Food Chemistry 127 (2011) 1268–1272
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Analytical Methods
Authentication of Atlantic salmon (Salmo salar) using real-time PCR Beatriz Herrero, Juan M. Vieites, Montserrat Espiñeira ⇑ Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 9 December 2010 Accepted 19 January 2011 Available online 25 January 2011 Keywords: Salmo salar Salmon TaqMan Real-time PCR PCR Identification
a b s t r a c t A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon ( Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primers-probe set was validated and further applied to 20 commercial samples labelled as salmon or S. salar in order to determinate if the species used for their manufacturing corresponded to this species. The methodology herein developed is useful to check the fulfilment of labelling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Atlantic salmon (Salmo salar) is a salmonid with greater commercial value. The current worldwide production of farmed S. salar exceeds one million tonnes. This farmed species constitute more than 90% of the farmed salmon market, and more than 50% percent of the total global salmon market. Despite the major markets for farmed Atlantic salmon are Japan, the European Union, and North America, the most rapidly growing supplier now is Chile, which has low labour and materials costs and can therefore effectively compete with traditional producing countries in distant markets (FAO). This massive aquaculture production of salmon has favoured the presence of multiples species in the markets. The main forms under which these species are marketed are whole, sliced, filleted, and as steaks, fresh or frozen. Among all the commercialised products it is worth pointing out smoked meat (mainly for the European market), paté, cans, marinated, and other products are progressively taking more important place in the fishing industry. The increase of processed products and the existence of a global market, may lead to substitutions of species in a deliberate form or non intentional form. In this sense, to guarantee the correct information to the consumers, the authorities of each region have established different regulations. For instance, in Europe the Regulation 104/2000 and 2065/2001 tries to protect the consumer’s rights (EU, 2001, 2006). The Codex Alimentarius Commission has also developed a standard outlining the main requirements for canned salmon (Codex, 1981). These regulations establish the ⇑ Corresponding author. Tel.: +34 986 469 301; fax: +34 986 469 269. E-mail address: [email protected] (M. Espiñeira). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.070
appropriate information about the commercial denomination and the scientific names of the species. In order to enforce labelling regulations and prevent species substitution is necessary to develop analytical methods that can be used to determine the genuine species included in any product, independently of the degree of transformation which it has undergone. The most recent and innovative are the genetic techniques, which permit seafood species identification thus granting the reliability of commercial transactions and avoiding fraud. In recent years, molecular authentication methodologies based on the PCR amplification have been developed and these methods have been successfully applied for species authentication of salmonids ( Carrera et al., 1998, 1999a,b, 2000; Russell et al., 2000). In this context, it worth highlighting the work developed by Espiñeira et al. for authentication of salmon, trout, and bream species in any seafood product, including those that have undergone intensive thermal treatment by means two methodologies alternatives PCR–RFLP and FINS (Espiñeira et al., 2009). A novel genetic technique for species identification is the application of specific DNA probes with the method of real-time PCR (RT-PCR). This methodology is based on the specific hybridization of a probe, which was designed for a certain species, with the DNA in the samples to be analysed. This technique is acquiring more importance due to its rapidity and sensitivity and has passed from being an almost exclusive method in microbiology to be mainly used for identification of fish species (Bayha et al., 2008; Dalmasso et al., 2007; Fox et al., 2005; Herrero et al., 2010; Lopez & Pardo, 2005; Trotta et al., 2005). In the present work one methodology based on species-specific RT-PCR assay was developed for the authentication of Atlantic
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salmon from other closely related species that are used as substitutes, in all kind of seafood products, including those that have undergone intensive thermal treatment. The profit from this methodology is, beside potential automation, that it can be applied by control laboratories without the need for great investment in equipment. Therefore, these molecular tools can be used for clarifying questions related to the correct labelling and traceability of commercial products that include salmon in their composition. 2. Materials and methods 2.1. Sample collection, storage and DNA extraction Samples of different salmon, trout and bream species were collected from several locations around the world (Table 1). When it was possible, the individuals were identified according to their morphological characters (Mackie, 1990). In other cases, authenticated fish tissues preserved in ethanol, were provided by Universities and research centres located around the world. Once identified, the samples were labelled and preserved at 80 °C. Moreover, 20 seafood products labelled as a type of salmon or S. salar were provided by import industries or purchased in supermarkets and shops from European countries and Chilean, in order to apply the developed methodology to commercial samples. Genomic DNA was extracted from 30 mg of muscle in fresh and frozen samples, according to the method described by Roger and Bendich with slight modifications (Roger, 1988). The obtained DNA was diluted in 100 ll of 1X Tris–EDTA (TE) buffer (Sigma). In the case of processed products used for the methodological validation and commercial samples DNA was extracted from 150 mg using the NucleoSpin Tissue kit (Macherey–Nagel), following the manufacturing instructions. The quality and quantity were determined by measuring the absorbance at 260 nm and the 260/280 nm and 234/260 ratios using a NanoDrop™ ND-1000 spectrophotometer (Thermo Scientific) (Winfrey et al., 1997). DNA extractions were appropriately labelled and stored at 80 °C for subsequent tasks. 2.2. Design of a specific RT-PCR method to detect and identify S. salar 2.2.1. Clonation and sequencing An ITS 1 fragment was amplified with primers described by (Pérez et al. (2008)). To obtain DNA sequences, PCR products were cloned into pGEM-T Easy Vector System II (Promega), following the supplier’s recommendations.
