120, 250 218, 227
146, 316 78, 84, 130, 158, 186 130, 163, 172 444 46, 132, 136, 167 46, 68, 105, 127, 189 146, 318 44, 135, 152 137, 291 138, 146, 189 135, 345 108, 112, 139 127, 163, 175 132, 337 233, 239 175, 295 481 149, 327 175, 296 440 129, 304 472 135, 340 136, 146, 153
121, 162, 184 124, 137, 166 73, 92, 282 78, 100, 124, 190 98, 127, 167 68, 153, 283 91, 103, 281 54, 106, 318 106, 369 486 86, 97, 127, 173 92, 291 87, 374 84, 373 45, 87, 144, 205 450 88, 376 91, 210 87, 374 85, 94, 257 86, 161, 226 102, 366 96, 285 92, 103, 281
The size variation for fragment, including variation due to DNA folding, different chips and gel matrices, was 10%. Nevertheless, the difference in fragment size did not affect the ability to identify individual species. 5. Conclusion The fish species in Taiwan Strait have a relatively big market in South China especially in Fujian province. The PCR-RFLP method used in this study allows for rapid, straightforward and efficient authentication of the widely consumed fish species in China market. This method was inexpensive, easy to use, less experimental procedures, provided an approach to solve the cases of mislabeling, fraud and substitution, and will play an important role on food rapid detection in import and export inspection. Acknowledgments This work was supported by grants (No. 2011IK245, 2013IK185) from General Administration of Quality Supervision, Inspection &
References Akasaki, T., Yanagimoto, T., Yamakami, K., Tomonaga, H., & Sato, S. (2006). Species identification and PCR-RFLP analysis of cytochrome b gene in cod fish (order Gadiformes) products. Journal of Food Science, 71, 190e195. Aranishi, F. (2005). Rapid PCR-RFLP method for discrimination of imported and domestic mackerel. Marine Biotechnology, 7, 571e575. Chen, T. Y., & Hwang, D. F. (2002). Electrophoretic identification of muscle proteins in 7 puffer species. Journal of Food Science, 67, 936e942. Chen, T. Y., Shiau, C. Y., Noguchi, T., Wei, C. I., & Hwang, D. F. (2003). Identification of puffer fish species by native isoelectric focusing technique. Food Chemistry, 83, 475e479. Chen, T. Y., Shiau, C. Y., Wei, C. I., & Hwang, D. F. (2004). Preliminary study on puffer fish proteome species identification of puffer fish by two-dimensional electrophoresis. Journal of Agricultural and Food Chemistry, 52, 2236e2241. Chen, W., Zhao, C., Shao, B., Jiang, S., Yan, C., Li, S., et al. (2010). Primer screening and optimization of PCR system for the detection of puffer fish. Chinese Journal of Food Science, 31, 376e381. Cui, Z., Liu, Y., Li, C. P., You, F., & Chu, K. H. (2009). The complete mitochondrial genome of the large yellow croaker, Larimichthys crocea (Perciformes, Sciaenidae): unusual features of its control region and the phylogenetic position of the Sciaenidae. Gene, 432, 33e43. Dooley, J. J., Sage, H. D., Clarke, M. A., Brown, H. M., & Garrett, S. D. (2005). Fish species identification using PCReRFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study. Journal of Agricultural and Food Chemistry, 53, 3348e 3357. Ishizaki, S., Yokoyama, Y., Oshiro, N., Teruya, N., Nagashima, Y., Shiomi, K., et al. (2006). Molecular identification of pufferfish species using PCR amplification and restriction analysis of a segment of the 16S rRNA gene. