Food Chemistry 135 (2012) 24392444

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Rapid method for controlling the correct labeling of products containing

common octopus (Octopus vulgaris) and main substitute species (Eledone
cirrhosa and Dosidicus gigas) by fast real-time PCR
Montserrat Espieira , Juan M. Vieites
Research Unit of Genomics and Proteomics Applied to Marine and Food Industry, ANFACO-CECOPESCA, Vigo, 36310 Pontevedra, Spain

a r t i c l e

i n f o

Article history:
Received 20 April 2012
Received in revised form 4 July 2012
Accepted 7 July 2012
Available online 20 July 2012
Keywords:
Octopus
Octopus vulgaris
Dosidicus gigas
Eledone cirrhosa
Fast real-time PCR
Authentication

a b s t r a c t
The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently
most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of
species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system
for the detection and identication of common octopus (Octopus vulgaris) and main substitute species
(Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed,
sensitivity and specicity in an homogeneous assay.
The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of
food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them.
The methodology herein developed is useful to check the fulllment of labeling regulations for seafood
products and to verify traceability in commercial trade and for sheries control.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
The common octopus, Octopus vulgaris, is a kind of octopus
cephalopod family Octopodidae that is found worldwide in tropical
and semitropical waters from near shore shallows to as deep as
200 m (Caddy, 1983).
The generic term octopus encompasses a range of soft mollusc
species included in the taxonomic order of the Octopods. Their
importance as a shery resource is widely recognized throughout
the world as a culinary product highly appreciated by the excellent
avor of their meat, its high content of calcium, vitamin A and B,
and low content in calories.
Fishing is carried out throughout the year and octopus products
can be found in worldwide markets, both fresh and frozen, cooked
or uncooked and also canned.
The high demand for octopus products has produced a gradual
increase in catches over the years resulting in a decrease in the
world population of this resource. This increase is reected by the
global catch data of octopuses from the FAO. In fact, it has grown
from capture values of 35,844 tons recorded in 1950 to values of
372,872 tons recorded in 2009 (FAO-FishStat, 2006). This has
prompted the authorities to establish closed seasons, minimum
sizes, Total Allowable Catches (TAC), number of traps and limited
Corresponding author. Tel.: +34 986 469 301; fax: +34 986 469 269.
E-mail address: montse@anfaco.es (M. Espieira).
0308-8146/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.07.056

working hours with the ultimate aim of ensuring the protection

and regeneration of the species. Also, to avoid unfair competition
and assure correct labeling, the authorities have established different regulations such as the European Commission Regulation 104/
2000 and 2065/2001 (Commission Regulation (EC) No. 2065/2001,
2001, Council Regulation (EC) No. 104/2000, 2000) which establish
the norms to apply over the commercial denomination and the scientic names of the species, and the Royal Decrees of 2004 (Royal
Decree (RD) 121/2004, 2004; Royal Decree (RD) 1702/2004, 2004)
where rules on the identication of shery products, aquaculture
and seafood are established, both for fresh, chilled or cooked and
frozen products. It is noted that for cephalopod commercial denomination, each country has its own legislation which inuences not
only the species sold, but the nal commercial formats. To this must
be added the great globalization of markets and the open borders
which permit species from many sources to be available to the consumer. All of this can lead to confusion with respect to the identication and labeling of products made from cephalopods (Herrero,
Lago, Vieites, & Espieira, 2012a).
For these reasons, it is necessary to develop methods that allow
species authentication in products regardless of the degree of processing that has been applied. In this sense, genetic techniques are
emerging as the best tool and present signicant advantages over
techniques based on protein analysis. These advantages include
the small amount of sample required for analysis, the greatest
degree of genetic variability examined and the possibility of

