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Food Control 22 (2011) 1015e1020

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

PCR-SSCP method for genetic differentiation of canned abalone and commercial

gastropods in the Mexican retail market
Fernando Aranceta-Garza, Ricardo Perez-Enriquez, Pedro Cruz*
Centro de Investigaciones Biolgicas de Noroeste (CIBNOR), Mar Bermejo 195, Playa Palo de Santa Rita, 23090 La Paz, B.C.S, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Traceability of canned abalone is not only being demanded to protect quality and certication of safety,
Received 9 August 2010 but also to uncover black market or fraudulent practices, such as canning other gastropods sold as
Received in revised form abalone, abalone style, or showing an abalone shell picture on the label. A forensic method for genetic
11 November 2010
differentiation of three species of abalone from other gastropods was developed with 18S rDNA analysis
Accepted 28 November 2010
by PCR-SSCP. A lysin gene marker was also developed to identify abalone species in Mexico. Both markers
worked correctly in raw, frozen, and canned products. In a forensic analysis, two out of seven canned
Keywords:
brands that claimed to contain abalone contained other gastropods. None of three other brands that
Mexican abalone
SSCP
showed the legend Abalone or an abalone shell picture contained abalone. The Chilean loco Con-
18S rDNA cholepas concholepas is the most common product used in abalone substitution. The lysine gene was
Lysin capable of separating canned abalone based on the species. Both methods proved reliable and useful for
Gastropods identifying abalone and can be used to certify the authenticity of the Mexican commercial product or
identify commercial fraud. By this genetic approach, the Mexican abalone industry could guarantee
authenticity.
2010 Elsevier Ltd. All rights reserved.

1. Introduction
With an estimated value of US$ 800 million, abalone is a valu-
able mollusk sold on the world market (Gordon & Cook, 2004). Its
high market value has been the basis of extensive commercial
exploitation for many years (Len-Carballo & Mucio-Diaz, 1996)
and this led to over-exploitation in several countries. To reverse
declining wild production, cultivation of abalone has increased,
with China and Taiwan as the main domestic producers (Gordon &
Cook, 2001).
The Mexican shery mainly exploits green abalone Haliotis ful-
gens and pink abalone Haliotis corrugata (Len-Carballo & Mucio-
Diaz, 1996), with an incipient aquacultural production of the red
abalone Haliotis rufescens. Most of the commercial product is can-
ned in brine, either whole (w84e90%) or in pieces (w10%). A small
proportion (5%) is sold fresh without the shell (CONAPESCA, 2003).
Removing the shell is a common practice in preparing gastropods
(abalone, clams, scallops, mussel), which is the only truly morpho-
logical character for identifying the species. This provides oppor-
tunities for commercial fraud by substituting lower quality or lower
priced products or illegal species (Supernault et al., 2009; Sweijd,
* Corresponding author. Tel.: 52 612 123 8484x3345; fax: 52 612 125 3625.
E-mail address: pcruz@cibnor.mx (P. Cruz).
Bowie, Marinaki, Harley, & Cook, 1998). Several canned products
originate in Mexico or other Latin-American countries that do not
contain abalone, but are labeled Abalone or abalone style are sold
on the Mexican retail market. One of the main non-abalone products
is the muricid Concholepas concholepas, commonly named loco in
Chile (Aguilera-Muoz, Valenzuela-Muoz, & Gallardo-Escrate,
2008).
The main worldwide challenge is certication of the quality and
safety of the products (Aguilera-Muoz et al., 2008). Since 2005,
food companies (including the aquaculture industry) have been
mandated to develop and implement protocols for identication
and traceability of their products (European Union regulation 178/
2002 and USA regulations under the Bio-Terrorism Law and the
COOL [Country of Origin Labeling] provision) to avoid fraud
(Thompson, Sylvia, & Morrissey, 2005). In canning seafood prod-
ucts, physically distinct characteristics are lost; hence the need to
rely on DNA tools for identication and certication (Bossier, 1999;
Lockley & Bardsley, 2000).
