Bull Vet Inst Pulawy 54, 49-53, 2010

PCR-RFLP ANALYSIS OF DNA FOR THE DIFFERENTIATION OF FISH SPECIES IN SEAFOOD SAMPLES

MOJMIR NEBOLA, GABRIELA BORILOVA, AND JANKA KASALOVA

Department of Meat Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno, 612 42 Brno, Czech Republic

nebolam@vfu.cz gborilova@vfu.cz

Received for publication November 4, 2009

Abstract Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis was used to identify fish

species and to perform their authentication in commercial seafood products. Universal primers were used for PCR amplification of 464-bp long fragments of the mitochondrial cytochrome b gene. The PCR products were digested with restriction enzymes AluI, HinfI, HaeIII, DdeI, NlaIII, HincII, and MboII. Sixty fish samples were obtained from the local markets in the Czech Republic. In 47 samples (78.3%), the results were in agreement with declarations of the producers and 10 samples (16.7%) contained other fish species. There were not great differences between fresh fillets (chilled, frozen etc.) and heat-processed foodstuffs (tinned, smoked, etc.). The correct designation was in 72.3% and 81.6% of samples, respectively. Even if in three cases the analysis was unsuccessful the method is useful for the control of the adulteration of food with fish tissue content and in this way it contributes to better consumer awareness.

Key words: fish, identification, PCR-RFLP, seafood products.

According to the European Union labelling

regulation (9), commercial and scientific names of fish should be included in the label of seafood products. Incorrect labelling may be the result of inter- and intraspecies substitutions in order to satisfy market demand for lesser-exploited fish species, and or to name less expensive or low-quality species as more expensive. The implication of incorrect labelling has not only an economic impact for the customer, but it may also have a negative sequel such as allergy or toxic syndromes. The use of biochemical markers, such as proteins, has provided a tool for controlling whether fish and their products are in compliance with labelling regulations (15). Protein electrophoresis and immunological methods are well-established procedures for the identification of raw fish (19).

More recently, DNA molecules have been the target compounds for species identification due to their high stability compared with proteins. DNA based–methods consist of the highly-specific amplification of one or more DNA fragments by means of PCR and in the analysis of the restriction fragment length polymorphism (RFLP). These methods are very simple, inexpensive, and promising techniques. The primers designed by Burgener and Hübner (5) amplify the mitochondrial cytochrome b gene, and subsequent RFLP analysis allows the identification of a broad spectrum of fish (Tuna, Swordfish, Salmon, Plaice, Sole). The method has been used for the identification of five groups of fish (11), and it has been tested in 12

European laboratories. The method is reported to have a 96% success rate in the identification of samples of unknown origin. Calo-Mata et al. (7) identified 16 species of gadoid fish by PCR and DNA sequencing. PCR-RFLP has also been described as useful technique for the differentiation between Salmon species (20), Flatfish (21), Sturgeon-caviar (14) and 23 Bony Fish over a wide range of species (22). These analyses were also accomplished on other genes (5S rRNA, 16S rRNA, etc.) for the detection of intraspecies and subspecies variations in Tuna (18), Hake (17), Calamare (8), and Mackerel (2). The aim of this work was the PCR-RFLP identification and differentiation of fish species in seafood products on the markets in the Czech Republic.

Material and Methods

Sixty fish samples belonging to the families Gadidae, Scombridae, Gemphylidae, Salmonidae, and Anguillidae were obtained on the local markets; 22 of them were fresh or frozen and 38 samples heat treated and processed minced products, fish smoked at ≥ 70°C, spreads, meal made to imitate crab or salmon, etc. DNA from fresh and frozen tissues was isolated using the standard DNeasy® Blood & Tissue Kit (Qiagen, Germany) and from processed food samples with the NucleoSpin® Tissue Kit (Macherey-Nagel, Germany). The primers H15149AD and L14735 used for the amplification of the 464 bp region of the

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cytochrome b gene were described by Burgener (6). The mixture contained 12.5 µl of PPP Master Mix (Top-Bio, Czech Republic), 10 pmol of each primer, and 100 ng of DNA template. PCR conditions: initial denaturation 94ºC (5 min), 35 cycles of denaturation 94ºC (90 s), annealing 50ºC (90 s), extension 72ºC (90 s), and final extension step 72ºC (7 min). The PCR products were digested by 10 U of restriction endonucleases (RE) in the 15 µl of reaction mixture. Restriction endonucleases AluI, HinfI, HaeIII, DdeI, NlaIII, HincII, and MboII (New England Biolabs) were chosen according to Wolf et al. (22) and Calo-Mata et al. (7). The PCR products and restriction fragments sizes were analysed by gel electrophoresis in 3% agarose gel stained with ethidium bromide.