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M. Espieira, J.M. Vieites / Food Chemistry 135 (2012) 24392444

analyzing samples subjected to different treatments. This is all due

to the high stability of DNA. There are several works which have
studied the identication of cephalopods by molecular methods
(Espieira, Vieites, & Santaclara, 2010; Santaclara, Espieira, & Vieites, 2007). These studies are complementary and both methods
described can be combined into a single global protocol allowing
the identication of the most commercially important species of
squid, octopus, cuttlesh, bobtail and bottle squid and all other
species that may be used as substitutes for higher value species
in processed food.
In contrast to conventional PCR techniques, real-time PCR is a
technology that has recently developed as a technique for the
authentication of species in food products (Herrero, Lago, Vieites,
& Espieira, 2011a, 2011b, 2012a, 2012b; Herrero, Madrin, Vieites, & Espieira, 2010; Herrero, Vieites, & Espieira, 2011a, 2011b;
Rojas et al., 2011; Trotta et al., 2005). Particularly relevant is the
study (Herrero et al., 2012a) that describes a methodology for controlling the correct labeling of products containing Loligo vulgaris.
This technique is characterized by real time measurement of PCR
products by the use of uorescent labels; thus it is inherently more
accurate than conventional PCR assays.
This technique is robust, sensitive and easily automatable. In
addition, the main advantage is its rapidity, especially in the case
of fast real-time PCR because this reduces the run time.
In this work, three methodologies based on fast-real time PCR
have been developed for the authentication of O. vulgaris and other
common substitute species such as Eledone cirrhosa and Dosidicus
gigas in order to identify fraud and assure the correct labeling of
products, including these that have undergone intensive treatments such as those involving high temperature and pressure.

Table 1
Samples included in this work.
Family

Species

Octopodidae

Bathypolypus bairdii
Bathypolypus pugniger
Eledone cirrhosa
Eledone moschata
Enteroctopus doeini
Octopus aegina
Octopus delippi
Octopus dollfusi
Octopus fangsiao
Octopus macropus
Octopus maya
Octopus membranaceus
Octopus mimus
Octopus vulgaris

Ommastrephidae

Dosidicus gigas
Illex argentinus
Illex coindetii
Illex illecebrosus
Nototodarus sloanii
Ommastrephes bartrami
Todarodes lippovae
Todarodes pacicus
Todarodes sagittatus
Todaropsis eblanae

Loliginidae

Loligo vulgaris
Loligo reynaudi
Loligo forbesii
Loligo gahi
Loligo opalescens
Loligo pealeii
Loligo plei
Loligo bleekeri
Alloteuthis media
Alloteuthis subulata
Loliolus japonica
Lolliguncula diomedeae
Lolliguncula panamansis
Uroteuthis chinensis
Uroteuthis duvauceli

Sepiidae

Cirroteuthidae

Sepia aculeata
Sepia apama
Sepia bertheloti
Sepia dollfusi
Sepia elegans
Sepia esculenta
Sepia hierredda
Sepia lycidas
Sepia ofcinalis
Sepia orbignyana
Sepia pharaonis
Sepietta oweniana
Inioteuthis japonica
Rossia palpebrosa
Sepiola atlantica
Sepiola rondeletii
Cirroteuthis mulleri

Gonatidae

Gonatus fabricii

2. Materials and methods

2.1. Sample collection, storage and DNA extraction
Specimens obtained to develop this methodology have been
previously authenticated both morphologically and genetically in
studies carried out by Espieira et al. (2010) and Santaclara et al.
(2007). Regarding O. vulgaris; E. cirrhosa; D. gigas, specimens and
samples (an average of 35) belonging to the complete geographical
distribution range for each species have been used to carry out this
methodology (Table 1).
Additionally, the methodologies developed were applied to 34
seafood products purchased in supermarkets and shops in Spain
(Table 2). DNA extraction was performed on 30 mg of muscle tissue (fresh and frozen) and 300 mg of processed products according
to the protocol described by Herrero et al. (2011b). The quality and
concentration of DNA 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 Scientic)
(Winfrey, Rott, & Wortman, 1997). DNA extractions were appropriately labeled and stored at 80 C for subsequent tasks.
2.2. Design of a specic fast real-time PCR method
2.2.1. Detection and identication method for common octopus
(O. vulgaris)
Sequences of the cytochrome oxidase subunit III (COIII) gene
were downloaded from the National Center for Biotechnology
Information (NCBI) (O. vulgaris (AJ250476), E. cirrhosa (X97949),
O. maya (GU362546) and O. mimus (AJ250480)). From the resulting
alignment, specic primers/probe set were designed by using Primer Express software (Applied Biosystems) (Table 3).
Also, a second specic primers/probe set that can help to improve the efciency and specicity of this methodology, and ampli-