Single-strand conformation polymorphism (SSCP) analysis is
a molecular technique that is very sensitive for detecting a single
base exchange or a few differences between sequences of short DNA
fragments, ranging from 100 to 400 bp (Hayashi, 1996; Lessa &
Applebaum, 1993). This capability relies on two principles: (1)
Single-strand DNA fragment conformation (secondary structure) is

0956-7135/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2010.11.025
 Author's personal copy
1016 F. Aranceta-Garza et al. / Food Control 22 (2011) 1015e1020
dependent on the sequence nucleotides (primary structure), and (2)
Minor changes in the sequence affects mobility of the single-strand
DNA fragment (Lessa & Applebaum, 1993). This leads to different
sequences migrating unevenly and presenting distinct banding
patterns.
Our objective was to create molecular tools that can differen-
tiate abalone (Haliotis spp.) from other commercial fresh, frozen,
and canned gastropods based on 18S rDNA and identify specic
abalone product at the species level using the lysin gene.
2. Materials and methods
2.1. Sample collection and DNA extraction
Wild specimens of abalone (H. fulgens and H. corrugata) were
obtained from the State of Baja California, Mexico and cultivated
specimens (H. rufescens) from an aquaculture facility (Table 1). Three
gastropods (Fissurella volcano, Megathura crenulata, and Megastrea
undosa) were also collected in the same region. Samples of the
limpet Fissurella spp. and the rock snail C. concholepas were obtained
from Chile (Table 1). Retail markets and manufacturers were the
source of 13 brands of canned products (Table 2). A can may contain
two to eight whole individuals or 18e24 pieces. For sampling, two to
four individual meat samples were randomly selected from each
can. A piece of foot (fresh samples) or meat (canned samples) was
dissected and xed in 70% ethanol until DNA extraction.
Total genomic DNA from fresh abalone samples were isolated
from 0.1 g of muscular tissue (foot) using a protocol based on phe-
nolechloroform washes (Sweijd et al., 1998). For the other fresh
samples of gastropods, total genomic DNA was extracted using
a commercial kit (DNeasy Tissue, Qiagen, Valencia, CA) according to
the manufacturers instructions. The DNA from the canned samples
was extracted from 0.5 g of tissue, using the same commercial kit and
the protocol of Sweijd et al. (1998) when amplication failed with the
rst method. Each DNA extraction was quantied using a Lambda
ladder (Invitrogen, Carlsbad, CA) in 1% agarose gel electrophoresis.
2.2. Standardization of the 18S rDNA marker in fresh gastropod
tissue using SSCP
Based on 18S rDNA sequences reported for some gastropods
available in 2007 in the GenBank (H. rufescens, L78885; Haliotis
kamtschatkana, AY923882; Haliotis discus hannai, AY319433; Hal-
iotis tuberculata, AF120511; Fissurella virescens, AY923871; Dicathias
orbita, DQ916537, and Siratus beauii, U86313), specic primers were
designed (18SHal2 Forward: 50 -TTGGATAACTGTGGTAATTCTAGAGC
and 18SHal2 Reverse: 50 -CCGGAATCGAACCCTGAT) with the online
program Primer3 (http://frodo.wi.mit.edu/).
PCR amplications of the 18S rDNA gene were performed in
a thermocycler (Applied Biosystems, Foster City, CA) with a nal
volume of 25 mL containing 50e100 ng DNA template, 0.5 mM of each
primer, 0.2 mM dNTP, 1.5 mM MgCl2, 1 buffer, and 0.05 units mL1
polymerase (Platinum Taq-polymerase, Invitrogen). The cycling
Table 1
Fresh sample characteristics.
Species Common name N Origin
Haliotis corrugata Pink abalone 32 Mexico
H. fulgens Green abalone 45 Mexico
H. rufescens Red abalone 20 Mexico
Fissurella volcano Volcano limpet 3 Mexico
Megathura crenulata Keyhole limpet 5 Mexico
Megastrea undosa Panocha snail 5 Mexico
Concholepas concholepas Loco 8 Chile
Fissurella sp. Limpet 8 Chile
N Number of individuals assayed.
