The present invention relates to the technical field of DNA barcoding, particularly to a method for the identification of seafood species in samples comprising the steps of isolating DNA from a sample, amplifying fragments of the isolated DNA using specific primers, sequencing of the amplified DNA fragments, and identifying the seafood species through sequence comparison with reference sequences. Further provided herein is a primer library and a kit.
Food adulteration is a worldwide problem in various food products, e.g., in farm animal, wild animal, seafood, and also plant products. The term of food adulteration is not uniformly defined but in general, it describes misdeclaration of food intending to gain an economic benefit without limits (Robson, K. et al, 2021). Seafood has a high risk of fraud and seafood products are often mislabelled. According to Pardo, M. et al. (2016), up to 27% of the seafood is mislabelled worldwide. Food adulteration includes, but is not limited to, replacement (a (valuable) ingredient is replaced by one of a lower value), relabelled or incorrectly labelled food. Incorrect labelling can result when different local names are used for the same species, when the same name is used for different species, or due to translation errors.
However, correct labelling of seafood products is important for traceability issues, protection of endangered species, mitigation of illegal fishing, and for individual reasons of end consumers (Rodriguez, E. M. and Ortea, I., 2017).
Correct declaration of seafood is regulated in the European Union. Thereby, international and national regulations exist to ensure legal trade in seafood and seafood products. The EU directive 1379/2013 regulates market organization of fishery and aquaculture products, including correct declaration of seafood. To comply with legal regulations, labels must include both the local trade name in the official language(s) and the correct scientific Latin name (Regulation (EU) No 1379/2013; Regulation (EU) No 1169/2011).
Regardless of clear and strict requirements for species declaration, incorrect labelling of e.g., bivalve products, has repeatedly been detected in Europe (Naumann, G. et al., 2012; Fernandes, T. et al., 2020). In German and Swiss studies, more than half of the products declared to contain “Jakobsmuschel” (or “Jacobsmuschel”) were labelled incorrectly. Although the German name “Jakobsmuschel” (or “Jacobsmuschel”) may only be used for scallop species of the genus Pecten, species of other genera (particularly Placopecten and Mizuhopecten) were identified in these products (Naumann, G. et al., 2012; Stephan, R. et al., 2014).
Compliance with regulations is especially important since seafood is gaining importance in human nutrition. In 2019, 107.6 billion US $ were made with the marketing of seafood (crustaceans and molluscs), compared to 8.1 billion US $30 years ago. In 2019, 1.03 million tons of mussels, scallops, and oysters were caught in nature and more than 10 million tons were cultivated in aquaculture, earning a profit of millions of US dollars. Worldwide, 6.1 million tonnes of crustaceans were caught and 10.5 million tonnes were cultivated in 2019. In the same year, 6.4 million tonnes of molluscs were caught and 17.6 million tonnes were cultivated.
Crustaceans and molluscs are divided into numerous genera comprising a high number of species with a worldwide distribution. A class of molluscs are for example bivalves, wherein Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) are the most important bivalve species for human consumption. Each of these bivalve species is divided into several genera comprising a high number of species which makes correct identification of seafood species difficult using known methods.
In the case of seafood, especially for bivalves, morphological characteristics such as shell, colour and size may allow correct species classification. However, after shell removal or mechanical processing, classification by morphology may be hampered or even be impossible (Espiheira, M. et al., 2009; Fernandez, A. et al., 2000).
Recently, matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) has been shown to be suitable for accurate species identification of scallops (Stephan, R. et al., 2014). However, MALDI-TOF MS instruments are expensive and do not allow high throughput analysis. Therefore, this methodology is less applicable for routine analyses and for the fast identification of several seafood species.
DNA metabarcoding methods have been recently developed for the identification of mammalian and poultry species in food (Dobrovolny S. et al., 2019).
JP 2010004890 discloses primers and methods for detecting mackerel, salmon, abalone, squid, crab, and shrimp.
Marin A. et al. (2015) disclose the use of Pectinidae family-specific primers for amplifying a partial region at the 5′ end of the 16S rRNA gene as a barcoding tool for scallops.
Marin A. et al. (2017) disclose a decaplex PCR assay for the detection of scallop species with species-specific primers targeting the variable 5′ end of the 16S rRNA gene.
