PCR-RFLP of the mitochondrial cytochromeoxidase gene: a simple method fordiscrimination between Atlantic salmon ( Salmosalar) and rainbow trout ( Oncorhynchus mykiss)Esther Carrera, Teresa Garcıa,* Ana Cespedes, Isabel Gonzalez, Alicia Fernandez,Pablo E Hernandez and Rosario MartınDepartamento de Nutricion y Bromatologıa III, Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain

Abstract: A DNA-based method (PCR-RFLP) has been developed for discrimination between Atlantic

salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). The polymerase chain reaction

(PCR) was used for ampli®cation of a 464 bp fragment of the mitochondrial cytochrome oxidase

subunit II (COII) gene. Digestion of the products with endonucleases Nci I and Sau 3AI, followed by

agarose gel electrophoresis of the digested products, yielded speci®c restriction pro®les that enabled

direct visual identi®cation of the species analysed. This PCR-RFLP methodology allowed clear

discrimination of Atlantic salmon and rainbow trout samples both in raw and smoked products.

# 1999 Society of Chemical Industry

Keywords: ®sh species differentiation; COII gene; PCR; RFLP; Salmo salar; Oncorhynchus mykiss

INTRODUCTIONThe problem of substitution or adulteration of costly

®sh by a cheaper one, whether by accident or

intention, is not a new one. Moreover, determining

the species of origin in processed products is an

integral part of food regulatory control. In Europe,

rainbow trout (Oncorhynchus mykiss) and Atlantic

salmon (Salmo salar) are commonly used for the

manufacture of smoked products that are dif®cult to

identify because of their lack of distinct morphological

characteristics.

A variety of molecular techniques have been

employed for ®sh species identi®cation. These tech-

niques include electrophoresis of proteins,1,2 liquid

chromatography,3 High Performance Liquid Chro-

matography,4 and immunoassays.5±8 Despite their

individual merits, these methods rely upon the analysis

of sarcoplasmic proteins and heat processing may alter

the results obtained.

Deoxyribonucleic acid (DNA) is a highly stable

molecule9 and its analysis is a promising method for

animal species identi®cation in foods.10±13 DNA can

be isolated from any animal tissues, such as muscle,

blood, bones or fat tissues14 and can be analysed using

techniques such as DNA±DNA hybridization and the

polymerase chain reaction (PCR), that detect the

presence of species-speci®c DNA sequences in food

products. In particular, PCR techniques have a high

potential because of their increased sensitivity and

speci®city.15

In recent years, DNA analysis has been conducted

on many species of marine ®sh. The mitochondrial

genome (mtDNA) has become very popular for

evolutionary and population genetic studies because

of its high copy number, its ease of isolation from the

nuclear genome, its small size (16,500 (�500bp)) and

its rapid accumulation of mutations.16±18 However,

despite the generally fast substitution rate of mtDNA

of animals, conserved areas can be identi®ed and

primers can be designed that have applicability for a

wide taxonomic range.19 Thus, single band ampli®ca-

tion products can be obtained from a wide range of

animals using universal primers. The sequence analy-

sis of these amplicons can be used for interspeci®c20±22

and intraspeci®c identi®cation of animals and for their

detection in manufactured food products.10

The purpose of this work was to develop a simple

method for discrimination between Atlantic salmon

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1654±1658 (1999)

* Correspondence to: T Garcıa, Departamento de Nutricion y Bromatologıa III, Facultad de Veterinaria, Universidad Complutense, 28040Madrid, SpainE-mail: tgarcia@eucmax.sim.ucm.esContract/grant sponsor: Comision Interministerial de Ciencia y Tecnologıa, Spain; contract/grant number: ALI95-0306Contract/grant sponsor: Ministerio de Educacion y Ciencia, SpainContract/grant sponsor: Comunidad Autonoma de Castilla-La Mancha(Received 3 July 1998; revised version received 21 December 1998; accepted 6 May 1998)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1654

and rainbow trout, based on PCR ampli®cation and

restriction site analysis of a conserved region of the

mitochondrial cytochrome oxidase gene.

MATERIALS AND METHODSDNA extractionTotal cellular DNA was extracted from raw and

smoked ®sh samples by the method of DeSalle etal23 as previously described.21

DNA was obtained from muscle samples from two

different individuals of raw Atlantic salmon (S salar)and raw rainbow trout (O mykiss), and also from six

different commercial samples of smoked salmon and

trout. The commercial brands of smoked salmon

analysed were all from Spain: Ahumados DomõÂnguez

(AlcorcoÂn), Skandia (MaÂlaga), Valkiria (MaÂlaga), El

Rey Vikingo (MaÂlaga), Wonder (Parla) and Pescader-

õÂas CorunÄesas (Madrid). The brands of smoked trout

were also from Spain: EurosalmoÂn (AlcorcoÂn), Pryca

(Madrid), Skandia (MaÂlaga), Ahumados DomõÂnguez

(AlcorcoÂn), Wonder (Parla) and PescaderõÂas CorunÄe-

sas (Madrid).