Subsequently, sequencing reactions of both DNA strands were carried out with the primers described previously in a final volume of 10 ll with BigDye Terminator cycle sequencing ready reaction v1.1 (Applied Biosystems) and sequenced on an ABI Prism 3130 (Applied Biosystems). Thermal cycle sequencing reaction and the subsequent sequencing products’ cleanup by ethanol precipitation were carried out in accordance with the manufacturer’s instructions (Applied Biosystems). Next, these sequences were analysed with Sequencing Analysis Software v5.3.1. (Applied Biosystems) and aligned with Clustal W (Thompson et al., 1997) available in the programme BioEdit 7.0 (Hall, 1999).The nucleotide sequences obtained were submitted to the GeneBank database of the National Centre for Biotechnology Information (NCBI).
2.2.2. Design of a specific assay From the sequences of the ITS 1 gene two internal primers and probe were designed: S. SALAR-F (50 -AGT GAT CCA CCG CTA AGA30 ), S. SALAR-R (50 -ACT ATG ACC TCT CGC TCT G-30 ) and a LNA™ probe labelled with S. SALAR PROBE (50 -FAM TTG TCC AGT TTT ACG CAG A-30 TAM), the LNA nucleotides are underlined. The PCR reactions were carried out in a total volume of 20 ll containing 250 ng of DNA template, 10 ll of SsoFast™ Probes Supermix (BIO-RAD), the amount of primers and probe that were optimised and molecular biology grade water (Eppendorf) up to adjust to the final volume. Optimal amount of primers and probe were evaluated by preparing dilution series. A common range of working stock concentrations of 50, 125 and 250 nM of each primer and the LNA™ TaqMan™ probe were used to determine the optimal concentrations. The reactions were performed in iQ 96-well PCR plates (BIORAD) covered with iCycler iQ™ Optical Tape (BIO-RAD) and reactions were run in triplicate on BIO-RAD iCycler iQ™ real-time PCR instrument. The annealing temperature of real-time PCR assay is one of the most critical parameters for reaction specificity. To find the optimal annealing temperature of reaction, a range of temperatures were tested with the following thermal cycling protocol: 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s and 60–64 °C for 30 s. To differentiate the positive signal observed in species belonging to genus Salmo in RT-PCR, restriction maps of the DNA sequences obtained of Salmo spp were generated using Webcutter 2.0 software. The enzyme Bfa I was selected for its ability to generate a characteristic restriction profile with band sizes easily distinguishable on agarose gels.
Table 1 Samples included in this work and collection locations. Family
Salmonidae
Bramidae
a b
Scientific name
Common namea
Samplesb
Location
Salmo salar Salmo trutta Oncorhynchus clarki Oncorhynchus mykiss Oncorhynchus tshawytscha Oncorhynchus nerka Oncorhynchus gorbuscha Oncorhynchus kisutch Oncorhynchus masou Oncorhynchus keta Salvelinus namaycush Brama brama Brama australis Brama japonica
Atlantic salmon Brown trout Cutthroat trout Rainbow trout Chinook salmon Kokanee Humpbacked salmon Coho salmon Cherry salmon Keta salmon Great lake trout Atlantic pomfret South rays bream Pacific pomfret
10 10 3 6 2 3 3 4 3 4 3 3 3 1
USA, ESP, IRL CAN, ITA, NOR ESP, UK, CAN, NOR USA ESP, FRA RUS CAN, CHN CAN, USA, CHN CAN JAP, CHN CAN, RUS USA ESP CHI CHN
Only it is shown one of the possible common names for each species. Each sample included between 1 and 5 individuals.
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Table 2 Commercial samples analysed. Product
Species declared
Species identified by sequencing
Species identified by method of this work
Average Ct value
Smoked, vacuum-packed Smoked, vacuum-packed Smoked, vacuum-packed Smoked Salmon in an Orange Sauce Smoked Salmon with fine herbs Canned with vegetal oil Canned with vegetal oil Natural canned (with water and salt) Pickled (canned) Carpaccio Carpaccio Marinated (canned) Frozen Salmon fillets Frozen salmon slices Frozen salmon slices Fresh salmon Fresh salmon Roe Pate Pate
S. salar S. salar S. salar S. salar S. salar Salmon S. salar Salmon S. salar Salmon Salmon S. salar S. salar S. salar S. salar S. salar S. salar Salmon Salmon Salmon
S. trutta S. salar S. salar O. mykiss S. salar S. trutta S. salar O. keta S. salar O. gorbuscha S. salar S. salar S. salar S. salar S. salar S. salar S. salar S. salar O. mykiss S. salar
Negative Positive Positive Negative Positive Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive
15,2 15,5 15,1 32,2 14,7 18,5 19,7 33,8 19,4 36,1 15,1 18,1 13,2 13,8 14 13,5 13 14,5 33 18
2.2.3. Specificity and sensitivity of the method The specificity of the assay was evaluated by testing the amplification of DNA from different salmonid, bream and trout species (Table 1). A serial dilution of DNA of S. salar was performed with DNA of other species (Table 1) in levels ranging from 50 ng to 10 pg and the fluorescence signal was determined. The dilutions were prepared by adding S. salar DNA and DNA from different species until completing the final amount of 50 ng. All measurements were performed in triplicate from three processed samples independently. The limit of detection (LOD) was established as the lowest concentration of DNA of S. salar which yields a fluorescent signal significantly different from the negative control.
