Verification of authenticity: A rapid identification method for commercial scallop species through multiplex real-time PCR
Introduction
Mislabelling and food fraud is a recognized issue especially for seafood products due to their high diversity, often similar morphology, or even loss of morphological properties through processing (Verrez-Bagnis et al., 2018; Wisniewski & Buschulte, 2019). Incorrect labelling and adulteration of seafood products may have several negative implications such as deception of consumers, lowering of the product quality, contribution to illegal, unreported, and unregulated (IUU) fishing, or may even constitute a risk to the consumer's health (Agnew et al., 2009; Griffiths et al., 2014). In order to prevent food fraud and mislabelling, the European regulation EU No. 1379/2013 on Common Market Organisation was established which determines commercial and scientific designations with further information on labels of raw, thawed, unprocessed or slightly processed fishery products (European Comission, 2013).
Scallops, belonging to the diverse family Pectinidae, are among the top fifteen of the most consumed seafood products in Europe with 0.34 kg per capita in 2016 (EUMOFA, 2018). There are approximately 50 genera and over 250 species with a cosmopolitan distribution in this family, of which around 18 scallop species are of high commercial interest (Marin, Fujimoto, & Arai, 2015). Together with the roes, called “coral”, the meat of the single adductor muscle is sold as high-prized seafood delicacy. On the European market and especially on the German market, the species King scallop Pecten maximus (Linnaeus, 1758), Great Atlantic scallop Placopecten magellanicus (Gmelin, 1791), and Japanese scallop Mizuhopecten yessoensis (Jay, 1857) are of major commercial importance. The global capture production for these species were 59,992 t, 20,6177 t, and 22,3216 t in 2016, respectively (FAO, 2020). A further species of Pecten spp., P. jacobaeus (St. Jame's scallop) has only a small contribution to European scallop landings, mainly from Mediterranean, Adriatic, and Aegean ports (Duncan, Brand, Strand, & Foucher, 2016).
Scallops are usually marketed fresh or frozen either in shells or as adductor muscle meats with or without the roe. Since scallops are often sold having the shells and morphological characteristics removed and they comprise rather expensive products, there is a high potential for mislabelling and food fraud. For the practical implementation of EU No. 1379/2013, each European country is required to draw up and publish an official list on commercial designations and scientific names. Pursuant to the Regulation on Fish Labelling (Federal Law Gazette, 2002/BGBl. I p. 3363), the German Federal Office for Agriculture and Food is competent for the compilation and management of a directory of commercial designations of seafood species. Accordingly, only species of Pecten spp. are allowed to be labelled as “Jakobsmuschel” (=King scallop) or “Pilgermuschel”. Other members of the family Pectinidae can be sold under the designation “Kammmuschel”/scallop, with the extension of few allowed specifications such as “Japanische Kammmuschel”/Japanese scallop for M. yessoensis as an example. Mislabelling and adulteration of scallop species has already been described in several studies (Manthey-Karl, Lehmann, Ostermeyer, Rehbein, & Schröder, 2015; Näumann, Stumme, & Rehbein, 2012; Stephan et al., 2014).
Due to the absence of morphological characteristics in many scallop products, molecular methods are needed to examine the authenticity of the species. Several DNA-based methods have been presented for scallop species identification including mainly Sanger sequencing of mitochondrial fragments such as a 16S rRNA gene and cytochrome oxidase subunit I (COI) (Feng, Li, Kong, & Zheng, 2011; Marin et al., 2015; Marín, Fujimoto, & Arai, 2013; Marin, Villegas-Llerena, Fujimoto, & Arai, 2017; Näumann et al., 2012; Wen et al., 2017). Additionally, a matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was designed for the identification of scallop species (Stephan et al., 2014). Disadvantages of these methods are that Sanger sequencing is time-consuming and can take up several days if external sequencing services are needed and MALDI-TOF requires the generation of a database comprising various available spectra as reference for comparison.
An increasingly common method for species identification and for testing food authenticity is real-time PCR, including the use of dual labelled probes, which has some advantages due to its sensitivity, specificity, and it allows a rapid identification within a few hours since no post-PCR treatment is required (reviewed in Hellberg & Morrissey, 2011). Depending on the available channels of the real-time thermocycler, differently fluorescence-labelled species-specific probes can be combined simultaneously to a multiplex assay.
Despite their commercial importance, relatively few publications on scallop identification techniques are available. Therefore, the aim of the present study was the development of a rapid method that allows the identification of three market relevant scallop species/genera within a few hours. In the present study, three main commercial species/genera Pecten spp., Placopecten magellanicus and Mizuhopecten yessoensis were selected for the development of primers and probes. In addition, the newly designed multiplex real-time PCR method was validated on commercial samples in order to provide a general overview on the current status of correct labelling of scallop products on the German market.
Section snippets
Scallop samples and DNA extraction
A total of 56 scallop samples were analysed. Samples of target species and non-target species were either provided by German seafood traders and processors, by partners of the SEATRACES project (Instituto de Investigaciones Marinas (IIM-CSIC), Muséum National d'Histoire Naturelle (MNHN), Laboratoire SCL de Marseille (SCL)), and company Escal (Strasbourg, France), or samples were purchased at German supermarkets and fish mongers. Scallop samples were bought fresh, frozen, canned, or were
Results and discussion
Mislabelling and substitution of scallop species as a common phenomenon have already been described in previous studies, which are possibly related to economic benefits and the similar morphology of scallop products. However, correct labelling is important to comply with the European regulations and is essential as scallop species differ in value and marketability (Stephan et al., 2014). Therefore, the aim of the present study was the development and validation of a multiplex real-time PCR
Conclusion
The present study confirms the results of previous investigations identifying a relatively high mislabelling rate of scallop species which may have implications on the economy and consumer's trust. Therefore, methods to examine the labelling of these products are desirable that could facilitate regular inspections by food monitoring authorities. The newly designed multiplex TaqMan real-time PCR assay constitutes a rapid and cost-effective method with a high throughput of samples for the
CRediT authorship contribution statement
Regina Klapper: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft, Writing - review & editing. Ute Schröder: Conceptualization, Funding acquisition, Investigation, Methodology, Validation, Writing - review & editing.
Declaration of competing interest
The authors declare no conflict of interest.
Acknowledgments
We thank Roswitha Koch for technical assistance and Dr. Ronald Maul as well as the anonymous reviewers for valuable comments on the manuscript. We further like to thank Carmen G. Sotelo, Amaya Velasco (IIM-CSIC), Solène Avignon (MNHN), Julien Landuré (SCL), and company Escal for providing reference samples. This work was carried out with funding from the EU Interreg Atlantic Area programme, project “SEATRACES”, number EAPA_87/2016.
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