Table 2
Labeling and origin of the brands that were analyzed.
Brand Label name Origin
Cedmex Abalone Mexico
Calmex Abalone Mexico
Rey del Mar Abalone Mexico
Oro del Pacico Cultured abalone (H. rufescens) Mexico
Unlabeleda Abalone Mexico
Porto Novo Abalone Mexico
Don Efe Abalone Peru
Atlantis Abalone (Concholepas concholepas) Chile
Fresca Mar Abalone (limpet) Mexico
Precious Giant top shellsh Mexico
Don Faus Abalone (limpet) Mexico
Island pacic Sea snail abalone style Mexico
a
Can without label provided by shermen of the cooperative Punta Abreojos.
program was: 94  C for 4 min, 30 cycles (94  C for 45 s, 60  C for 45 s,
72  C for 45 s), followed by a nal extension of 72  C for 10 min. Each
PCR product of the amplication was sequenced (Macrogen, Seoul,
Korea), analyzed, and edited using genetic software Sequencher v.4.6
(Genes Codes, Ann Arbor, MI). All edited consensus sequences were
aligned using the ClustalW program (Thompson, Higgins, & Gibson,
1994) and the homologies were analyzed using the program Bio-
Edit v.7.0.5.3. (Hall, 1999). The BLAST program in the GenBank (NCBI)
was used to verify correspondence with the species sequences
(Altschul, Gish, Miller, Myers, & Lipman, 1990). The sequences were
deposited in the GenBank with the following accession numbers:
Haliotis corrugata HM775288, H. fulgens HM775289, H. rufescens
HM775290, C. concholepas HM775291, Fissurella sp. HM775292,
F. volcano HM775293, M. crenulata HM775294, M. undosa HM775295.
The PCR products from the fresh samples (Table 1) were analyzed
by SSCP analysis to obtain the control band pattern (or genotype) for
each species. The procedure was done as follows: 5 mL PCR product
were mixed with 5 mL denaturing solution containing 95% form-
amide, 10 mM NaOH, 0.01% bromophenol blue, and 0.01% xylene
cyanol, denatured for 8 min at 95  C, then immediately cooled on
ice for 4 min, followed by electrophoretic analysis through a 10%
non-denaturing polyacrilamide gels (40% acrylamide/37.5:1 bis-
acrylamide) in an electrophoresis chamber (D-CODE, Universal
Detection Mutation System, Bio-Rad, Hercules, CA) that was previ-
ously cooled to 4  C using an external thermostatic circulating
system (Model 1166, VWR, Niles, IL). Each electrophoresis was run at
10e15 W for 21 h. SSCP bands were visualized (SybrGold, Invi-
trogen, Carlsbad, CA) using a multi-view scanner (FMBIO III, Hitashi
Solutions America, South San Francisco, CA).
2.3. Standardization of partial lysin gene marker in three abalone
species using SSCP
Based on sequences of lysin in the GenBank (H. corrugata
AF076835 and HM775297, H. fulgens AF076832, and H. rufescens
AF076824), primers were designed with the online program
Primer3 (http://frodo.wi.mit.edu/) to amplify a small fragment
(144e150 bp) of the lysin gene. The sequences were aligned using
BioEdit v.7.0.5.3. (Hall, 1999) to design primers within the
conserved regions (LisHal5-F: 50 -GGACGAGTAGACCGTCTTGG and
LisHal5-R: 50 -CATGAGGTACTGGCGATCAA).