cations of the partial 16S RNA gene fragment were carried out
using the primers and the protocol described previously by Bonnaud, Boucherrodoni, and Monnerot (1994, 1997). PCR products
were sequenced in both directions to avoid sequencing errors
using the same primers of PCR amplication. DNA automatic
sequencing in an ABI Prism 3130 (Applied Biosystems) was
performed in both strands in a nal volume of 10 ll with BigDye
Terminator cycle sequencing ready reaction v1.1 (Applied Biosystems). Thermal cycle sequencing reaction and the subsequent
sequencing product cleanup by ethanol precipitation were carried
out in accordance with the manufacturers instructions (Applied
Biosystems). Raw data were analyzed with Sequencing Analysis

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M. Espieira, J.M. Vieites / Food Chemistry 135 (2012) 24392444

Table 2
Commercial samples analyzed with the developed methodologies.
Product

Canned

Prepared
Dishes

Frozen

Fresh

Octopus. Seafood delicacies

Octopus in oil
Octopus in olive oil
Octopus in olive oil
Octopus in olive oil
Octopus medallions in olive oil
Octopus-squid in olive oil
Octopus in Galician sauce
Octopus in Galician sauce
Octopus in garlic sauce
Octopus pieces in garlic sauce
Octopus-squid in garlic sauce
Octopus-squid in marinara sauce
Octopus-squid in marinara sauce
Octopus-squid in marinara sauce
Galician-style octopus
Galician-style octopus
Octopus tentacles
Octopus tentacles in pieces
Octopus tentacles in pieces
Octopus in garlic sauce
Octopus salpicon
Octopus in vinaigrette
Octopus pie
Seafood cocktail
Cooked octopus
Octopus
Octopus
Octopus
Octopus
Octopus
Octopus
Octopus
Octopus

Species labeled

Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Dosidicus gigas
Unidentied
Unidentied
Dosidicus gigas
49% Squid and 22% Octopus
Unidentied
Dosidicus gigas
Dosidicus gigas
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Unidentied
Octopus spp.
Octopus vulgaris
Octopus vulgaris
Octopus spp.
Octopus spp.
Unidentied
Octopus spp.
Unidentied
Unidentied

Software v 5.3.1 (Applied Biosystems) and these sequences were

analyzed with Chromas 1.45 software (Mc Carthy, 1996) and
aligned with CLUSTAL_W (Thompson, Gibson, Plewniak, Jeanmougin, & Higgins, 1997) available in the program BioEdit 7.0 (Hall,
1999). The nucleotide sequences obtained were submitted to the
GeneBank database of the NCBI.
From the sequences of the 16S RNA gene, two internal primers
and probe were designed inside this region by using Primer Express software (Applied Biosystems) (Table 3).
2.2.2. Detection and identication method for E. cirrhosa
Amplications of the partial 16S RNA gene fragment were carried out using the primers and the protocol described above for
the design of the detection and identication methodology for O.
vulgaris. Finally, two internal primers and probe were also designed inside this region using Primer Express software (Applied
Biosystems) (Table 3).
2.2.3. Detection and identication method for D. gigas
Primer ITS5 and ITS4 described by White, Bruns, Lee, and Taylor
(1990) were used to amplify the ITS1-5.8S-ITS2 region.
To obtain DNA sequences, PCR products were cloned into
pGEM-T Easy Vector System II (Promega), following the suppliers
recommendations. Subsequently, sequences were performed as
previously described. From the sequences of the ITS 1 gene two
internal primers and probe were designed inside this region by
using Primer Express software (Applied Biosystems) (Table 3).
2.3. Design of a specic assays
Independent PCRs for each of the three designs were carried
out in a total volume of 20 lL containing 250 ng of DNA template

Species identied by sequencing

Octopus vulgaris
Dosidicus gigas
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Eledone cirrhosa
Dosidicus gigas
Octopus vulgaris
Octopus vulgaris
Dosidicus gigas
Dosidicus gigas
Octopus vulgaris
Dosidicus gigas
Dosidicus gigas
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Octopus maya
Octopus maya
Octopus maya
Octopus mimus
Octopus mimus
Eledone cirrhosa