2.4. Application of method to commercial products After the validation of the method developed in the present work, this was applied to 20 products labelled as salmon or S. salar species. These products were acquired in supermarkets from Europe and Chile. The purpose of these analyses was to evaluate the situation regarding the labelling of these products on the market (Table 2).In order to ensure the proper working of method, the methodology described by Espiñeira et al. was applied (Espiñeira et al., 2009).
3. Results and discussion 3.1. Design of method specific to identify Atlantic salmon
2.3. Methodological validation Individuals from different species were authenticated on the basis of their morphological traits. Then the main treatments applied to commercial products were applied to them, these were canning, salting, smoking, marinating, and precooking (two processed products for each one of the kind of processed for every species included in Table 1). In addition with each treatment were used different kinds of sauces or condiments. Also, the same treatments were applied in mixtures of Atlantic salmon with other species to check the sensitivity and specificity of the developed method. The two treatments more aggressive are canned and smoked. The treatment applied to canned samples involved 121 °C of temperature and 1.2 bars of overpressure and the time varied depending on the size of the can. The smoking process combines two effects: on the one hand salting and drying steps and on the other the effect of temperature. The temperature corresponding to smoking of the fillets was raised to 121 °C until inside the product 60 °C were reached. The cooking time depended on the thickness of the fillets. All these treatments were carried out in the CECOPESCA (Spanish National Centre of Fish Processing Technology) pilot plant. The products were analysed with the methodology developed in the present work. The coincidence percentage between the species identified on the basis of morphological traits and the genetic methodology developed was calculated to establish the specificity of the method.
ITS 1 is located between the 18S rDNA (nuclear small-subunit rRNA gene) and 5.8S rDNA genes. This region has been used previously in the genetic identification of species, giving satisfactory results, in part, due to the characteristic mode of evolution, concerted evolution and the multicopy nature of this gene (Santaclara et al., 2006a,b). The TaqMan RT-PCR technology has recently gained wide acceptance in the identification of species. This system has been applied to the identification of microorganisms (Hanna et al., 2006), plants (Hird et al., 2003; Hernandez et al., 2005a,b), and animals (Dalmasso et al., 2007; Herrero et al., 2010; Jonker et al., 2008; Kesmen et al., 2009; Kumari, 2007; Laube et al., 2003; Lopez and Pardo, 2005; Trotta et al., 2005). Among the advantages of the real-time PCR technique it is worth highlighting its specificity, sensitivity, reproducibility and rapidity. This technique allows verifying the functioning of the PCR while it is running, saving the time from secondary visualisation or identification techniques of the PCR products. The optimisation of the PCR conditions allows granting highest level of sensitivity while maintaining the specificity of the technique. In this study, a method specific to identify Atlantic salmon was designed. It has been described a real-time PCR assay with TaqMan probes including Locked Nucleic Acid (LNA) bases to increase thermal stability and hybridization specificity. Nucleotidic sequences two or three clones for each sample were selected per species were obtained using the primers described by Pérez et al. (Pérez & Presa, 2008). Due to the intra individual length
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polymorphism of ITS 1 the direct sequencing is not possible (Santaclara et al., 2006a,b), therefore PCR products were cloned. From sequences obtained (accession numbers HQ260440HQ260451) one internal region for designing the primer/probe set S. SALAR was selected. This set generated PCR products of 131 bp allowing the detection of Salmo spp. The conditions that allow the best results to be obtained were established by means of primers and probe matrix. Concentrations of 125 nM for both primers and 250 nM for the probe yielded the best results in terms of specificity and sensitivity. When the positive signal is observed, RT-PCR followed by digestion using the selected specific restriction enzyme is carried out to confirm the identity of the PCR product. A fragment of 75 bp is observed when S. trutta is present in the sample, moreover, digestion may not happen when the sample is S. salar. In this study, intraspecific variability of the selected restriction target was not found in the samples analysed. 3.2. Specificity and sensitivity of the method The specificity of the primer/probe set S. SALAR was confirmed using genomic DNA from other trout and bream species from different geographical areas (Table 1). No cross-reactivity was detected with any of the tested samples. In this way the optimal annealing conditions were established at 60 °C in order to assure the higher specificity of the developed methodology (Fig. 1). The most important parameter for RT-PCR is the threshold cycle (Ct). The Ct is the point at which fluorescence is first detected as statistically significant above the baseline or background. It is inversely correlated to the logarithm of the initial copy number. The threshold should be set above the amplification baseline and within the exponential increase phase. The higher the initial amount of sample DNA, the sooner the accumulated product is detected in fluorescence plot, and the lower the Ct value (SigmaAldrich). It is necessary to find the lowest Ct value and the highest final fluorescence value by means of appropriate concentrations of primers and probe. In all samples that contained Salmo spp the Ct values obtained were 12 ± 2. On the other hand, in the cross-reactivity analysis no false positive results were observed, under the stringent assay conditions used, as documented by Ct values >27. The efficiency of the developed method was calculated based on the slope of the standard curve obtained using DNA serial dilutions
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with a template of a known concentration (tenfold dilutions from 10 ng to 10 pg) as templates for RT-PCR (Pfaffl, 2004). The amplification plot of the experiment using primer/probe set S. SALAR generated a slope of 3.40 or 97% efficiency, with a correlation coefficient of 0.999. The amplification efficiency (E) was calculated using the equation E = (10(1/slope)1) 100. These values of Ct and efficiency demonstrated the utility of the RT-PCR system to identify Salmo spp.The limit of detection of the developed RT-PCR assay revealed that at least 50 pg of the target DNA is necessary for positive results.