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 1, 139e144. Lin, W. F., & Hwang, D. F. (2007). Application of PCR-RFLP analysis on species identification of canned tuna. Food Control, 18, 1050e1057. Liu, S., Chen, L., Dai, F., & Zhuang, Z. (2010). Application of DNA barcoding gene COI for classifying family Sciaenidae. Oceanologia et Limnologia Sinica, 41, 223e232. Mendonca, F. F., Hashimoto, D. T., Porto-Foresti, F., Oliveira, C., Gadig, O. B. F., & Foresti, F. (2009). Identification of the shark species Rhizoprionodon lalandii and R. porosus (Elasmobranchii, Carcharhinidae) by multiplex PCR and PCR-RFLP techniques. Molecular Ecology Resources, 9, 771e773. Rasmussen, R. S., & Morrissey, M. T. (2008). DNA-Based methods for the identification of commercial fish and seafood species. Comprehensive Reviews in Food Science and Food Safety, 7, 280e295. Russell, V. J., Hold, G. L., Pryde, S. E., Rehbein, H., Quinteiro, J., Rey-Mendez, M., et al. (2000). Use of restriction fragment length polymorphism to distinguish between Salmon species. Journal of Agricultural and Food Chemistry, 48, 2184e 2188. Trotta, M., Schonhuth, S., Pepe, T., Cortesi, M. L., Puyet, A., & Bautista, J. M. (2005). Multiples PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. Journal of Agricultural and Food Chemistry, 53, 2039e2045. Wu, X., Liu, X., Cai, M., Ye, X., & Wang, Z. (2011). Genetic analysis of farmed and wild stocks of large yellow croaker Larimichthys crocea by using microsatellite markers. African Journal of Biotechnology, 10, 5773e5784.
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Food Control journal homepage: www.elsevier.com/locate/foodcont
Differentiation of fish species in Taiwan Strait by PCR-RFLP and lab-on-a-chip system Shuangya Chen a, *, Yongxiang Zhang b, Hong Li c, Jiahe Wang a, Weiling Chen a, Yu Zhou a, Shan Zhou d a
Xiamen Entry-Exit Inspection and Quarantine Technology Center, No. 2165, Jiangang Road, Haicang District, Xiamen 361026, China Xinglin Entry-Exit Inspection and Quarantine Bureau, Xiamen 361022, China China National Accreditation Service for Conformity Assessment, Beijing 100088, China d Agilent Technologies Co. Ltd., Beijing 100102, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 January 2014 Received in revised form 3 March 2014 Accepted 6 March 2014 Available online 27 March 2014
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-achip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-achip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the control of the adulteration of food with fish tissue content in Taiwan Strait. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Fish species identification Taiwan Strait PCR-RFLP Lab-on-a-chip
1. Introduction In recent years, more and more cases of adulteration including mislabeling, fraud and substitution with cheaper fish arose in the fish market. Hence, techniques that enable authentication of commercial fishery products are highly requested to guarantee accurate labeling and fraudulent substitution. Species identification of fish is typically based on morphological characteristics. However, most of the external features allowing morphological identification of whole fish are not apparent after processing. DNA and protein based methods are also used for fish identification. Protein method has less advantage on processed sample in which protein may be already denatured or degraded. DNA based methods tend to be more favorite and reliable due to their high specificity and sensitivity, strong stability and easy application. In particular, because of ease-of-use, low cost and repeatable, PCR-RFLP has become widely used for fish species identification, such as cod fish (Akasaki, Yanagimoto, Yamakami, Tomonaga, & Sato, 2006), mackerel (Aranishi, 2005), tuna (Lin & Hwang, 2007) and shark (Mendonca et al., 2009). The shortcomings of traditional electrophoresis include complicate preparation of the gel, long electrophoresis time and * Corresponding author. Tel.: þ86 592 3269929; fax: þ86 592 3269921. E-mail address: [email protected] (S. Chen). http://dx.doi.org/10.1016/j.foodcont.2014.03.019 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved.