PCR amplications were performed in a thermocycler with
a nal volume of 25 mL, containing 50e100 ng DNA template,
0.5 mM of each primer, 0.2 mM dNTP, 1.5 mM MgCl2, 1 buffer, and
0.05 units mL1 Platinum Taq-polymerase. The cycling program was
94  C for 4 min, 40 cycles (94  C for 45 s, 50  C for 60 s, 72  C for
45 s), and a nal extension at 72  C for 10 min. The PCR products
were sequenced (Macrogen, Seoul, Korea), edited, and analyzed
with BioEdit v.7.0.5.3 (Hall, 1999) obtaining the sequences for each
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F. Aranceta-Garza et al. / Food Control 22 (2011) 1015e1020
abalone species (GenBank accession numbers: H. corrugata
HM775298, HM775299, H. fulgens HM775300eHM775304,
H. rufescens HM775305). LisHal5 PCR products were analyzed by
SSCP to obtain the abalone species specic banding pattern using
the same methodology and specications as in Section 2.2.
2.4. Certication of canned products using partial 18S rDNA and
lysin gene markers
Canned products (Table 2) were rst analyzed with the 18S
rDNA marker to discriminate between abalone and other gastro-
pods. PCR amplications were analyzed and compared with the
DNA-SSCP banding pattern of fresh samples as controls. In the case
of canned products with non-matching control banding patterns,
the PCR products were sequenced (Macrogen), edited, analyzed,
and resolved using a BLAST (NCBI) search. For canned products
identied as abalone using the 18S marker, lysin gene PCR products
were analyzed using the SSCP protocol to discriminate among the
three abalone species using fresh specimens as controls.
3. Results and discussion
The two-step method for genetic differentiation of the three
Mexican abalone species from other commercial gastropods
worked correctly for fresh and canned samples.
Fig. 1. 18S rDNA consensus sequences from fresh gastropod samples. Haliotis sp. is the consensus sequence from red, pink, and blue abalone.
1017
3.1. Standardization of the 18S rDNA marker in fresh gastropod
tissue using SSCP
High quality amplications were obtained for the 18S rDNA
marker in the standard fresh samples, with PCR products of
266e277 pb. The alignment of the sequences was 197 bp, including
insertions/deletions (Fig. 1), and the inter-species divergence
percentages were between 0.06% and 22.9%. The partial 18S rDNA
marker contained 46 informative sites and no intraspecic poly-
morphisms among all gastropod samples (Fig. 1). Moreover, abalone
(Haliotis spp.) sequences were identical, permitting discrimination
from the rest of the gastropod samples. The absence of intraspecic
variability in this marker was also observed for 53 commercial
bivalve species (Espieira, Gonzlez-Lavn, Vieites, & Santaclara,
2009) and for Mytilus spp. (Bendezu, Slater, & Carney, 2005).
The Blast search showed high matching probabilities of each
species PCR products with their respective putative GenBank
sequences, either at the species level (M. undosa) or at the genus level
(Haliotis spp., Fissurella spp.). This means that the 18S rDNA fragment
can be used to identify these three genera, especially Haliotis.
The 18S rDNA had been used in forensic studies as a genetic
marker for the identication of commercial canned or fresh mollusk
species of bivalves (Espieira et al., 2009), mussels (Santaclara et al.,
2006), oysters (Klinbunga et al., 2003), and abalone (Naganuma,
Hisadome, Shiraishi, & Kojima, 1998); as an ecological marker for
the genetic identication of European scallops and mussels larvae
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1018 F. Aranceta-Garza et al. / Food Control 22 (2011) 1015e1020

Fig. 2. Different 18S rDNA-SSCP banding patterns observed (10 bp DNA ladder). MU Megastrea undosa; MC Megathura crenulata; F Fissurella sp.; Cc Concholepas con-
cholepas; U giant top shellsh canned product, no fresh ngerprint; H Haliotis spp.; Fv Fissurella volcano.

(Bendezu et al., 2005); and as a phylogenetic marker in gastropods,
successfully separating the abalones from the other mollusks
(Geiger & Thacker, 2005; Yoon & Kim, 2005; 2007).
