Differentiation of Fish Species in Taiwan Strait by PCR-RFLP and Lab
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Food Control 44 (2014) 26e34 Contents lists available at ScienceDirect 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 b Xinglin Entry-Exit Inspection and Quarantine Bureau, Xiamen 361022, China c China National Accreditation Service for Conformity Assessment, Beijing 100088, China d Agilent Technologies Co. Ltd., Beijing 100102, China article info abstract Article history: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a- Received 9 January 2014 chip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 Received in revised form groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 3 March 2014 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested Accepted 6 March 2014 with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion Available online 27 March 2014 were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-a- chip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the Keywords: fi Fish species identification control of the adulteration of food with sh tissue content in Taiwan Strait. Ó Taiwan Strait 2014 Elsevier Ltd. All rights reserved. PCR-RFLP Lab-on-a-chip 1. Introduction large sampling volume. On the contrary, the new lab-on-a-chip system has advantages in simple and convenient manipulation In recent years, more and more cases of adulteration including and speed. Moreover, the lab-on-a-chip system is more secure due mislabeling, fraud and substitution with cheaper fish arose in the to eliminating the gel staining step and it has been used for dif- fish market. Hence, techniques that enable authentication of ferentiation of white fish (Dooley, Sage, Clarke, Brown, & Garrett, commercial fishery products are highly requested to guarantee 2005). accurate labeling and fraudulent substitution. Taiwan Strait is a 180-km-wide, 360-km-long, and 60-m-deep Species identification of fish is typically based on morphological channel separating Mainland China and Taiwan, and links the East characteristics. However, most of the external features allowing China Sea with the South China Sea. As an important fishing morphological identification of whole fish are not apparent after resource, there are more than 700 ocean fish species. Among them processing. DNA and protein based methods are also used for fish over one hundred are important commercial species and most of identification. Protein method has less advantage on processed them belong to Order Perciformes. Due to big variety in quality and sample in which protein may be already denatured or degraded. price, differentiate fish with similar morphology but different in DNA based methods tend to be more favorite and reliable due to commercial value is an important issue. their high specificity and sensitivity, strong stability and easy The purpose of the present work was to discriminate fish species application. In particular, because of ease-of-use, low cost and in Taiwan Strait using PCR-RFLP approach and lab-on-a-chip sys- repeatable, PCR-RFLP has become widely used for fish species tem, and offer a rapid, ease-of-use, safe and effective analysis identification, such as cod fish (Akasaki, Yanagimoto, Yamakami, method for label certification. Tomonaga, & Sato, 2006), mackerel (Aranishi, 2005), tuna (Lin & Hwang, 2007) and shark (Mendonca et al., 2009). 2. Material and methods The shortcomings of traditional electrophoresis include complicate preparation of the gel, long electrophoresis time and 2.1. Samples * Corresponding author. Tel.: þ86 592 3269929; fax: þ86 592 3269921. A total of 62 species (Table 1) were purchased from seafood E-mail address: [email protected] (S. Chen). markets of Xiamen, China, and taxonomically identified by an http://dx.doi.org/10.1016/j.foodcont.2014.03.019 0956-7135/Ó 2014 Elsevier Ltd. All rights reserved. S. Chen et al. / Food Control 44 (2014) 26e34 27 Table 1 Fish species collected for PCR-RFLP differentiation study. Order Family Species Order Family Species Perciformes Serranidae Epinephelus akaara Perciformes Priacanthidae Priacanthus tayenus Epinephelus moara Lutjanidae Lutjanus malabaricus Epinephelus fuscoguttatus Stromateidae Pampus argenteus Epinephelus coioides Formionidae Formio niger Epinephelus awoara Carangidae Trachinotus blochii Epinephelus quoyanus Decapterus maruadsi Promicrops lanceolatus Selaroides leptolepis Plectropomus