large sampling volume. On the contrary, the new lab-on-a-chip system has advantages in simple and convenient manipulation and speed. Moreover, the lab-on-a-chip system is more secure due to eliminating the gel staining step and it has been used for differentiation of white fish (Dooley, Sage, Clarke, Brown, & Garrett, 2005). Taiwan Strait is a 180-km-wide, 360-km-long, and 60-m-deep channel separating Mainland China and Taiwan, and links the East China Sea with the South China Sea. As an important fishing resource, there are more than 700 ocean fish species. Among them over one hundred are important commercial species and most of them belong to Order Perciformes. Due to big variety in quality and price, differentiate fish with similar morphology but different in commercial value is an important issue. The purpose of the present work was to discriminate fish species in Taiwan Strait using PCR-RFLP approach and lab-on-a-chip system, and offer a rapid, ease-of-use, safe and effective analysis method for label certification. 2. Material and methods 2.1. Samples A total of 62 species (Table 1) were purchased from seafood markets of Xiamen, China, and taxonomically identified by an
S. Chen et al. / Food Control 44 (2014) 26e34
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Table 1 Fish species collected for PCR-RFLP differentiation study. Order
Family
Species
Order
Family
Species
Perciformes
Serranidae
Epinephelus akaara Epinephelus moara Epinephelus fuscoguttatus Epinephelus coioides Epinephelus awoara Epinephelus quoyanus Promicrops lanceolatus Plectropomus leopardus Plectropomus maculatus Cromileptes altivelis Lateolabrax japonicus Siniperci chuatsi Larimichthys crocea Nibea albiflora Otolithoides biauritus Chrysochir aureus Sciaenops ocelcatus Larimichthys polyactis Collichthys niveatus Collichthys lucidus Johnius grypotus Hapalogenys mucronatus Hapalogenys nigripinnis Plectorhinchus cinctus Parapristipoma trilineatum Acanthopagrus latus Acanthopagrus schlegel Parargyrops edita Pagrosomus major Rhabdosargus globiceps Oplegnathus fasciatus
Perciformes
Priacanthidae Lutjanidae Stromateidae Formionidae Carangidae
Priacanthus tayenus Lutjanus malabaricus Pampus argenteus Formio niger Trachinotus blochii Decapterus maruadsi Selaroides leptolepis Psenopsis anomala Nemipterus marginatus Scolopsis vosmeri Cheilinus undulatus Signus fuscescens Leiognathus equulus Echeneis naucrates Gerres filamentosus Kyphosus bigibbus Microcanthus strigatus Takifugu oblongus Takifugu xanthopterus Lagocephalus gloveri Lagocephalus wheeleri Lagocephalus lunaris Narke japonica Rhynchobatus djiddensis Raja porosa Dasyatis zugei Chiloscyllium plagiosum Mustelus griseus Scorpaenopsis neglecta Sebastiscus marmoratus Elentheronema tetradactylum
Sciaenidae
Coryphaenidae
Sparidae
Oplegnathidae
ichthyologist at Third Institute of Oceanography, State Oceanic Administration, P. R. China. Three individuals per species were tested. Fish muscle samples from live or frozen specimens of each species were obtained freshly and immediately processed by cutting small muscle portions (1e2 g) and subsequently put them at 20 C until use. 2.2. DNA extraction Total genomic DNA was extracted from fish muscle. Fresh and frozen pieces of samples (100e400 mg) were homogenized in liquid N2. DNA was extracted using Qiagen DNeasy Blood & Tissue Kit (Spin-Column Protocol) and eluted in ultrapure water. Concentrations (ng/mL) of DNA were assessed at 260 nm using a Thermo nanodrop 2000C spectrophotometer.