3.3. Methodological validation The aim of the methodological validation was to check whether the manufacturing process which processed food underwent had no influence on the detection of S. salar. The elaborated products in the pilot plant of CECOPESCA were analysed by the proposed methodology. The different treatments of transformation applied, allow the evaluation of the correct PCR amplification when these process are applied in these products. S. salar was detected in all these samples that contained this species independently of the transformation process. The DNA fragmentation in fresh or frozen fish is not significant. However, in the case of fishes that have undergone different treatments, the thermal and pressure processing generates DNA fragmentation. This was the case of cans or smoked products, where fragments of little sizes were formed (Espiñeira et al., 2009). DNA fragmentation will make the increment of the Ct value in these products. The average Ct values were 18 for cans and pate products and 15 for smoked products. Despite this, Ct was always less than 27 when S. salar was present and did not produce any false negatives, in any case. Therefore, the RT-PCR system might be applied to fresh, frozen, precooked and canned fish.
3.4. Application to commercial products Twenty fresh, frozen, precooked and canned fish were tested for the identification of S. salar by using the method proposed in the present study (Table 2). To evaluate the degree of incorrect labelling found in the market, a study was carried out, it is worth highlighting that, of all the samples tested, the scientific name of the species did not
Fig. 1. Specificity of the RT-PCR assay. (A) Amplification pattern showed by salmon species, with Ct values about 12 ± 2. (B) Amplification pattern showed by others species with Ct values >27.
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appear on seven of the labels (only appeared the commercial denomination ‘‘Salmon’’). In 5% of the products analysed, the name of the species displayed on the label was not in agreement with the species contained, as determined by genetic analysis using the methodology herein developed (Table 2) and confirmed by sequencing with the methodology developed by (Espiñeira et al. (2009)) showing concordant results both methods. Altogether, this work describes the development of a method for the detection of S. salar in fish products. RT-PCR has become an important technique in many fields of the food industry. Although this technique is more costly than traditional PCR, this is offset by savings in subsequent costs and time (the entire procedure can be completed within 4 h.), as post PCR processing steps are no longer required for detection of the PCR product. Also, other advantages of this technique include reliability, sensitivity and specificity. All these characteristics are turning it into a very appropriate tool for the authentication of S. salar in all kind of seafood products. The possible applications of this method is the following: normative control of raw and processed products, particularly the authenticity of imported species; the verification of the traceability of different fishing batches along the commercial chain; correct labelling and protection of the consumer’s rights; fair competence among fishing operators; and the fisheries’ control. On the other side as laid down in Regulation (EC) No 999/2001 of the European parliament and of the Council and Commission regulation (EC) No 710/2009 shall have the feed coming from a species not serve for the supply of the same species for the prevention, control and eradication of certain transmissible spongiform encephalopathies (EU, 2009). Therefore, the technique developed in this work will allow feed control and compliance with these regulations dismissing the presence of S. salar in feed for their own food, since this species is one of the largest aquaculture productions. Acknowledgement We also thank Paloma Morán (Universidad de Vigo), Elson Leal (Instituto de Fomento Pesquero, Chile), Mª Angelica Larrain (Universidad de Chile) Robert Devlin and David Patterson (Fisheries and Oceans, Canada), Kristi Millar and Tobi Ming (Pacific Biological Station, Canada) for providing some of the samples included in this work. References Bayha, K. M., Graham, W. M., et al. (2008). Multiplex assay to identify eggs of three fish species from the northern Gulf of Mexico, using locked nucleic acid Taqman real-time PCR probes. Aquatic Biology, 4, 65–73. Carrera, E., Garcia, T., et al. (1999a). PCR-RFLP of the mitochondrial cytochrome oxidase gene: A simple method for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Journal of the Science of Food and Agriculture, 79(12), 1654–1658. Carrera, E., Garcia, T., et al. (1999b). Salmon and trout analysis by PCR-RFLP for identity authentication. Journal of Food Science, 64(3), 410–413. Carrera, E., Garcia, T., et al. (2000). Differentiation of smoked Salmo salar, Oncorhyncus mykiss and Brama raii using the nuclear marker 5S rRNA. International Journal of Food Science and Technology, 35, 401–406. Carrera, E., Garcia, T., et al. (1998). Identification of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene. Journal of Food Protection, 61(4), 482–486. Codex standard for canned salmon (CODEX STAN 3-1981). Dalmasso, A., Fontanella, E., et al. (2007). Identification of four tuna species by means of real-time PCR and melting curve analysis. Veterinary Research Communication, 31(1), 335–357.