The standard banding pattern of each fresh sample obtained
from the SSCP was unique to each gastropod species (Fig. 2),
generating a genetic ngerprint that consisted of 2 or 3 diagnostic
bands that unequivocally distinguish the Mexican abalones from the
other gastropods. The SSCP technique has been used for authenti-
cation of cod (Comi, Iacumin, Rantsiou, Cantoni, & Cocolin, 2005),
tuna (Rehbein, Mackie et al., 1999), and sturgeon caviar (Rehbein,
Kress, & Schmidt, 1999); for identication of crustacean larvae in
farm-raised shrimp (Khamnamtong, Klinbunga, & Menasveta,
2005), and for identication of bivalves (Fernndez et al., 2002).
3.2. Standardization of partial lysin gene marker in three abalone
species using SSCP
The LisHal5 primers consistently amplied a single PCR product
in all fresh and canned abalone samples of the species we studied,
with a similar fragment size among them (155 bp). The resulting
sequences showed 12 polymorphic sites where four species-
specic variations were clearly distinguished among the three
species (Fig. 3). Identity percentages among the sequences were
above 94.8%, which is very conservative, but with enough poly-
morphic divergence (3.3e5.2%) to discriminate among the three
species. In other studies, a large fragment of the lysin marker
(470e524 bp) was capable of distinguishing between Northern
Hemisphere and Southern Hemisphere abalone only by their
amplication products (Supernault et al., 2009); also, it was used to
resolve the phylogenetic relationships among seven California
abalone species (Lee & Vacquier, 1992) and on a global scale (Lee &
Vacquier, 1995) and claim that abalone sperm-lysin have species-
specic sequences that increase the usefulness for species
identication.
The PCR-SSCP banding patterns of the three abalone species
were obtained (Fig. 4), with each consisting of 3 or 4 diagnostic
bands that easily distinguished between abalone species. Intra-
specic variability in the sequences of H. fulgens and H. corrugata

Fig. 3. Consensus sequences for the three abalone species showing the polymorphic sites on each LisHal5 sequence. R A G; W A T; Y C T; M A C; and K G T.
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F. Aranceta-Garza et al. / Food Control 22 (2011) 1015e1020 1019

Fig. 4. SSCP banding patterns of the LisHal5 fragment in the same three Mexican abalone species with a 10 bp DNA ladder. Hc H. corrugata (pink abalone); Hf H. fulgens (green
abalone); and Hr H. rufescens (red abalone).

(Fig. 3) was reected in minor variations in each species banding claimed to contain abalone. Of the latter ten, seven brands claimed
pattern but did not affect diagnosis (Fig. 4). There are no SSCP studies to contain only abalone and three brands stated in small letters
for abalone, but other approaches to identication have been used, below the Abalone legend or in the list of ingredients to contain
such as DNA RAPD, which is used in Thailand (Klinbunga et al., 2004) C. concholepas or limpet (Table 3).
and California (Marn, Haye, Marchant, & Winkler, 2007); restriction When the genetic 18S rDNA-SSCP patterns of the seven brands
fragment length polymorphism (RFLP) in southern hemisphere that claimed to exclusively contain abalone were compared with
abalone (Elliott, Bartlett, Evans, & Sweijd, 2002); and direct the banding pattern of the fresh product control, ve indicated
sequencing in Japanese (Naganuma et al., 1998) and American abalone (Table 3). The other two contained C. concholepas (Porto
abalone (Gruenthal & Burton, 2005). The mayor drawbacks of these Novo and Don Efe) or a banding pattern of an unknown gastropod
methods are poor reproducibility of RAPDs (Partis & Wells, 1996), (Porto Novo), for which no fresh or wild control were available for
RFLP detects differences in DNA sequences only when present at the our study (Table 3). This indicates that these brands are selling
specic endonuclease recognition site, and the lack of a sequencer in a product clearly different to what they claim.