Centrolophidae Nemipteridae Labridae Siganidae Leiognathidae Echeneidae Gerreidae Kyphosidae Tetraodontiformes
Tetraodontidae
Torpediniformes Rajiformes Rajiformes Rajiformes Orectolobiformes Carcharhiniformes Scorpaeniformes Scorpaeniformes Mugiliformes
Narkidae Rhynchobatidae Rajidae Dasyatidae Orectolobidae Triakidae Scorpaenidae Scorpaenidae Polynemidae
2.4. RFLP analysis PCR products were digested with three restriction enzymes DdeI, HaeIII and NlaIII (New England Biolabs, Inc.). Restriction reactions were carried out in 5 mL final volume containing 2.5 mL of PCR product, 0.5 mL of restriction enzyme, and 0.5 mL of restriction enzyme buffer supplied by the manufactures and incubated at 37 C for 2 h. Reactions were terminated by incubation at 65 C for 20 min. PCR products (5 mL) were mixed with 1 mL 60 mmol/L EDTA to achieve a final concentration of 10 mmol/L EDTA. 1 mL of reaction mix was loaded on to Agilent DNA 1000 chips and analyzed on the 2100 Bioanalyzer. RFLP profile was detected and compared with the positions of size markers. 3. Results
2.3. PCR amplification
3.1. DNA extraction and PCR amplification
Cyt b gene primers (Russell et al., 2000) named L14735 (50 AAAAACCACCGTTGTTATTCAACTA-30 ) and H15149 (50 -GCICCTCARAATGAYATTTGTCCTCA-30 ) were synthesized by Takara Co., Ltd., and used to amplify a fragment of 464 bp. The amplification reactions were carried out in 25 mL final reaction volume containing 12.5 mL of 2 PCR mix (Guangzhou Dongsheng Biotech Co., Ltd.), 1 mL of each primer (10 mM), 1 mL of template DNA (2.5e25.0 ng/mL) and 9.5 mL sterile distilled water. The amplification conditions according to Agilent DNA Fish ID Ensemble Protocol (Agilent Technologies Inc.) on BIO-RAD PTC-200 DNA Engine Thermal Cycler were: initial denaturation at 95 C for 5 min, 40 cycles of amplification (denaturation at 95 C for 30 s, annealing at 50 C for 30 s, and extension at 72 C for 30 s), and final extension at 72 C for 7 min. The PCR products were detected and compared with the positions of size markers using Agilent DNA 1000 Kit on the Agilent 2100 Bioanalyzer.
Among all the specimens, majorities (48 fish species) belong to Perciformes. Others belong to Tetraodontiformes, Rajiformes, Torpediniformes, Scorpaeniformes, Mugiliformes and Orectolobiformes. Most of Perciformes samples came from Serranidae, Sciaenidae, Sparidae and Coryphaenidae. All of the DNA extracts from 62 fish species produced 464 bp PCR fragment respectively, indicated that the DNA were successfully extracted and the template DNA could be used for PCR amplification. 3.2. RFLP analysis of fish species 3.2.1. RFLP analysis of groupers in family Serranidae Groupers are valuable fish species and in Taiwan Strait mostly point to fishes belongs to genera Epinephelus, Promicrops,
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Plectropomus and Cromileptes. Since there are huge price differences exist in different grouper species, meats of cheaper grouper are occasionally used to adulterate expensive ones, such as Cromileptes altivelis and Plectropomus leopardus. Sometimes, Lateolabrax japonicus and Siniperci chuatsi are also used to adulterate groupers. Previous publication showed that PCR approaches using 16S rRNA gene had been developed for grouper identification (Epinephelus and Mycteroperca species) from substitute species such as Nile perch (Lates niloticus) and wreck fish (Polyprion americanus) (Trotta et al., 2005). While some highly consumed expensive grouper species in China market, like P. leopardus and C. altivelis, were not belong to Epinephelus or Mycteroperca. Therefore this method was not appropriate in China market.