Species identied by Real Time PCR

O. vulgaris

E. cirrhosa

D. gigas

+

+
+
+
+


+
+


+


+
+
+
+
+
+
+
+
+
+
+
+
+













+


























+


+





+


+
+

+
+




















and 10 lL of TaqMan Fast Advanced Master Mix (Applied Biosystems). The amount of primers and probe were optimized and
the nal volume was adjusted with Ambion Nuclease-free water
(Ambion). The reactions were performed in triplicate on DNA
samples in MicroAmp Optical 96-well reaction plates (Applied
Biosystems) with MicroAmp optical caps (Applied Biosystems)
using the ViiA 7 Real-Time PCR System (Applied Biosystems).
Amplication was carried out with the following conditions:
95 C for 20 s, and 40 cycles each of 95 C for 1 s and 5060 C
for 20 s.
The specicity and sensitivity for each of the three designs were
independently evaluated. The specicity was evaluated by testing
the amplication of DNA from each species with the other outgroups (Table 1). The limit of detection (LOD) is dened as the lowest concentration of DNA which yields a uorescent signal signicantly different from the negative control. For this, a serial dilution
of DNA from each species was performed with DNA of other species (Table 1) in levels ranging from 250 ng to 0.001 pg and the
uorescence signal was determined for each of the three primerprobe set. Dilutions were prepared by adding DNA of target species
to DNA from different species until completing the nal amount of
250 ng. All measurements were performed in duplicate from three
samples independently.
2.4. Methodological validation
Individual samples of O. vulgaris, E. cirrhosa and D. gigas were
authenticated on the basis of their morphological features. Different products, e.g., rings, tubes and arms, all in different preparations (fried, frozen, coated in butter and canned) were elaborated
from these individuals. The most extreme treatment that was applied to the samples was the sterilization in a horizontal retort

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M. Espieira, J.M. Vieites / Food Chemistry 135 (2012) 24392444

Table 3
Primers and probes used in this work.
Oligonucleotide name

Sequence 50 30

OVUL F2
OVUL R2
OVUL 2
OVUL 16SF
OVUL 16SR
OVUL 16S
ELEDONE 16SF
ELEDONE 16SR
ELEDONE 16S
DGIG F
DGIG R
DGIG

CCC TAT TTA ATA CAG CAA TCT TA

GTT GCG GAT TTT AAA TCA TTA
6-FAM-TATCCGTCACCTGAGCACACC-BHQ1
GCT GCG GTA TTA TAA CTG
GAC AGT TAA ATT CTC GTC AA
6-FAM-ACTAAGGTAGCATAATAATTTGCCCT-BHQ1
ACC GAG AGT TTT GCT TAA
CAC CTA TCT GAG CT TCTG
6-FAM-TCC AAC ATC GAG GTC GCA ATC T-BHQ1
GGA AGT AAA AGT CGT AAC AAG
CCT TCT TCG GCA AAA GAG
6-FAM-TGA ACC TGC GGA AGG ATC ATT ACC-BHQ1