Espiñeira, M., Vieites, J. M., et al. (2009). Development of a genetic method for the identification of salmon, trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies. European Food Research Technology, 229, 785–793. EU, (2001). ‘‘Reglamento (CE) n° 2065/2001 de la Comisión, de 22 de octubre de 2001, por el que se establecen las disposiciones de aplicación del Reglamento (CE) n° 104/2000 del Consejo en lo relativo a la información del consumidor en el sector de los productos de la pesca y de la acuicultura. EU, (2006). Reglamento (CE) N° 104 / 2000 DEL CONSEJO de 17 de diciembre de 1999 por el que se establece la organización común de mercados en el sector de los productos de la pesca y de la acuicultura. EU (2001). Regulation (EC) No 999/2001 of the European. Parliament and of the Council of 22 May 2001 laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. EU (2009). 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Analytical Methods
Authentication of Atlantic salmon (Salmo salar) using real-time PCR Beatriz Herrero, Juan M. Vieites, Montserrat Espiñeira ⇑ Area of Molecular Biology and Biotechnology, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain
a r t i c l e
i n f o
Article history: Received 17 September 2010 Received in revised form 9 December 2010 Accepted 19 January 2011 Available online 25 January 2011 Keywords: Salmo salar Salmon TaqMan Real-time PCR PCR Identification
a b s t r a c t A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon ( Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primers-probe set was validated and further applied to 20 commercial samples labelled as salmon or S. salar in order to determinate if the species used for their manufacturing corresponded to this species. The methodology herein developed is useful to check the fulfilment of labelling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Atlantic salmon (Salmo salar) is a salmonid with greater commercial value. The current worldwide production of farmed S. salar exceeds one million tonnes. This farmed species constitute more than 90% of the farmed salmon market, and more than 50% percent of the total global salmon market. Despite the major markets for farmed Atlantic salmon are Japan, the European Union, and North America, the most rapidly growing supplier now is Chile, which has low labour and materials costs and can therefore effectively compete with traditional producing countries in distant markets (FAO). This massive aquaculture production of salmon has favoured the presence of multiples species in the markets. The main forms under which these species are marketed are whole, sliced, filleted, and as steaks, fresh or frozen. Among all the commercialised products it is worth pointing out smoked meat (mainly for the European market), paté, cans, marinated, and other products are progressively taking more important place in the fishing industry. The increase of processed products and the existence of a global market, may lead to substitutions of species in a deliberate form or non intentional form. In this sense, to guarantee the correct information to the consumers, the authorities of each region have established different regulations. For instance, in Europe the Regulation 104/2000 and 2065/2001 tries to protect the consumer’s rights (EU, 2001, 2006). The Codex Alimentarius Commission has also developed a standard outlining the main requirements for canned salmon (Codex, 1981). These regulations establish the ⇑ Corresponding author. Tel.: +34 986 469 301; fax: +34 986 469 269. E-mail address: [email protected] (M. Espiñeira). 0308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.01.070
appropriate information about the commercial denomination and the scientific names of the species. In order to enforce labelling regulations and prevent species substitution is necessary to develop analytical methods that can be used to determine the genuine species included in any product, independently of the degree of transformation which it has undergone. The most recent and innovative are the genetic techniques, which permit seafood species identification thus granting the reliability of commercial transactions and avoiding fraud. In recent years, molecular authentication methodologies based on the PCR amplification have been developed and these methods have been successfully applied for species authentication of salmonids ( Carrera et al., 1998, 1999a,b, 2000; Russell et al., 2000). In this context, it worth highlighting the work developed by Espiñeira et al. for authentication of salmon, trout, and bream species in any seafood product, including those that have undergone intensive thermal treatment by means two methodologies alternatives PCR–RFLP and FINS (Espiñeira et al., 2009). A novel genetic technique for species identification is the application of specific DNA probes with the method of real-time PCR (RT-PCR). This methodology is based on the specific hybridization of a probe, which was designed for a certain species, with the DNA in the samples to be analysed. This technique is acquiring more importance due to its rapidity and sensitivity and has passed from being an almost exclusive method in microbiology to be mainly used for identification of fish species (Bayha et al., 2008; Dalmasso et al., 2007; Fox et al., 2005; Herrero et al., 2010; Lopez & Pardo, 2005; Trotta et al., 2005). In the present work one methodology based on species-specific RT-PCR assay was developed for the authentication of Atlantic
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salmon from other closely related species that are used as substitutes, in all kind of seafood products, including those that have undergone intensive thermal treatment. The profit from this methodology is, beside potential automation, that it can be applied by control laboratories without the need for great investment in equipment. Therefore, these molecular tools can be used for clarifying questions related to the correct labelling and traceability of commercial products that include salmon in their composition. 2. Materials and methods 2.1. Sample collection, storage and DNA extraction Samples of different salmon, trout and bream species were collected from several locations around the world (Table 1). When it was possible, the individuals were identified according to their morphological characters (Mackie, 1990). In other cases, authenticated fish tissues preserved in ethanol, were provided by Universities and research centres located around the world. Once identified, the samples were labelled and preserved at 80 °C. Moreover, 20 seafood products labelled as a type of salmon or S. salar were provided by import industries or purchased in supermarkets and shops from European countries and Chilean, in order to apply the developed methodology to commercial samples. Genomic DNA was extracted from 30 mg of muscle in fresh and frozen samples, according to the method described by Roger and Bendich with slight modifications (Roger, 1988). The obtained DNA was diluted in 100 ll of 1X Tris–EDTA (TE) buffer (Sigma). In the case of processed products used for the methodological validation and commercial samples DNA was extracted from 150 mg using the NucleoSpin Tissue kit (Macherey–Nagel), following the manufacturing instructions. The quality and quantity were determined by measuring the absorbance at 260 nm and the 260/280 nm and 234/260 ratios using a NanoDrop™ ND-1000 spectrophotometer (Thermo Scientific) (Winfrey et al., 1997). DNA extractions were appropriately labelled and stored at 80 °C for subsequent tasks. 2.2. Design of a specific RT-PCR method to detect and identify S. salar 2.2.1. Clonation and sequencing An ITS 1 fragment was amplified with primers described by (Pérez et al. (2008)). To obtain DNA sequences, PCR products were cloned into pGEM-T Easy Vector System II (Promega), following the supplier’s recommendations.