Results and Discussion Sixty different seafood products containing

meat of commercial fish species of the families Gadidae, Scombridae, Gemphylidae, Salmonidae, and Anguillidae were subjected to DNA analysis by PCR-RFLP methods. Twenty-two samples were frozen fillets and 38 food products subjected to technological treatments.

Species identification of the family Gadidae. Restriction enzymes DdeI, NlaIII, and MboII were used for the analysis of 22 samples: eight samples were chilled or frozen, 14 heat-treated fish, spreads, and processed minced products (e.g. Surimi fingers, Codfish a la salmon). Profiles obtained corresponded to four species: Atlantic Cod (Gadus morhua), Alaska Pollock (Theragra chalcogramma), Blue Whiting (Micromesistius poutassou), and Poor Cod (Trisopterus minutus) (7, 22). The digestion with endonuclease NlaIII enabled differentiating Blue Whiting and Poor Cod but the similarity of Atlantic Cod and Alaska Pollock profiles often made difficult the recognition of small fragments. The DdeI and MboII restriction patterns permit the differentiation of all fish species analysed (Fig. 1a).

Fig. 1. PCR/RFLP analysis of family Gadidae with DdeI a) and MboII b). M – molecular weight marker, 1 - G. morhua, 2 - T. chalcogramma, 3 - M. poutassou, 4 - T. minutus.

Restriction profiles using MboII were published

only for Atlantic Cod (22) but it enables us to distinguish remaining three species: Alaska Pollock (300 bp, 130 bp), Blue Whiting (300 bp, 170 bp), and Poor Cod (300 bp, 170 bp, 90+80 bp).

From 22 fish samples belonging to the family Gadidae, in five cases mislabelled species were detected: three fillets – a fresh fish on ice labelled as Coal Fish (P. virens), corresponds to Alaska Pollock (T. chalcogramma), Alaska Pollock (T. chalcogramma) corresponds to Blue Whiting (M. poutassou), and English Whiting (M. merlangus) was in fact Poor Cod (T. minutus). Two technologically-treated products - Cod fish á la salmon and Cod fish á la homer labelled as T. chalcogramma were detected as M. poutassou.

Species identification of the families Scombridae and Gempylidae. Twenty samples were used to identify the most common species of these families: Atlantic Mackerel (Scomber scombrus) - 19 samples (five frozen, eight cooked-tinned, six smoked), Escolar (Lepidocybium flavobrunneum) - one sample (smoked) and Atlantic Bluefin Tuna (Thunnus thynnus) - nine samples (three frozen, five cooked-tinned, one smoked).

For the identification of the Mackerel and Escolar RE HincII, HinfI and MboII were used. Only two restriction profiles were found (Table 1, Figs 2a - c).

No information was found in the literature about PCR-RFLP identification of this part of the cytochrome b gene. Aranishi (2) developed the PCR-RFLP analysis after amplification of the nuclear 5S ribosomal DNA nontranscribed spacer and Infante et al. (12) multiplex-PCR assay of the mitochondrial NADH dehydrogenase subunit 5. For that reason, the PCR products were sequenced (Macrogen Corp.) and the sequences were compared to the NCBT database (1). The RFLP patterns of the majority of samples correspond to S. scombrus AB120717.1. and two samples were identified as Ruvettus pretiosus EF988662.1. (Oilfish), which belongs to the family Gemphylidae. Analysis of this PCR product with RE used is therefore suitable for thr differentiation of both species.

False declaration was confirmed in the products smoked mackerel fillet (declared as L. flavobrunneum – Escolar) and Titbit mackerel (declared as Mackerel). In the second case the replacement of fish even from other families took place. Such mislabelling may be dangerous for the consumers because of their high oil content.

Restriction analysis of samples declared as Thunnus thynnus (Alantic Bluefin Tuna) after digestion with NlaIII, AluI, HincII, HinfI, MboII, HaeIII, and DdeI are shown in Fig. 3. The profiles correspond to Wolf et al. (22) and demonstrated this species in all products.