leopardus Centrolophidae Psenopsis anomala Plectropomus maculatus Nemipteridae Nemipterus marginatus Cromileptes altivelis Scolopsis vosmeri Lateolabrax japonicus Labridae Cheilinus undulatus Siniperci chuatsi Siganidae Signus fuscescens Sciaenidae Larimichthys crocea Leiognathidae Leiognathus equulus Nibea albiflora Echeneidae Echeneis naucrates Otolithoides biauritus Gerreidae Gerres filamentosus Chrysochir aureus Kyphosidae Kyphosus bigibbus Sciaenops ocelcatus Microcanthus strigatus Larimichthys polyactis Tetraodontiformes Tetraodontidae Takifugu oblongus Collichthys niveatus Takifugu xanthopterus Collichthys lucidus Lagocephalus gloveri Johnius grypotus Lagocephalus wheeleri Coryphaenidae Hapalogenys mucronatus Lagocephalus lunaris Hapalogenys nigripinnis Torpediniformes Narkidae Narke japonica Plectorhinchus cinctus Rajiformes Rhynchobatidae Rhynchobatus djiddensis Parapristipoma trilineatum Rajiformes Rajidae Raja porosa Sparidae Acanthopagrus latus Rajiformes Dasyatidae Dasyatis zugei Acanthopagrus schlegel Orectolobiformes Orectolobidae Chiloscyllium plagiosum Parargyrops edita Carcharhiniformes Triakidae Mustelus griseus Pagrosomus major Scorpaeniformes Scorpaenidae Scorpaenopsis neglecta Rhabdosargus globiceps Scorpaeniformes Scorpaenidae Sebastiscus marmoratus Oplegnathidae Oplegnathus fasciatus Mugiliformes Polynemidae Elentheronema tetradactylum ichthyologist at Third Institute of Oceanography, State Oceanic 2.4. RFLP analysis Administration, P. R. China. Three individuals per species were tested. Fish muscle samples from live or frozen specimens of each PCR products were digested with three restriction enzymes species were obtained freshly and immediately processed by cut- DdeI, HaeIII and NlaIII (New England Biolabs, Inc.). Restriction re- ting small muscle portions (1e2 g) and subsequently put them actions were carried out in 5 mL final volume containing 2.5 mLof at À20 C until use. 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 2.2. DNA extraction 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 Total genomic DNA was extracted from fish muscle. Fresh and to achieve a final concentration of 10 mmol/L EDTA. 1 mL of reaction frozen pieces of samples (100e400 mg) were homogenized in mix was loaded on to Agilent DNA 1000 chips and analyzed on the 2100 Bioanalyzer. RFLP profile was detected and compared with the liquid N2. DNA was extracted using Qiagen DNeasy Blood & Tissue Kit (Spin-Column Protocol) and eluted in ultrapure water. Con- positions of size markers. centrations (ng/mL) of DNA were assessed at 260 nm using a Thermo nanodrop 2000C spectrophotometer. 3. Results 2.3. PCR amplification 3.1. DNA extraction and PCR amplification Cyt b gene primers (Russell et al., 2000) named L14735 (50- Among all the specimens, majorities (48 fish species) belong to AAAAACCACCGTTGTTATTCAACTA-30) and H15149 (50-GCICCTCAR- Perciformes. Others belong to Tetraodontiformes, Rajiformes, Tor- AATGAYATTTGTCCTCA-30) were synthesized by Takara Co., Ltd., and pediniformes, Scorpaeniformes, Mugiliformes and Orectolobi- used to amplify a fragment of 464 bp. The amplification reactions formes. Most of Perciformes samples came from Serranidae, were carried out in 25 mL final reaction volume containing 12.5 mLof Sciaenidae, Sparidae and Coryphaenidae. 2 Â PCR mix (Guangzhou Dongsheng Biotech Co., Ltd.), 1 mL of each All of the DNA extracts from 62 fish species produced 464 bp primer (10 mM), 1 mL of template DNA (2.5e25.0 ng/mL) and 9.5 mL PCR fragment respectively, indicated that the DNA were success- sterile distilled water. The amplification conditions according to fully extracted and the template DNA could be used for PCR Agilent DNA Fish ID Ensemble Protocol (Agilent Technologies Inc.) on amplification. BIO-RAD PTC-200 DNA Engine Thermal Cycler were: initial dena- turation at 95 C for 5 min, 40 cycles of amplification (denaturation at 3.2. RFLP analysis of fish species 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 3.2.1. RFLP analysis of groupers in family Serranidae detected and compared with the positions of size markers using Groupers are valuable fish species and in Taiwan Strait Agilent DNA 1000 Kit on the Agilent 2100 Bioanalyzer. mostly point to fishes belongs to genera Epinephelus, Promicrops, 28 S. Chen et al. / Food Control 44 (2014) 26e34 Plectropomus and Cromileptes. Since there are huge price differ- In present study, we collected 12