Subsequently, sequencing reactions of both DNA strands were carried out with the primers described previously in a final volume of 10 ll with BigDye Terminator cycle sequencing ready reaction v1.1 (Applied Biosystems) and sequenced on an ABI Prism 3130 (Applied Biosystems). Thermal cycle sequencing reaction and the subsequent sequencing products’ cleanup by ethanol precipitation were carried out in accordance with the manufacturer’s instructions (Applied Biosystems). Next, these sequences were analysed with Sequencing Analysis Software v5.3.1. (Applied Biosystems) and aligned with Clustal W (Thompson et al., 1997) available in the programme BioEdit 7.0 (Hall, 1999).The nucleotide sequences obtained were submitted to the GeneBank database of the National Centre for Biotechnology Information (NCBI).
2.2.2. Design of a specific assay From the sequences of the ITS 1 gene two internal primers and probe were designed: S. SALAR-F (50 -AGT GAT CCA CCG CTA AGA30 ), S. SALAR-R (50 -ACT ATG ACC TCT CGC TCT G-30 ) and a LNA™ probe labelled with S. SALAR PROBE (50 -FAM TTG TCC AGT TTT ACG CAG A-30 TAM), the LNA nucleotides are underlined. The PCR reactions were carried out in a total volume of 20 ll containing 250 ng of DNA template, 10 ll of SsoFast™ Probes Supermix (BIO-RAD), the amount of primers and probe that were optimised and molecular biology grade water (Eppendorf) up to adjust to the final volume. Optimal amount of primers and probe were evaluated by preparing dilution series. A common range of working stock concentrations of 50, 125 and 250 nM of each primer and the LNA™ TaqMan™ probe were used to determine the optimal concentrations. The reactions were performed in iQ 96-well PCR plates (BIORAD) covered with iCycler iQ™ Optical Tape (BIO-RAD) and reactions were run in triplicate on BIO-RAD iCycler iQ™ real-time PCR instrument. The annealing temperature of real-time PCR assay is one of the most critical parameters for reaction specificity. To find the optimal annealing temperature of reaction, a range of temperatures were tested with the following thermal cycling protocol: 95 °C for 2 min followed by 40 cycles of 95 °C for 5 s and 60–64 °C for 30 s. To differentiate the positive signal observed in species belonging to genus Salmo in RT-PCR, restriction maps of the DNA sequences obtained of Salmo spp were generated using Webcutter 2.0 software. The enzyme Bfa I was selected for its ability to generate a characteristic restriction profile with band sizes easily distinguishable on agarose gels.
Table 1 Samples included in this work and collection locations. Family
Salmonidae
Bramidae
a b
Scientific name
Common namea
Samplesb
Location
Salmo salar Salmo trutta Oncorhynchus clarki Oncorhynchus mykiss Oncorhynchus tshawytscha Oncorhynchus nerka Oncorhynchus gorbuscha Oncorhynchus kisutch Oncorhynchus masou Oncorhynchus keta Salvelinus namaycush Brama brama Brama australis Brama japonica
Atlantic salmon Brown trout Cutthroat trout Rainbow trout Chinook salmon Kokanee Humpbacked salmon Coho salmon Cherry salmon Keta salmon Great lake trout Atlantic pomfret South rays bream Pacific pomfret
10 10 3 6 2 3 3 4 3 4 3 3 3 1
USA, ESP, IRL CAN, ITA, NOR ESP, UK, CAN, NOR USA ESP, FRA RUS CAN, CHN CAN, USA, CHN CAN JAP, CHN CAN, RUS USA ESP CHI CHN
Only it is shown one of the possible common names for each species. Each sample included between 1 and 5 individuals.
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Table 2 Commercial samples analysed. Product
Species declared
Species identified by sequencing
Species identified by method of this work
Average Ct value
Smoked, vacuum-packed Smoked, vacuum-packed Smoked, vacuum-packed Smoked Salmon in an Orange Sauce Smoked Salmon with fine herbs Canned with vegetal oil Canned with vegetal oil Natural canned (with water and salt) Pickled (canned) Carpaccio Carpaccio Marinated (canned) Frozen Salmon fillets Frozen salmon slices Frozen salmon slices Fresh salmon Fresh salmon Roe Pate Pate
S. salar S. salar S. salar S. salar S. salar Salmon S. salar Salmon S. salar Salmon Salmon S. salar S. salar S. salar S. salar S. salar S. salar Salmon Salmon Salmon
S. trutta S. salar S. salar O. mykiss S. salar S. trutta S. salar O. keta S. salar O. gorbuscha S. salar S. salar S. salar S. salar S. salar S. salar S. salar S. salar O. mykiss S. salar
Negative Positive Positive Negative Positive Positive Positive Negative Positive Negative Positive Positive Positive Positive Positive Positive Positive Positive Negative Positive
15,2 15,5 15,1 32,2 14,7 18,5 19,7 33,8 19,4 36,1 15,1 18,1 13,2 13,8 14 13,5 13 14,5 33 18
2.2.3. Specificity and sensitivity of the method The specificity of the assay was evaluated by testing the amplification of DNA from different salmonid, bream and trout species (Table 1). A serial dilution of DNA of S. salar was performed with DNA of other species (Table 1) in levels ranging from 50 ng to 10 pg and the fluorescence signal was determined. The dilutions were prepared by adding S. salar DNA and DNA from different species until completing the final amount of 50 ng. All measurements were performed in triplicate from three processed samples independently. The limit of detection (LOD) was established as the lowest concentration of DNA of S. salar which yields a fluorescent signal significantly different from the negative control.