In present study, we collected 12 Serranidae species, including six Epinephelus species, two Plectropomus species, Promicrops lanceolatus, C. altivelis, L. japonicus and S. Chuatsi, and used a PCR-RFLP method to discriminate them. The restriction patterns of 12 fish species were shown in Fig. 1 and mean fragment sizes were shown in Table 2. Combined three restriction patterns, all the species except Epinephelus akaara and Epinephelus moara showed obvious differences in the patterns, suggesting the PCR-RFLP method is useful for differentiating L. japonicus and S. Chuatsi from grouper species, as well as differentiating C. altivelis and P. leopardus from other cheaper groupers. However, E. akaara and E. moara had same patterns, indicated these two grouper could not be distinguished by three enzymes. Furthermore, TaqI, MseI, EcoRI, BamHI and XhoI enzymes could not
Fig. 1. PCR-RFLP profiles of Serranidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, E. akaara; 2, E. moara; 3, E. fuscoguttatus; 4, E. coioides; 5, E. awoara; 6, E. quoyanus; 7, E. lanceolatus; 8, P. leopardus; 9, P. maculatus; 10, C. altivelis; 11, L. japonicus; 12, S. chuatsi.
S. Chen et al. / Food Control 44 (2014) 26e34 Table 2 PCR-RFLP fragment sizes of 12 Serranidae species. Sample
E. akaara E. moara E. fuscoguttatus E. coioides E. awoara E. quoyanus E. lanceolatus P. leopardus P. maculatus C. altivelis L. japonicus S. chuatsi
29
Table 3 PCR-RFLP fragment sizes of 12 species belong to Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae.
Fragment size/bp DdeI
HaeIII
NlaIII
447 446 194, 269 447 462 457 451 469 115, 340 122, 330 89, 117, 259 447
119, 169, 177 119, 170, 178 178, 288 180, 295 117, 174 38, 182, 251 48, 133, 160 39, 103, 127, 189 38, 102, 128, 187 59, 75, 103, 233 114, 321 138, 147, 188
180, 285 181, 287 182, 284 182, 286 183, 280 495 181, 286 90, 101, 152 88, 98, 244 479 446 108, 373
Sample
H. mucronatus H. nigripinnis P. cinctus P. trilineatum A. latus A. schlegel P. edita P. major R. globiceps O. fasciatus P. tayenus L. malabaricus
Fragment size/bp DdeI
HaeIII
NlaIII
95, 123, 255, 261 122, 327 89, 120, 215 456 65, 392 462 169, 298 166, 256 396 200, 245 150, 289 458
182, 287 129, 342 129, 333 47, 134, 293 463 463 130, 149, 191 129, 148, 191 128, 332 131, 335 119, 130, 151 126, 150, 176
92, 106, 127, 168 95, 105, 297 184, 279 485 184, 285 88, 110, 270 94, 107, 291 91, 105, 287 82, 89, 285 102, 146, 186 92, 105, 256 364
Fig. 2. PCR-RFLP profiles of 12 fish species belong to Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). M, Molecular mass marker; 1, H. mucronatus; 2, H. nigripinnis; 3, P. cinctus; 4, P. trilineatum; 5, A. latus; 6, A. schlegel; 7, P. edita; 8, P. major; 9, R. globiceps; 10, O. fasciatus; 11, P. tayenus; 12, L. malabaricus.
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differentiate E. akaara and E. Moara either (data not shown), implied that other gene or other method such as DNA sequence analysis may be chosen to discriminate E. akaara and E. moara. 3.2.2. RFLP analysis of 12 morphologically similar species in family Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae Some fish species from the family Coryphaenidae, Sparidae, Oplegnathidae, Priacanthidae and Lutjanidae have a joint name “bream” in China and have similar morphological characteristics, which result in false identification, especially when the fish were
processed into filets. 12 common fish species (Hapalogenys mucronatus, Hapalogenys nigripinnis, Plectorhinchus cinctus, Parapristipoma trilineatum, Acanthopagrus latus, Acanthopagrus schlegel, Parargyrops edita, Pagrosomus major, Rhabdosargus globiceps, Oplegnathus fasciatus, Priacanthus tayenus and Lutjanus malabaricus) in the Xiamen fish market coming from five different fish families were chosen to compare with the PCR-RFLP files. PCR-RFLP patterns generated with enzymes DdeI, HaeIII and NlaIII were shown in Fig. 2 and mean fragment sizes were shown in Table 3. The great difference of the patterns showed that these morphologically similar species could be discriminated with three
Fig. 3. PCR-RFLP profiles of Sciaenidae species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, L. crocea; 2, N. albiflora; 3, O. biauritus; 4, C. aureus; 5, S. ocelcatus; 6, L. polyactis; 7, C. niveatus; 8, C. lucidus; 9, J. grypotus.)