Primers design and PCR amplificationCytochrome oxidase gene sequences available from

GenBank database were used for the design of primers

CYTO-1: 5'-TCCTTCATTTTCACGACC-3' and

CYTO-2: 5'-TGCGTCCATTTTTACACC-3'. Se-

quence analysis of the cytochrome oxidase gene and

primer design were performed with the aid of the

Wisconsin Package, Version 9.0 (Genetics Computer

Group, Madison, WI).

The PCR ampli®cation reactions were performed in

a total volume of 50ml. Each reaction mixture

contained 2±20ng of template DNA, 50pmol of each

primer, 200mM of each dNTP and 2U of DynaZyme

II DNA polymerase (Finnzymes Oy, Espoo, Finland)

in a reaction buffer containing 10mM Tris-HCl, pH

8.8, 1.5mM MgCl2, 50mM KCl and 0.1% Triton X-

100. The PCR was carried out in a Progene Thermal

Cycler (Techne Ltd, Cambridge, UK). PCR condi-

tions were as follows: a denaturation step of 94°C for

3min, followed by 35 cycles consisting of 30s at 94°C,

30s at 49°C and 1min at 72°C.

PCR products (8ml) were mixed with 2ml of gel

loading solution (Sigma Chemical Co, St. Louis, MO,

USA), loaded in a 1.5% D-1 agarose gel (Hispanlab S

A., Alcobendas, Spain) containing 1mgmlÿ1 of ethi-

dium bromide in Tris-acetate buffer (0.04M Tris-

acetate, 0.001M EDTA, pH 8.0) and electrophoresed

at 100V for 1h. The resulting DNA fragments were

visualized by UV transillumination and analysed using

Geldoc 1000UV Fluorescent Gel Documentation

System-PC (BIORAD, Hercules, CA, USA).

Clean-up and sequencing of the PCR productsThe PCR product (120ml) was loaded in a 1% LM-2

agarose gel (Hispanlab) containing 1mgmlÿ1 ethidium

bromide in Tris-acetate buffer and electrophoresed at

80V for 90min. The DNA of the band was excised

under UV light, puri®ed using the QIAquick Gel

Extraction Kit Protocol (QIAGEN, GmbH, Ger-

many), and resuspended in 30ml sterile water. The

concentration and quality of the puri®ed DNA were

estimated by ¯uorescence comparison of 1ml of the

puri®ed PCR product with the bands of a standard

(Mass Ruler, BIORAD, Hercules, CA), following

agarose electrophoresis in a gel stained with ethidium

bromide. A Geldoc 1000 System-PC (BIORAD) was

used for image analysis.

Puri®ed PCR products were sequenced at the

Centro de Investigaciones BioloÂgicas, Consejo Super-

ior de Investigaciones Cienti®cas (Madrid, Spain)

using the CYTO-1 primer and the dRhodamine

Terminator Cycle Sequencing Ready Reaction Kit

(Perkin-Elmer/Applied Biosystems Division, Foster

City, CA), in a ABI PRISM Model 377 DNA

Sequencer (Perkin-Elmer). Sequences were analysed

and prepared for publication with the help of the

Wisconsin Package, Version 9.0.

Restriction site analysis of the PCR productsCrude PCR products were digested with endonu-

cleases Nci I and Sau 3AI (New England BioLabs,

Beverly, MA, USA) without further puri®cation.

Digests were performed in a total volume of 20ml

including 14ml of ampli®ed DNA, 10U of enzyme and

1:10 dilution of the manufacturer's recommended

10� digestion buffer. Digestion reactions were in-

cubated for 3h at 37°C. Eight microlitres of the

digested PCR products were loaded in a 3% MS-8

agarose gel (Hispanlab) containing ethidium bromide

(1mgmlÿ1) and electrophoresed for 1h at 100V. After

electrophoresis, the sizes of the resulting fragments

were estimated by comparison with a commercial

molecular size marker, 100bp ladder (GibcoBRL, Life

Technologies, Maryland, USA).

RESULTS AND DISCUSSIONIn this work, we have focused on the study of part of

the mitochondrial cytochrome oxidase II gene for

Figure 1. Electrophoretic analysis of the COII PCR products obtained from:(1) raw salmon; (2) smoked salmon; (3) raw trout and (4) smoked trout.M=Molecular weight marker, 100 bp ladder.

J Sci Food Agric 79:1654±1658 (1999) 1655

PCR discrimination of salmon and trout

discrimination between Atlantic salmon and rainbow

trout. A preliminary search for salmon and trout

mitochondrial gene sequences in the nucleotide

databases showed suf®cient nucleotide substitutions

in the COII gene to allow differentiation between

species. Based on published COII sequences (Gen-

Bank accession number L29771 for rainbow trout,

U12145 and L04501 for Atlantic salmon), primers

CYTO-1 and CYTO-2 were designed to amplify a

464-bp fragment of the gene. PCR reactions using

primers CYTO-1 and CYTO-2 yielded a single 464-

bp ampli®ed fragment for all the salmon and trout

DNA samples extracted from raw or smoked muscle

(Fig 1).