Species identification of the families Salmonidae and Anguillidae. To the most consumed fish from family Salmonidae belongs Humpback (Onconrhynchus gorbuscha), Salmon (Salmo salar), Chum Salmon (Oncorhynchus keta), and Rainbow Trout (Oncorhynchus mykkis). An important representative of Anguillidae family is European Eel (Anguilla anguilla). Eight samples of the family Salmonidae (six frozen, two tinned) and one sample of smoked European Eel were analysed using RE HinfI, MboII, and HaeIII (Fig. 4), and NlaIII and DdeI (Fig. 5).

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Table 1 Restriction profiles of Scomber scombrus and Ruvettus pretiosus

Restriction endonucleases HincII HinfI MboII Scomber scombrus 464 bp 260 bp, 220 bp 270 bp, 170 bp Ruvettus pretiosus 370 bp, 90 bp 220 bp, 200 bp, 60 bp 464 bp

Fig. 2. PCR - RFLP patterns of Ruvettus pretiosus and Scomber scombrus following digestion with HincII (a), HinfI (b) and MboII (c). M – molecular weight marker, 1, 2 – R. pretiosus, 3, 4 – S. scombrus.

Fig. 3. PCR - RFLP patterns of T. thynnus. M - molecular weight marker; 1 – NlaIII, 2 – AluI, 3 – HincII, 4 – HinfI, 5 – MboII, 6 – HaeIII, 7 – DdeI.

Fig. 4. PCR - RFLP patterns of family Salmonidae following digestion with HinfI (a), MboII (b), and HaeIII (c). M - marker 50 bp, 1 – PCR, 2 – O. keta, 3 – O. gorbusha, 4 – S. salar, 5 – A. anguilla, 6 – O. mykkis.

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Fig. 5. PCR - RFLP patterns of family Salmonidae following digestion with NlaIII (a), DdeI (b).

M - marker 50 bp, 1 – PCR, 2 – O. keta, 3 – O. gorbusha, 4 – S. salar, 5 – A. anguilla, 6 – O. mykkis. The restriction profiles correspond to Wolf et

al. (22) and Hold et al. (11). Combined analysis with more RE is necessary. HinfI is the best enzyme for S. salar identification. To distinguish O. gorbusha, O. Keta, and O. Mykkis, the enzymes MboI or NlaIII and RE HaeIII for A. anguilla determination are the most convenient.

In three samples, the declaration did not correspond with our findings. The product marked as Salmon fillet (S. salar) contained tissue of O. keta and Chum Salmon steak (O. keta) and Norwegian salmon (S. salar) were in fact Humback (O. gorbusha).

All results from the analysis of 60 samples can be summarised consecutively: PCR amplification was succesful in 95% of samples. Only from three cooked samples was no PCR product obtained. Although DNA exhibits high thermal stability, it is known that heat processing with overpressure may cause DNA degradation affecting the quality of DNA and possibly the presence of additives may inhibit DNA polymerase.

In 47 samples (78.3%), the results were in agreement with declarations and only 10 samples (16.7%) contained fish species other than the producer claimed. Correct designation was in 72.3% of fresh and 81.6% of processed samples. In recent years, German food-control laboratories have established proof of a significant number of cases (60%) of incorrectly labelled Flatfish on the market (10). In Spain, from 70 fish fillet samples, 58 were incorrectly labelled (3) and from 37 analysed meals from school lunch rooms and restaurants only nine samples were correct (4). In Italy, 84% of surimi-based fish products were prepared with species different from those declared (16). In Canada and the USA, 25% of the seafood samples from markets and restaurants were potentially mislabeled (23). Ling et al. (13) analysed 30 fish-steaks from markets in China and 43% samples declared as “cod” were mislabelled and contained meat of Escolar or Oilfish.

In conclusion, it could be assumed that the situation on the markets in the Czech republic could be better than in other countries. But the consumption of seafood, and consequently the importation of fish products, is relatively low. Nevertheless the change in consumers’ attitudes towards health and nutrition and

increased demand for seafood and derived products requires enhanced attention to seafood authentication and food safety

Acknowledgments: This study was

supported by grant No. MSM 6215712402 of the Ministry of Education, Youth, and Sports, of the Czech Republic.

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