S. Chen et al. / Food Control 44 (2014) 26e34 Table 4 PCR-RFLP fragment sizes of 9 Sciaenidae species. Sample
L. crocea N. albiflora O. biauritus C. aureus S. ocelcatus L. polyactis C. niveatus C. lucidus J. grypotus
Fragment size/bp DdeI
HaeIII
NlaIII
121, 332 125, 318 116,317 468 445 440 441 446 166, 302
131, 342 127, 159, 174 73,111,125,143 124, 133, 180 75, 117, 130, 146 128, 338 126, 336 130, 335 476
80, 92, 107, 219 102, 365 90,96,119,164 105, 370 91, 105, 285 90, 100, 287 88, 98, 286 91, 103, 285 187, 283
enzymes. Among them, the patterns of P. edita and P. major were very similar, the only difference between them was that PCR product of P. edita was digested to two fragments of around 169 bp and 298 bp by DdeI, while PCR product of P. major was digested to two fragments of around 166 bp and 256 bp. 3.2.3. RFLP analysis of 9 Sciaenidae species Large yellow croaker (Larimichthys crocea) is an economically important marine fish in China. Due to its high price, other cheaper fish species such as Nibea albiflora were found to be dyed with stain and labeled as yellow croaker by some vendors, especially in those frozen products. Thus, it is necessary to identify L. crocea from morphologically similar species. Some researches on genetic analysis of L. crocea have been reported (Cui, Liu, Li, You, & Chu, 2009; Wu, Liu, Cai, Ye, & Wang, 2011). However, the study on identification method of this species is very limited so far. Here, we collected 9 Sciaenidae species (L. crocea, N. albiflora, Otolithoides biauritus, Chrysochir aureus, Sciaenops ocelcatus, Larimichthys polyactis, Collichthys niveatus, Collichthys lucidus and Johnius grypotus) from Xiamen market and intended to use PCR-RFLP method to discriminate large yellow croaker from other croakers. PCR-RFLP patterns generated with enzymes were shown in Fig. 3 and mean fragment sizes were shown in Table 4. The result indicated that L. crocea could be easily discriminated from other eight fish species with three enzymes. While L. polyactis, C. niveatus and C. lucidus had the same RFLP patterns, indicating that they could not be discriminated by this method. Liu, Chen, Dai, and Zhuang (2010) compared 75 COI gene sequences (650 bp) of 30 species of Sciaenidae and found that the genetic differentiation (0.004) between C. niveatus and C. lucidus
31
has not yet reached the level of species variation, which is different from the morphological conclusion. The small difference in COI gene sequences between two species also suggested that DNA method using COI gene would be insufficient for discriminate them. With the present result, other fast-evolving genes such as NADH dehydrogenase (ND) gene or internal transcribed spacer (ITS) sequence may be better for the discrimination of L. polyactis, C. niveatus and C. lucidus. 3.2.4. RFLP analysis of 5 puffer species The meat of puffer fish is delicious, nutritious and has high economic value. So that puffer fish are known as “the king of fish”. However, deaths by puffer fish toxin which has high toxicity, had been reported all over the world every year. There are more than forty puffer fish species in China, only seven or eight species among them are safe to eat. In order to confirm the puffer species of the food and protect the health of consumers, simple and effective identification method is needed to differentiate puffer fish species. Due to geography and temperature reasons, the distribution of puffer fish species is limited. In Taiwan Strait the most common puffer species are low toxic Takifugu oblongus, Takifugu xanthopterus, Lagocephalus gloveri, Lagocephalus wheeleri and toxic Lagocephalus