Sequencing of the COII PCR products (Fig 2) and

comparison of our sequences with those obtained from

the databases has been used in this work to ®nd

nucleotide substitutions useful for the speci®c identi-

®cation of salmon and trout samples. In the 428bp

region sequenced (excluding primers), 40 nucleotides

(9.3%) were found to be different between the PCR

products of salmon and trout (Fig 2). However, while

sequencing speci®c DNA regions is a valuable tech-

nique in phylogenetic and population studies24±26 it is

costly and time-consuming and might be inappropri-

ate for food analysis of a large number of samples.

Several authors14,21,22,27,28 have determined the dis-

criminatory power of restriction fragment length

polymorphisms analysis of amplicons (PCR-RFLP)

obtained from mitochondrial DNA for animal species

identi®cation. With this approach, amplicons are not

sequenced but are digested with restriction endonu-

cleases. Therefore, sequencing may be useful for

selection of diagnostic sites and speci®c endonu-

cleases, but only variations within the recognition

sequences of the selected enzymes are used for species

identi®cation. Base substitutions at the position of the

restriction sites are detectable as the difference of

restriction fragment length on agarose gel electrophor-

esis.

Figure 2. DNA sequences of part of the cytochrome oxidase II gene fromtwo Atlantic salmon and two rainbow trout aligned with the homologousrainbow trout (accession number L29771) and Atlantic salmon (accessionnumbers U12145 and LO4501) sequences obtained from GenBank. Nci I(5'-CCCGG-3') and Sau 3Al (5'-GATC-3') restriction sites are shown withshadow. Bold type nucleotides in trout sequence indicate the position ofprimers CYTO-1 and CYTO-2 used for PCR amplification.

Figure 3. Restriction profiles of the COII PCR products obtained fromsamples of (1–3) raw salmon; (4–6) smoked salmon; (7–9) raw trout and(10–12) smoked trout. Samples in lines are: 1, 4, 7, 10 undigested; 2, 5, 8,11 digested with Nci I and 3, 6, 9, 12 digested with Sau 3Al. M=molecularweight marker 100 bp ladder.

1656 J Sci Food Agric 79:1654±1658 (1999)

E Carrera et al

Detailed comparison of the restriction maps of the

sequences obtained in this work and those from the

GenBank/EMBL databases showed that at least two

restriction endonucleases (Nci I and Sau 3AI) were

theoretically suitable for identi®cation of COII PCR

products obtained from salmon and trout DNA.

Figure 3 shows the results obtained following restric-

tion analysis of the salmon and trout PCR products.

The Nci I site present in the salmon samples yielded

two fragments of 133bp and 330bp. As expected, Nci I

did not cleave the PCR products of rainbow trout. In

the trout samples, the two restriction sites for the

enzyme Sau 3AI yielded three fragments of 108bp,

127bp and 229bp. However, Sau 3AI did not cleave

Atlantic salmon PCR products.

The restriction pro®les obtained for all the six

commercial samples of smoked salmon and trout are

in agreement with those obtained from raw samples of

these species. Results did not show intraspeci®c

polymorphism for the two restriction endonucleases

tested within the samples analysed. We have also

searched the GenBank/EMBL databases for se-

quences of the COII gene from other Salmo and

Oncorhynchus species. However, they were not avail-

able and it was not possible to compare their

restriction maps with those of Salmo salar and

Onchorhynchus mykiss.In previous work,21 PCR-RFLP of a conserved

region of the cytochrome b gene was used for

discrimination between S salar and O mykiss. How-

ever, primers CYTO-1 and CYTO-2 designed in this

work allow perfect hybridization with the template

sequences, while PCR priming with universal primers

CYTB-1 and CYTB-229 may give weaker results for Ssalar and O mykiss DNA. Moreover, differences

among the nucleotide sequences of S salar and Omykiss COII fragment were found to be higher than

those obtained with the cytochrome b fragment (9.3%

vs. 7.5% nucleotides). Thus, although both mitochon-

drial markers (cytochrome b and cytochrome oxidase

II) are appropriate for a clear discrimination between Ssalar and O mykiss, the COII gene fragment ampli®ed

and sequenced in this work could be considered more

speci®c.

PCR-RFLP of the mitochondrial COII gene is a

powerful technique for discrimination between raw

and smoked samples of Atlantic salmon and rainbow

trout. Unambiguous interpretation of the results may

be achieved visually without the need for computer

analysis. Compared to alternative techniques such as

direct sequencing of PCR products or conventional

mtDNA analysis, PCR-RFLP offers the advantages of

being simpler, cheaper and especially useful for

routine analysis of large number of samples.

ACKNOWLEDGEMENTSThis work was supported by grant No ALI95-0306

from the ComisioÂn Interministerial de Ciencia y

TecnologõÂa (CICYT) of Spain. E. Carrera, I GonzaÂlez

and A FernaÂndez are recipients of fellowships from the

Ministerio de EducacioÂn y Ciencia (Spain). A

CeÂspedes is a recipient of a fellowship from the

Comunidad AutoÂnoma de Castilla-La Mancha.

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