2.4. Application of method to commercial products After the validation of the method developed in the present work, this was applied to 20 products labelled as salmon or S. salar species. These products were acquired in supermarkets from Europe and Chile. The purpose of these analyses was to evaluate the situation regarding the labelling of these products on the market (Table 2).In order to ensure the proper working of method, the methodology described by Espiñeira et al. was applied (Espiñeira et al., 2009).
3. Results and discussion 3.1. Design of method specific to identify Atlantic salmon
2.3. Methodological validation Individuals from different species were authenticated on the basis of their morphological traits. Then the main treatments applied to commercial products were applied to them, these were canning, salting, smoking, marinating, and precooking (two processed products for each one of the kind of processed for every species included in Table 1). In addition with each treatment were used different kinds of sauces or condiments. Also, the same treatments were applied in mixtures of Atlantic salmon with other species to check the sensitivity and specificity of the developed method. The two treatments more aggressive are canned and smoked. The treatment applied to canned samples involved 121 °C of temperature and 1.2 bars of overpressure and the time varied depending on the size of the can. The smoking process combines two effects: on the one hand salting and drying steps and on the other the effect of temperature. The temperature corresponding to smoking of the fillets was raised to 121 °C until inside the product 60 °C were reached. The cooking time depended on the thickness of the fillets. All these treatments were carried out in the CECOPESCA (Spanish National Centre of Fish Processing Technology) pilot plant. The products were analysed with the methodology developed in the present work. The coincidence percentage between the species identified on the basis of morphological traits and the genetic methodology developed was calculated to establish the specificity of the method.
ITS 1 is located between the 18S rDNA (nuclear small-subunit rRNA gene) and 5.8S rDNA genes. This region has been used previously in the genetic identification of species, giving satisfactory results, in part, due to the characteristic mode of evolution, concerted evolution and the multicopy nature of this gene (Santaclara et al., 2006a,b). The TaqMan RT-PCR technology has recently gained wide acceptance in the identification of species. This system has been applied to the identification of microorganisms (Hanna et al., 2006), plants (Hird et al., 2003; Hernandez et al., 2005a,b), and animals (Dalmasso et al., 2007; Herrero et al., 2010; Jonker et al., 2008; Kesmen et al., 2009; Kumari, 2007; Laube et al., 2003; Lopez and Pardo, 2005; Trotta et al., 2005). Among the advantages of the real-time PCR technique it is worth highlighting its specificity, sensitivity, reproducibility and rapidity. This technique allows verifying the functioning of the PCR while it is running, saving the time from secondary visualisation or identification techniques of the PCR products. The optimisation of the PCR conditions allows granting highest level of sensitivity while maintaining the specificity of the technique. In this study, a method specific to identify Atlantic salmon was designed. It has been described a real-time PCR assay with TaqMan probes including Locked Nucleic Acid (LNA) bases to increase thermal stability and hybridization specificity. Nucleotidic sequences two or three clones for each sample were selected per species were obtained using the primers described by Pérez et al. (Pérez & Presa, 2008). Due to the intra individual length
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polymorphism of ITS 1 the direct sequencing is not possible (Santaclara et al., 2006a,b), therefore PCR products were cloned. From sequences obtained (accession numbers HQ260440HQ260451) one internal region for designing the primer/probe set S. SALAR was selected. This set generated PCR products of 131 bp allowing the detection of Salmo spp. The conditions that allow the best results to be obtained were established by means of primers and probe matrix. Concentrations of 125 nM for both primers and 250 nM for the probe yielded the best results in terms of specificity and sensitivity. When the positive signal is observed, RT-PCR followed by digestion using the selected specific restriction enzyme is carried out to confirm the identity of the PCR product. A fragment of 75 bp is observed when S. trutta is present in the sample, moreover, digestion may not happen when the sample is S. salar. In this study, intraspecific variability of the selected restriction target was not found in the samples analysed. 3.2. Specificity and sensitivity of the method The specificity of the primer/probe set S. SALAR was confirmed using genomic DNA from other trout and bream species from different geographical areas (Table 1). No cross-reactivity was detected with any of the tested samples. In this way the optimal annealing conditions were established at 60 °C in order to assure the higher specificity of the developed methodology (Fig. 1). The most important parameter for RT-PCR is the threshold cycle (Ct). The Ct is the point at which fluorescence is first detected as statistically significant above the baseline or background. It is inversely correlated to the logarithm of the initial copy number. The threshold should be set above the amplification baseline and within the exponential increase phase. The higher the initial amount of sample DNA, the sooner the accumulated product is detected in fluorescence plot, and the lower the Ct value (SigmaAldrich). It is necessary to find the lowest Ct value and the highest final fluorescence value by means of appropriate concentrations of primers and probe. In all samples that contained Salmo spp the Ct values obtained were 12 ± 2. On the other hand, in the cross-reactivity analysis no false positive results were observed, under the stringent assay conditions used, as documented by Ct values >27. The efficiency of the developed method was calculated based on the slope of the standard curve obtained using DNA serial dilutions
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with a template of a known concentration (tenfold dilutions from 10 ng to 10 pg) as templates for RT-PCR (Pfaffl, 2004). The amplification plot of the experiment using primer/probe set S. SALAR generated a slope of 3.40 or 97% efficiency, with a correlation coefficient of 0.999. The amplification efficiency (E) was calculated using the equation E = (10(1/slope)1) 100. These values of Ct and efficiency demonstrated the utility of the RT-PCR system to identify Salmo spp.The limit of detection of the developed RT-PCR assay revealed that at least 50 pg of the target DNA is necessary for positive results.