lunaris. In present study, PCR-RFLP method and lab-on-achip system were applied to discriminate these five puffer species. Isoelectric focusing electrophoresis (IEF), sodium dodecyl sulfate e polyacrylamide gel electrophoresis (SDS-PAGE) and twodimensional electrophoresis (2DE) techniques using protein molecules were applied to identify puffer fish species (Chen & Hwang, 2002; Chen, Shiau, Noguchi, Wei, & Hwang, 2003; Chen, Shiau, Wei, & Hwang, 2004). PCR-RFLP was also reported using mitochondrial 16S rRNA gene to differentiate nine puffer fish species collected from the coastal area of Okinawa Islands in Japan (Ishizaki et al., 2006). A PCR method was also established for detecting puffer fish, while this method can not differentiate puffer fish species (Chen et al., 2010). PCR-RFLP patterns generated with enzymes DdeI, HaeIII and NlaIII were shown in Fig. 4 and mean fragment sizes were shown in Table 5. Results showed that patterns generated with enzymes DdeI were different between Takifugu and Lagocephalus. T. oblongus and T. xanthopterus both generated only one band using DdeI enzyme, suggesting that no DdeI site existed in the PCR amplicon. While all three Lagocephalus species generated two bands bigger than 100 bp.
Fig. 4. PCR-RFLP profiles of puffer species obtained on microchips. PCR products were digested by restriction enzymes (M, Molecular mass marker; 1, T. oblongus; 2, T. xanthopterus; 3, L. gloveri; 4, L. wheeleri; 5, L. lunaris.).
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Table 5 PCR-RFLP fragment sizes of 5 puffer species. Sample
T. oblongus T. xanthopterus L. gloveri L. wheeleri L. lunaris
Fragment size/bp DdeI
HaeIII
NlaIII
375 372 215, 236 178, 238 123, 325
63, 75, 305 138, 296 130, 299 119, 132, 183 45, 128, 255
467 460 476 71, 83, 93, 106, 164 126, 343
NlaIII profiles had three types, one band for T. oblongus, T. xanthopterus and L. gloveri, several small bands for L. wheeleri, and two bands of around 126 bp and 343 bp for L. lunaris. For HaeIII profiles, T. xanthopterus and L. gloveri produced similar patterns different from other samples. According to the above data, none of the puffer species could be identified only by one enzyme. Combined three enzymes patterns, these puffer species could be differentiated from each other.
3.2.5. RFLP analysis of 24 other commercial ocean fish species in Xiamen market It is significant to expand the range of fish species to verify the practicality of this PCR-RFLP method. Twenty four other commercial ocean fish species in Xiamen market were collected. Among the species, 15 samples belong to Perciformes. PCR-RFLP patterns were shown in Fig. 5. Other 9 samples belong to other orders and PCRRFLP patterns of these fishes were shown in Fig. 6. Mean fragment sizes of all 24 species were shown in Table 6. The results showed that profiles of each species had specificity and these 24 species could be differentiated via three enzymes. 4. Discussion The cytochrome b (cyt b) gene contains both slowly and rapidly evolving codon positions, as well as more conservation and more reliable regions. Therefore, this gene has been considered one of the most frequently used gene for phylogenetic studies at the species or genus level (Rasmussen & Morrissey, 2008). In the present study,
Fig. 5. PCR-RFLP profiles of 15 Perciformes species obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, P. minor; 2, F. niger; 3, T. blochii; 4, D. maruadsi; 5, S. leptolepis; 6, P. anomala; 7, N. marginatus; 8, C. undulatus; 9, S. fuscescens; 10, L. equulus; 11, E. naucrates; 12, S. vosmeri; 13, G. filamentosus; 14, K. bigibbus; 15, M. strigatus.)
