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Food Control 19 (2008) 10961099

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Short communication

Application of multiplex PCR for the identication of grouper meals

in the restaurant industry
Luis Asensio *
Departamento de Nutricion, Bromatologa y Tecnologa de los Alimentos, Facultad de Farmacia, Universidad San Pablo
CEU, 28668 Boadilla del Monte, Madrid, Spain
Received 23 May 2007; received in revised form 30 October 2007; accepted 7 November 2007

Abstract
Detection of sh species adulteration in the restaurant industry is important for consumer protection and condence, and for an accurate implementation of the traceability for successful regulatory food controls. In this study, 37 purported grouper (Epinephelus marginatus) meals (20 from school and university lunch rooms and 17 from restaurants) from Madrid have been analysed by using multiplex
PCR technology. Species-specic primers of the 5S rDNA gene (designed previously in another work) were used obtaining specic DNA
fragments that could authenticate grouper meals; only 9 out of 37 samples were conrmed as authentic grouper. This genetic marker
could be very useful for the accurate authentication of grouper meals in the restaurant industry.
2007 Elsevier Ltd. All rights reserved.
Keywords: Grouper; Restaurant industry; Multiplex PCR; Authentication

1. Introduction
Identication of sh species for the restaurant industry
can be uncertain whenever the usual external characteristics such as skin pigmentation, shape, size and appearance
are removed on cooking processing and only a portion of
esh is available. For this reason, when sh is cooked
opportunities for substitution or adulteration increase
(Mackie, 1996). Mislabelling in this industry could be
harming consumer condence in sh and shery products.
It may happen for two reasons:
 Fish may be incorrectly identied at capture or wholesale
and incorrect name is carried on and used at the point of
sale. When restaurateurs purchase the sh species, they
do not have the expertise to correctly identify species.
 Restaurateurs could substitute or rename sh species in
order to get a better price or to meet consumer demand
for a particular species.
*

Tel.: +34 91 372 64 49; fax: +34 91 351 04 75.

E-mail address: lasen.fcex@ceu.es

0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2007.11.002

In this context, grouper (Epinephelus marginatus) is a

delicate taste sh with high cost, popularity and demand.
Grouper llets in the marketplace and grouper meals at
restaurants are subjected to substitution using cheaper sh
species such as Nile perch (Lates niloticus). Moreover,
grouper can be substituted by wreck sh (Polyprion americanus): this species, which is similar to grouper, is renamed
as grouper to make it sound more appealing to consumers.
Therefore, analytical methods able to identify sh species are essential to prevent wilful or unintentional substitution of high priced sh with lower priced species in the
restaurant industry.
Although protein based methods such as immunological, electrophoretic and chromatographic techniques are
of considerable value in certain instances, some proteins
are denatured, and therefore other techniques such as
DNA-based methods are more adequate for the analysis
of cooked sh. DNA molecule is stable at high temperature
and the information content of DNA is higher than proteins (Lockley & Bardsley, 2000a; Woolfe & Primrose,
2004). DNA-based methods developed for sh identication include sequencing of polymerase chain reaction

L. Asensio / Food Control 19 (2008) 10961099

(PCR)-amplied genomic and mitochondrial DNA fragments, PCR-restriction fragment length polymorphism
(PCR-RFLP), PCR-single strand conformation polymorphism (PCR-SSCP), random amplied polymorphic
DNA (RAPD) and multiplex PCR, among others (Asensio
Gil, 2007).
Among these methods multiplex PCR is very rapid, specic, reliable and easy to perform. Prior sequence knowledge is required in order to design specic primers; this
permits the size of the product to be predicted, so that identication is conrmed if an appropriately sized amplicon is
seen on a gel (Lockley & Bardsley, 2000a). This method
oers a promising alternative for detection of sh species
adulteration in the restaurant industry.
On the basis of this information, a multiplex PCR has
been used for the identication of grouper meals in the
Madrid restaurant industry. This methodology has been
based on PCR amplication of species-specic fragments
in the 5S rDNA gene.
2. Materials and methods
2.1. Samples collection and DNA extraction
Authentic specimens of grouper, wreck sh and Nile
perch were obtained from local markets from Madrid.
Every species was morphologically identied according to
the keys of Froese and Pauly (2007) and Muus, Nielsen,
Dahlstrom, and Nystrom (1998).
On the other hand, 37 sh meals samples were collected
at random in dierent places of the Madrid restaurant
industry; 20 of them from school and university lunch
rooms and 17 from restaurants. These samples appeared
in the menu as roasted, grilled or fried grouper. Approximately, 1 g of each sample was collected from the meals.
Genomic DNA was extracted from sh muscle samples,
according to a previously described procedure (Cespedes
et al., 1998).
2.2. Multiplex PCR
The set of primers used for PCR amplication of DNA
were the forward primer 5S1 (50 -TACGCCCGATCTCGTCCGATC-30 ), designed by Penda`s, Moran, Freije, and
Garca-Vazquez (1994), and the specically reverse primers
5SG (50 -CTTAATGCACATATGCTCACTGAC-30 ), 5SW
(50 -CCTCTGTGCTATAAGTTGGACCT-30 ) and 5SP (50 TACGCTGACGTGCAGATGCA-30 ) designed by Asensio et al. (2001). These primers were mixed in the same ratio
and used together for the multiplex PCR of this study.
Double-stranded amplications were carried out in a
nal volume of 25 ll containing 2 mM MgCl2, 10 pmol
of each primer (5S1, 5SG, 5SW and 5SP), 510 ng of template DNA, and 2 U of DNA polymerase (Roche, Mannheim, Germany) in a reaction buer containing 75 mM
TrisHCl, pH 9.0, 50 mM KCl, 20 mM (NH4)2SO4 and
0.001% bovine serum albumin.

