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Development of a qPCR method for the identification and quantification of two closely related tuna species, bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) in canned tuna Daline BOJOLLY, Périne Doyen, Bruno Le Fur, Urania Christaki, Veronique Verrez-Bagnis, and Thierry GRARD J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04713 • Publication Date (Web): 13 Jan 2017 Downloaded from http://pubs.acs.org on January 15, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
Full title: Development of a qPCR method for the identification and quantification of two closely related tuna species, bigeye tuna (Thunnus obesus) and yellowfin tuna (Thunnus albacares) in canned tuna Daline Bojollya,f,h, Périne Doyena,b,c,d,e, Bruno Le Furh, Urania Christakif, Véronique Verrez-Bagnisg, Thierry Grarda,b,c,d,e* a
Univ. Littoral Côte d’Opale, EA 7394 – ICV – Institut Charles Viollette, USC Anses –
ULCO, F-62200 Boulogne-sur-Mer, France b
c
Univ. Artois, F-62000 Arras, France
d
e
Univ. Lille, F-59000 Lille, France
INRA, France
ISA, F-59000 Lille, France
f
Laboratoire d’Océanologie et de Géosciences, UMR 8187 (ULCO, Lille 1, CNRS),
Wimereux, France g
Ifremer, F-44300 Nantes, France
h
PFINV, F-62200 Boulogne-sur-Mer, France
E-mail of each author:
[email protected] [email protected] [email protected] [email protected] [email protected] *Corresponding author: Thierry Grard Tel: +33 3 21 99 45 20; Fax: +33 3 21 99 45 08. E-mail address:
[email protected] ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
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Abstract
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Bigeye tuna (Thunnus obesus) and yellowfin tuna (T. albacares) are among the most widely
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used tuna species for canning purpose. Not only is substitution, but also mixing of tuna
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species is prohibited by the European regulation for canned tuna products. However, as
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juveniles of bigeye and yellowfin tunas are very difficult to distinguish, unintentional
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substitutions may occur during the canning process. In this study, two mitochondrial markers
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from NADH dehydrogenase subunit 2, and cytochrome c oxidase subunit II genes were used
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to identify bigeye tuna and yellowfin tuna, respectively, utilizing TaqMan qPCR
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methodology. Two different qPCR based methods were developed to quantify the percentage
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of flesh of each species used for can processing. The first one was based on absolute
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quantification using standard curves realized with these two markers; the second one was
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founded on relative quantification with the universal 12S rRNA gene as the endogenous gene.
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Based on our results, we conclude that our methodology could be applied to authenticate these
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two closely related tuna species when used in a binary mix in tuna cans.
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Keywords: tuna, authentication, quantification, canned products, TaqMan, qPCR, species
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identification, bigeye tuna (Thunnus obesus), yellowfin tuna (Thunnus albacares)
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Introduction
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Recent years have been marked by various food scandals such as 'Horsegate' (meat
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adulteration crisis in Europe in 2013), and the results of fish products mislabeling studies1, 2.
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These events have highlighted the need: to set up an enhanced traceability system to improve
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quality process in industries; to reinforce consumer protection; and to reduce fraudulent
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activities. Seafoods are in the 'top ten' food products that are most likely to be subject of
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fraud3. Among these is one of the major marine species captured in 20124, tuna, which
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represents an important economic value in the canning industry. According to the European
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legislation, mixing of tuna species in tuna cans is strictly forbidden5. However, substitutions
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between tuna species could appear during the filleting and canning process when external
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morphological characteristics, such as dorsal fins or finlets, are removed and fillets with
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similar appearance and texture are obtained. Furthermore, difficulties exist in identifying
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juveniles of bigeye tuna (Thunnus obesus, Lowe, 1839) and yellowfin tuna (Thunnus
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albacares, Bonnaterre, 1788) due to their similarity 6. Moreover, these two species are often
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captured together from the same schools, particularly around the fish aggregation devices
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(FADs)7, which are extensively used nowadays, resulting in potential substitutions. It was
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these observations that led this study to focus on these two particular species.
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Several methods have been developed to identify species when the flesh is raw or cooked.
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Prior to the 1990s, the most used technique was the isoelectric, which focused on proteins
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(IEF) based on sarcoplasmic protein profiles8. However, sterilization, which takes place
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during canning process, induces an irreversibly denaturation of proteins9. Furthermore, IEF is
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sometimes of no value in discriminating fish species within families such as scombridae
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(mackerels, tunas, and bonitos)9. DNA however, represents a great advantage over proteins as
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it is stable at high temperatures, is present in all cells of the animal, and is endowed with a
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greater variability linked to genetic codes10. Consequently, biomolecular DNA techniques to
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identify fish species, including tuna species, have been developed for more than 20 years11.
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DNA degrades during heat treatment into small fragments12, but these are nevertheless,
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informative enough to differentiate even closely related tuna species11,
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regions of both nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) can be amplified by
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a polymerase chain reaction (PCR)14, mtDNA has preferentially been used for authentication
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of the species, in particular when the starting sample has suffered intense heat treatments15.
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Concerning canned tuna, most studies show a preference to mtDNA in relation to nDNA
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because of its relative abundance and its circular structure, which provides greater resistance
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. Although the
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to thermal degradation15,
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the processing of canned tuna, highly degrades DNA, cleaving it into tiny fragments. The
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average size of DNA fragments for canned products is between