3.3. Methodological validation The aim of the methodological validation was to check whether the manufacturing process which processed food underwent had no influence on the detection of S. salar. The elaborated products in the pilot plant of CECOPESCA were analysed by the proposed methodology. The different treatments of transformation applied, allow the evaluation of the correct PCR amplification when these process are applied in these products. S. salar was detected in all these samples that contained this species independently of the transformation process. The DNA fragmentation in fresh or frozen fish is not significant. However, in the case of fishes that have undergone different treatments, the thermal and pressure processing generates DNA fragmentation. This was the case of cans or smoked products, where fragments of little sizes were formed (Espiñeira et al., 2009). DNA fragmentation will make the increment of the Ct value in these products. The average Ct values were 18 for cans and pate products and 15 for smoked products. Despite this, Ct was always less than 27 when S. salar was present and did not produce any false negatives, in any case. Therefore, the RT-PCR system might be applied to fresh, frozen, precooked and canned fish.
3.4. Application to commercial products Twenty fresh, frozen, precooked and canned fish were tested for the identification of S. salar by using the method proposed in the present study (Table 2). To evaluate the degree of incorrect labelling found in the market, a study was carried out, it is worth highlighting that, of all the samples tested, the scientific name of the species did not
Fig. 1. Specificity of the RT-PCR assay. (A) Amplification pattern showed by salmon species, with Ct values about 12 ± 2. (B) Amplification pattern showed by others species with Ct values >27.
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appear on seven of the labels (only appeared the commercial denomination ‘‘Salmon’’). In 5% of the products analysed, the name of the species displayed on the label was not in agreement with the species contained, as determined by genetic analysis using the methodology herein developed (Table 2) and confirmed by sequencing with the methodology developed by (Espiñeira et al. (2009)) showing concordant results both methods. Altogether, this work describes the development of a method for the detection of S. salar in fish products. RT-PCR has become an important technique in many fields of the food industry. Although this technique is more costly than traditional PCR, this is offset by savings in subsequent costs and time (the entire procedure can be completed within 4 h.), as post PCR processing steps are no longer required for detection of the PCR product. Also, other advantages of this technique include reliability, sensitivity and specificity. All these characteristics are turning it into a very appropriate tool for the authentication of S. salar in all kind of seafood products. The possible applications of this method is the following: normative control of raw and processed products, particularly the authenticity of imported species; the verification of the traceability of different fishing batches along the commercial chain; correct labelling and protection of the consumer’s rights; fair competence among fishing operators; and the fisheries’ control. On the other side as laid down in Regulation (EC) No 999/2001 of the European parliament and of the Council and Commission regulation (EC) No 710/2009 shall have the feed coming from a species not serve for the supply of the same species for the prevention, control and eradication of certain transmissible spongiform encephalopathies (EU, 2009). Therefore, the technique developed in this work will allow feed control and compliance with these regulations dismissing the presence of S. salar in feed for their own food, since this species is one of the largest aquaculture productions. Acknowledgement We also thank Paloma Morán (Universidad de Vigo), Elson Leal (Instituto de Fomento Pesquero, Chile), Mª Angelica Larrain (Universidad de Chile) Robert Devlin and David Patterson (Fisheries and Oceans, Canada), Kristi Millar and Tobi Ming (Pacific Biological Station, Canada) for providing some of the samples included in this work. References Bayha, K. M., Graham, W. M., et al. (2008). Multiplex assay to identify eggs of three fish species from the northern Gulf of Mexico, using locked nucleic acid Taqman real-time PCR probes. Aquatic Biology, 4, 65–73. Carrera, E., Garcia, T., et al. (1999a). PCR-RFLP of the mitochondrial cytochrome oxidase gene: A simple method for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Journal of the Science of Food and Agriculture, 79(12), 1654–1658. Carrera, E., Garcia, T., et al. (1999b). Salmon and trout analysis by PCR-RFLP for identity authentication. Journal of Food Science, 64(3), 410–413. Carrera, E., Garcia, T., et al. (2000). Differentiation of smoked Salmo salar, Oncorhyncus mykiss and Brama raii using the nuclear marker 5S rRNA. International Journal of Food Science and Technology, 35, 401–406. Carrera, E., Garcia, T., et al. (1998). Identification of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) by using polymerase chain reaction amplification and restriction analysis of the mitochondrial cytochrome b gene. Journal of Food Protection, 61(4), 482–486. Codex standard for canned salmon (CODEX STAN 3-1981). Dalmasso, A., Fontanella, E., et al. (2007). Identification of four tuna species by means of real-time PCR and melting curve analysis. Veterinary Research Communication, 31(1), 335–357.
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