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PCR amplication was performed in a GeneAmp PCR

System 9700 Thermal Cycler (Applied Biosystems, Foster
City, CA). Thirty ve cycles of amplication with the following step-cycle prole were carried out: strand denaturation at 94 C for 45 s, primer annealing at 65 C for 45 s
and primer extension at 72 C for 45 s. An initial denaturation at 94 C for 3 min, and a nal extension at 72 C for
5 min, improved the product yielded. The amplication
products were tested in a 1.5% D1 (Pronadisa, Torrejon
de Ardoz, Spain) agarose gel, containing ethidium bromide
(1 lg/mL) in Trisacetate buer (0.04 M Trisacetate,
0.001 M EDTA, pH 8.0). DNA fragments were visualized
by UV transillumination and analysed using Geldoc 1000
UV Fluorescent Gel Documentation System-PC (Bio-Rad
Laboratories, Hercules, CA).
3. Results and discussion
Grouper (E. marginatus) is a high quality sh species frequently substituted in the marketplace. As there was no
study previously related to the analysis of grouper meals
in the restaurant industry, the purpose of this study has
been the application of the multiplex PCR for the authentication of grouper meals in this industry. Due to its high
cost, when grouper loses its morphological characteristics
during cooking process is highly susceptible to be substituted for cheap sh species such as Nile perch (L. niloticus)
in meals.
In search for fast and simple genetic techniques, multiplex PCR has gained acceptance among sh species identication methods because it is very rapid, specic, reliable
and easy to perform. Multiplex PCR reactions have been
described for sh and shery products identication
such as black caviar (DeSalle & Birstein, 1996), at sh
species (Cespedes et al., 1999), tuna and bonito (Lockley
& Bardsley, 2000b), cod, haddock and whiting (Taylor,
Fox, Rico, & Rico, 2002) and swordsh meat (Hsieh,
Chai, Cheng, Hsieh, & Hwang, 2004). Some of these
works have reported the analysis of processed shery
products.
In this context and in agreement with the results
obtained in a previously reported work (Asensio et al.,
2001), the combination of dierent primers specic for
grouper (5SG), Nile perch (5SP) and wreck sh (5SW),
along with the 5S1 oligonucleotide (forward primer),
allowed the amplication of specic regions of the 5S
rDNA gene for these three sh species used as standards
in this work: a 323 bp fragment from grouper DNA, a
471 bp fragment from wreck sh DNA and a 185 bp fragment from Nile perch DNA (Fig. 1A, lines 13). When the
meal samples, collected from the restaurant industry, were
analysed by multiplex PCR the amplicons obtained were
compared to those standards to determine meals authenticity. After this analysis, 9 out of 37 samples analysed corresponded to be grouper, 6 of them were wreck sh, 19 were
Nile perch and 3 unknown (no amplication was obtained)
(Fig. 1, lines 440).

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L. Asensio / Food Control 19 (2008) 10961099

Fig. 1. Electrophoretic analysis of the 5S rDNA amplicons from the multiplex PCR. (A) Samples are: 1 = grouper; 2 = wreck sh; 3 = Nile perch; 4
20 = restaurant meal samples and (B) Samples are: 2140 = meal samples from school and university lunch rooms. M indicates 1 kb plus DNA ladder
(Gibco BRL, Life Technologies, Inc., Rockville, MD) for molecular weight marker and NC corresponds negative control.

Likewise, in the samples collected from the restaurants,

9 were grouper, 6 were wreck sh, 1 was Nile perch and 1
an unknown sample (Fig. 1A). In these restaurants the
fraud was lower than school and university lunch rooms
since only one sample from a restaurant turned out to be
Nile perch and in another restaurant the sample was
unknown: in this last case, probably a cheap sh species
could be used. Moreover, since grouper and wreck sh
quality and price are very similar, this would not be a real
fraud since wreck sh is renamed as grouper to make it
sound more appealing to consumers.
On the other hand, all the samples obtained from school
and university lunch rooms were incorrectly named in the
menus since none of these samples was grouper. Almost
the majority of these samples corresponded to be Nile
perch (Fig. 1B) which is the sh species most used as a substitute for grouper in Spain. Moreover, the 2 samples
unknown probably were other cheap sh species used as
a substitute for grouper.
In conclusion, multiplex PCR based on 5S rDNA gene,
is a powerful technique that has been proved to be very
useful to detect mislabelling meals in the restaurant industry. It has the potential to produce considerable savings of
time and eort within the laboratory without compromising test utility. The use of this technology in this work is
a good example of applied research and its direct impact
on the lives and health of consumers.

Acknowledgements
The author is grateful to Rosario Martn, Isabel Gonzalez and Teresa Garca (Departamento de Nutricion, Bromatologa y Tecnologa de los Alimentos de la Facultad de
Veterinaria de la UCM) for the work developed previously
related to the design of species-specic primers from grouper, wreck sh and Nile perch.
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