any genetic laboratory will delay forensic results for days or weeks. It is important to mention that in three brands (Atlantis, Fresca
With LisHal5-SSCP, using PCR and polyacrilamide gel submarine Mar, and Don Faus) that contained gastropods other than abalone,
electrophoresis, results are available in two days. the labels stated Abalone and contained a picture of an abalone
shell. These actions could mislead consumers who think they are
3.3. Certication of canned products using partial 18S rDNA and buying abalone; this could be considered commercial fraud. Simi-
lysin gene markers larly, the label of the Precious brand claims that it contains Giant Top
Shellsh, a common name for M. undosa, but neither the sequence
Of the 12 brands of canned gastropods that we analyzed, two nor SSCP banding pattern of the canned product matched this
claimed to contain either giant top shellsh or sea snail and ten species. International labeling standards in food products are
dictated by the Codex Alimentarius Food Labeling Committee, which
states that labels of sh products must contain the common and
Table 3 scientic name to avoid commercial fraud (Espieira et al., 2009).
SSCP forensic analysis for identity of gastropods and abalone in canned products Identication of abalone species in commercial products is
based on 18S rDNA and lysin genes, respectively.
gaining importance to control abalone poaching (Sweijd et al., 1998)
Brand N Label legend Forensic diagnosis and for validating differentials in price, based on the species (see
18S rDNA lysin Food Price SearcheFood & Agri. Products Daily Market Price in
Cedmex 2 Abalone H 1 Hc/1 Hf China; http://www.21food.com/news/foodprice.jsp?sdate2010-
Calmex 2 Abalone H 1 Hc Hf/1 Hf 05-11&categorySeafood&productAbalone).
Rey del Mar 4 Abalone H 1 Hc/3 Hf The LisHal5 primers successfully amplied samples to obtain an
Oro del Pacico 1 Abalone H 1 Hr SSCP species-specic banding pattern for abalone sold in cans,
Cooperativa Punta 2 Abalone H 2 Hc
Abreojos
allowing correct species identication of each (Table 3). However,
Porto Novo 2 Abalone Cc/U some canning companies do not strictly follow rules about what
Don Efe 1 Abalone Cc species they put into a can, since species mixing was observed in
Atlantis 1 Abalone Cc some cans. The brand Oro del Pacico, containing H. rufescens, was
(C. concholepas)
the only brand with the common and scientic name correctly
Fresca Mar 1 Abalone (limpet) U
Precious 1 Giant top shellsh U labeled. Abalone sperm-lysin, as a genetic marker, was used in
Don Faus 1 Abalone (limpet) Cc another forensic abalone study in South Africa, where it success-
Island Pacic 2 Sea snail Cc fully identied three abalone species involved in poaching (Sweijd
N number of cans; H Haliotis; Hc Haliotis corrugata, Hf H. fulgens; Hr H. et al., 1998); it proved to be a good genetic marker for identication
rufescens; Cc Concholepas concholepas; U unknown, fresh sample unavailable. at the species level.
 Author's personal copy
1020 F. Aranceta-Garza et al. / Food Control 22 (2011) 1015e1020
We concluded that the methods are reliable and useful for rapid
identication (two days) of Mexican abalone products and can
distinguish abalone at the species level (three days). The methods
can genetically identify raw, frozen, and canned products and the
approach can be used to certify authenticity of Mexican commercial
products or identify commercial fraud. The Mexican abalone
industry could then guarantee the authenticity of the Mexican
products.
Acknowledgments
The authors are grateful to E. Serviere and A. Mazariegos for
providing fresh samples of the Mexican gastropods and to E. Gon-
zlez-Poblete and M. Campalans-Barnier for providing the Chilean
gastropods. G. Ponce-Daz, L. R. Cruz-lvarez, and members of the
shermen cooperatives Buzos y Pescadores, Emancipacin, Baha
Tortugas, Pescadores Nacionales de Abuln, Punta Abreojos, and Leyes
de Reforma provided the canned products. S. vila-lvarez facili-
tated the work at the Aquaculture Genetics Laborator at CIBNOR.
I. Fogel of CIBNOR provided editorial improvements. This study was
funded by CONACYT (grants 2008-79482 and SAGARPA-2004-C01-
82) to R. Perez-Enriquez and SEMARNAT-CONACYT (grant 2004-
C01-153) to G. Ponce-Daz.
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