S. Chen et al. / Food Control 44 (2014) 26e34
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Fig. 6. PCR-RFLP profiles of 9 fish species in other order obtained on microchips. PCR products were digested by restriction enzymes (A, DdeI; B, HaeIII; C, NlaIII). (M, Molecular mass marker; 1, N. japonica; 2, R. djiddensis; 3, R. porosa; 4, D. zugei; 5, C. Plagiosum; 6, M. griseus; 7, S. neglecta; 8, S. marmoratus; 9, E. tetradactylum.)
we investigated the PCR-RFLP profiles of fish species in Taiwan Strait using cyt b gene to discriminate them. The result shows that the RFLP profile is specific, effective, rapid and suitable for food inspection. Although the method described above is useful for identification purposes, it is not suited for samples containing a mixture of different species as profiles become too complex to interpret. The method is not ideally appropriate for heavily processed products either, such as canned samples. A smaller (<200 bp) PCR target would be better suited for canned food.
Compare with conventional gel electrophoresis, lab-on-a-chip system made RFLP profiles more accurate, sensitive, rapid and easier to perform. DNA fragments greater than 25 bp of chip 1000 were expected detected using lab-on-a-chip system. However, some small fragments lower than 50 bp were inconsistently observed. This was probably because they were too close to the sizing limit (25 bp) or did not fluoresce sufficiently to be detected due to low concentration. These small fragments displayed low peak height on the electropherogram and need to be manually adjusted.
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Quarantine of the People’s Republic of China (AQSIQ) and a grant (No. 3502Z20134055) from Science and Technology Program of Xiamen City.
Table 6 PCR-RFLP fragment sizes of 24 fish species. Sample
P. minor F. niger T. blochii D. maruadsi S. leptolepis P. anomala N. marginatus C. undulatus S. fuscescens L. equulus E. naucrates S. vosmeri G. filamentosus K. bigibbus M. strigatus N. japonica R. djiddensis R. porosa D. zugei C. plagiosum M. griseus S. neglecta S. marmoratus E. tetradactylum
Fragment size/bp DdeI
HaeIII
NlaIII
465 454 161, 281 120, 341 161, 318 469 448 467 114, 122, 214 120, 296 510 482 479 481 121, 332 142, 337 479 490 478 488 236 444 92, 120, 250 218, 227
146, 316 78, 84, 130, 158, 186 130, 163, 172 444 46, 132, 136, 167 46, 68, 105, 127, 189 146, 318 44, 135, 152 137, 291 138, 146, 189 135, 345 108, 112, 139 127, 163, 175 132, 337 233, 239 175, 295 481 149, 327 175, 296 440 129, 304 472 135, 340 136, 146, 153
121, 162, 184 124, 137, 166 73, 92, 282 78, 100, 124, 190 98, 127, 167 68, 153, 283 91, 103, 281 54, 106, 318 106, 369 486 86, 97, 127, 173 92, 291 87, 374 84, 373 45, 87, 144, 205 450 88, 376 91, 210 87, 374 85, 94, 257 86, 161, 226 102, 366 96, 285 92, 103, 281
The size variation for fragment, including variation due to DNA folding, different chips and gel matrices, was 10%. Nevertheless, the difference in fragment size did not affect the ability to identify individual species. 5. Conclusion The fish species in Taiwan Strait have a relatively big market in South China especially in Fujian province. The PCR-RFLP method used in this study allows for rapid, straightforward and efficient authentication of the widely consumed fish species in China market. This method was inexpensive, easy to use, less experimental procedures, provided an approach to solve the cases of mislabeling, fraud and substitution, and will play an important role on food rapid detection in import and export inspection. Acknowledgments This work was supported by grants (No. 2011IK245, 2013IK185) from General Administration of Quality Supervision, Inspection &
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