steel-air, at 115 C for 20 min, with 1.2 bars of overpressure and in

cooking water for 30 min.
These sample treatments were performed in the pilot plant of
ANFACO-CECOPESCA (Spanish National Association of Seafood Producers National Technological Centre for Seafood and Aquaculture Products Preservation).
Products were analyzed in the same way as the standard species used to develop the methodologies described. The results obtained using morphological features were compared to those
obtained by means of the application of the methodology developed to determine the specicity of the methods.
2.5. Application of real-time PCR to commercial products
After the validation of the methods developed, they were applied to 34 products labeled as octopus. These products were acquired in supermarkets in Spain. The purpose of these analyses
was to evaluate the situation regarding the labeling of these products (Table 3).
In order to ensure the proper working of real-time PCR, the
methodology described by Espieira et al. was applied (Espieira
et al., 2010).
3. Results and discussion
3.1. Design of fast real-time PCR method specic to detect and identify
O. vulgaris, E. cirrhosa and D. gigas
Rapid identication of octopus species is an important issue for
the shery industry, not only for traceability control in the food
chain, but also in the production and release of manufactured
products.
To obtain reliable results, it is necessary to optimize the reaction. Optimization leads to the highest level of sensitivity while
maintaining the specicity of the technique. These conditions were
established by mixing different concentrations of primers and
probes. Concentrations of 900 nM for each primer and 250 nm
for each probe yielded the highest endpoint uorescence and the
lowest threshold cycle.
The specicity of each primer/probe set was conrmed using
genomic DNA from other species from different geographical areas.
No cross-reactivity was detected with any of the tested samples
(Table 1).
The optimal annealing temperature should be determined
empirically because it depends on the melting temperature of each
primer/probe set (Winfrey et al., 1997). In the present work, for
each method this temperature was different (Table 3).
The threshold cycle (Ct) is an important parameter for real-time
PCR. The Ct depends on the starting template copy number, the
efciency of PCR amplication, efciency of cleavage or hybridiza-

Gene

Amplicon size

Tm

COIII

111 bp

55 C

16S

109 bp

58 C

16S

153 bp

58 C

ITS 1

116119 bp

60 C

Table 4
Efciency and sensibility.
Oligonucleotide name

LDO

Efciency

Slope

R2

OVUL F2
OVUL R2
OVUL 2
OVUL 16SF
OVUL 16SR
OVUL 16S
ELEDONE 16SF
ELEDONE 16SR
ELEDONE 16S
DGIG F
DGIG R
DGIG

0.05 pg/nl

85.15%

3.73

0.996

0.05 pg/nl

99%

3.34

0.999

5 pg/nl

91.53%

3.543

0.989

0.5 pg/nl

105%

3.1868

0.999

tion of the uorogenic probe and the sensitivity of detection (Bustin, 2004).
In all samples of O. vulgaris, E. cirrhosa and D. gigas, the Ct values
obtained were 25 2 and 22 2 for O. vulgaris designs, COIII and
16S RNA gene specic designs respectively, and 18 2 and 15 2
respectively for E. cirrhosa and D. gigas specic designs when
100 ng of fresh and frozen template were used. In processed products, this Ct was higher but always less than 32 due to the DNA
fragmentation, in all designs. All depends on the degree of processing of each sample analyzed, and which depends in turn on temperature, pressure and additives added to the sample, so the Ct
value will vary as a function of these parameters without being
able to dene a representative mean value. Also, in the cross-reactivity analysis, no false positive results were observed, even under
the most severe conditions used in the test. This was documented
by Ct values, where Ct values found were always >40 in all non-target species under study for each design.
Robust and precise assays are usually correlated with high PCR
efciency. For each of the three designs, the efciency was calculated based on the slope of the standard curve obtained using
DNA serial dilutions with template of known concentration (from
250 to 0.025 ng) as templates for real-time PCR (Bustin, 2004).
The efciency (E) of each reaction was determined by the following
equation E = (10(1/slope)  1)  100 (Table 4 and Fig. 1).
With respect to efciency, the optimal assay values range between 90% and 110%. The value of the efciency obtained for the
specic assay of O. vulgaris with the primer/probe set designed
from COIII was outside these values (85.15%), while that obtained
with the set designed from 16S RNA gene was better (99%). It
should be noted also that although the value of R2 was optimal
for both the primer/probe set designed from COIII as created from
the 16S gene (0.996 and 0.999 respectively), these criteria established that the primers/probe set designed from 16S gene was better and, therefore, that the specic assay for O. vulgaris designed
with this set was also better.

M. Espieira, J.M. Vieites / Food Chemistry 135 (2012) 24392444

2443

Fig. 1. Amplication plots of the 10 dilution series of the D. gigas target with primer/probe set DGIG.

The aim of the species identication methodologies was not to

nd small traces of an ingredient or substance in a sample and subsequently therefore the LDO was not a critical point for the method. However, the detection limit was calculated for each one of the
designs in order to show the high sensitivity of these methodologies (Table 4).
3.2. Methodological validation
The aim of the methodological validation was to check whether
the manufacturing process inuenced the detection of target species. The elaborated products in the pilot plant of ANFACO-CECOPESCA were analyzed using the proposed methodology.
The results obtained with the application of the fast real-time
PCR method developed were in agreement with those that were
based on morphological characteristics. Therefore, the specicity
of the method is 100% when applied to these kinds of products.
3.3. Application to commercial products
Commercial products were acquired from supermarkets in
Spain. Here, this taxonomic group is a valuable resource due to
its high demand. Canned varieties constitute one of the main commercial formats. In response, since 1984 Spain has legislated products sold under this format (Annex 4, Royal Decree (RD) 1521/
1984 of 1 August 1984 approving the technical-sanitary regulation
of shery and aquaculture establishments and shery and aquaculture products intended for human consumption, 1984). The application of the real-time PCR method developed in the present study
to 34 commercial samples conrmed level of compliance with the
labeling regulations for cephalopods.
Due to high demand and current stocks of this natural resource,
fresh octopus is scarce in the market so it is usual that closed seasons are established. One consequence of these closed seasons is a
raised commercial value for octopus products. As a result, it was
difcult to acquire fresh products for this work. Consequently,
the number of fresh octopus samples was lower than that of other
products, but this fact was not of great importance because the

identication in fresh products by morphological features was

successful.
It is important also to note that labeling regulations do not require the particular species to be declared. This explains the reasons for the vast majority of products (67.6%) failing to supply
such information. This percentage is higher in fresh products
(100%) and prepared dishes (100%) than in canned (66.6%) and frozen products (25%) (Table 2). In the rst case, the information is
usually limited to the commercial name of the product and the
capture area, while in frozen octopus its scientic name usually appears, but in prepared and canned dishes, even when the species
from which the product has been manufactured is not included,
the commercial name is also indicated as well as commercial format (integer, tentacles, in pieces, etc.) as well as the various additives used as sauces and condiments. It is also important to note
that all prepared dishes, even if the species was not declared, were
prepared from O. vulgaris.
Within frozen products there were 5 commercial samples that
could not be identied by the technique developed in this work.
After proceeding according to the methodology of Espieira et al.
(2010), it was conrmed that this was because none of the 5 samples belonged to any of the 3 species under study; 3 of them were
identied by FINS technique as O. maya, and the other 2 as O.
mimus (Table 2).
The seas off the coast of Galicia in Northwest Spain are cool and
rich in nutrients. For this reason, O. vulgaris from this region is
highly appreciated. Despite the absence of an ofcial certicate
of designation of origin of the Galician octopus (Galician Ras
Octopus), this denomination is used to legally protect certain
foods that are produced in a given area, thus adding value to the
product and providing access to national and international
markets.
Galician octopus is the traditional way of preparing octopus in
typical Galician fairs and pilgrimages and is normally associated
with O. vulgaris species. Reviewing market products, all but one
of the products labeled as Galician-style octopus or Octopus in
Galician sauce contain O. vulgaris. This exception was a product
described as Octopus in Galician sauce and, despite being correctly labeled as D. gigas in the ingredient listing, this species is

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M. Espieira, J.M. Vieites / Food Chemistry 135 (2012) 24392444

not an octopus, so this ambiguity between the commercial name of

the product and the labeled and identied species, has the potential to mislead consumers.
Finally, as demonstrated during the methodology validation,
the level of processing applied to a sample affects the value of its
Ct. The greater the degree of processing a sample has undergone,
the higher the Ct.
In conclusion, the results suggest that the fast real-time PCR
technique developed here represents a useful tool for the authentication of species such as O. vulgaris, E. cirrhosa and D. gigas in different products including processed products that have undergone
aggressive treatments such as canning. It presents advantages in
terms of accuracy, sensitivity, specicity, dynamic range, highthroughput capacity and absence of post-PCR manipulation, making it different from other techniques such as conventional PCR.
The possible applications of these methods are to authenticate
imported species, to verify the traceability of products in commercial trade and to ensure the correct application of food labeling
regulations.
Acknowledgment
This work was funded by Grant 10MMA004CT from Consellera
de Innovacin e Industria de la Xunta de Galicia.
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