Wen J. et al. (2017) discuss the species characterization of cephalopod products by DNA barcoding and phylogenetic analysis using CO/and 16SrRNA genes.
Spielmann G. et al. (2019) disclose the comparison of three DNA marker regions for their suitability to identify food relevant crustaceans of the order Decapoda.
Sun S. et al. (2021) describe the application of DNA barcoding for the identification of dried shellfish products such as scallop, squid, octopus, and cuttlefish.
Klapper R. et al. (2021) disclose the identification of commercial scallop species through multiplex real-time PCR.
Gense K. et al. (2021) disclose a DNA metabarcoding method for the identification of bivalve species in seafood products.
However, methods which provide comprehensive information on the plurality of seafood species which are present in a food sample are still missing. Thus, there is an urgent need in the field for improved means of identifying several different seafood species in complex and processed foodstuffs suitable for food authentication in routine analysis.
It is the objective of the present invention to provide improved means and methods for the identification of several different seafood species in food samples.
The objective is solved by the subject matter of the present invention.
The present invention provides a method which is highly suitable for the identification of seafood species of different origin and processing degree in complex food samples. The inventors of the present invention surprisingly discovered a library of primers which can be incorporated in a fast and reliable metabarcoding method for the identification of a plurality of seafood species in food samples. Thereby, the specific primer sequences of the library are found particularly suitable for combining the primers in the amplification step since they are suitable for amplification at similar conditions such as at similar or even identical temperatures. Temperatures used in a PCR reaction highly depend on the specific characteristics of the primer sequences used in the reaction and may vary even for slightly altered primer sequences. For example, primers with different melting temperatures can hardly be combined successfully in one PCR reaction as the annealing temperature of the PCR reaction depends on the melting temperature of the used primers. Another advantage of the invention described herein is that the method is highly specific based on the specific primer sequences and the length of the primers. This invention successfully identifies seafood species with a low number of PCR cycles, even in highly processed food. The low PCR cycles decrease unspecific PCR results and thus lead to highly reliable results, especially by preventing false positive results.
According to the invention, there is provided a method for identifying seafood species in a sample, comprising the steps of
Specifically, amplifying of fragments of DNA in step b) is performed by a polymerase chain reaction (PCR), preferably by a PCR comprising 25-30 cycles, more preferably by 25 cycles, and preferably at an annealing temperature of 60-65° C., more preferably at an annealing temperature of 62° C.
Specifically, amplifying fragments of the DNA in step b) is performed with at least 1, 2, 3, 4, 5, 6, or 7 primer sets.
Specifically, the amplified DNA fragments of step b) are 16S rDNA fragments, preferably the amplified DNA fragments comprise 120 bp-220 bp of the 16S rDNA.
Specifically, the reference sequences of seafood species comprise the DNA sequence of the 16S rDNA of said seafood species.
More specifically, the reference sequences are any one of SEQ ID NOs: 28 to 1153, or any combinations thereof.
Specifically, the identified species of the family of Crustacean are selected from the following Group 1: Varuna litterata, Hemisquilla ensigera, Gonodactylus smithii, Pullosquilla thomassini, Chorisquilla trigibbosa, Telmessus acutidens, Lithodes aequispinus, Panulirus echinatus, Jasus paulensis, Jasus caveorum, Parastacus pilimanus, Parastacus brasiliensis, Parastacus defossus, Parastacus nicoleti, Gonodactylus graphurus, Jasus lalandii, Lopholithodes mandtii, Lithodes spp., Lithodes maja, Jasus edwardsii, Panulirus regius, Panulirus pascuensis, Panulirus laevicauda, Panulirus gracilis, Panulirus guttatus, Panulirus femoristriga, Chionoecetes spp., Paralomis granulosa, Panulirus spp., Scyllarus arctus, Palinurus elephas, Episesarma mederi, Austropotamobius torrentium, Cycloachelous granulatus, Eriocheir recta, Cervimunida johni, Achelous floridanus, Portunus sayi, Portunus anceps, Palinurus mauritanicus, Palinurus charlestoni, Pseudosquilla ciliata, Pleuroncodes monodon, Portunus ventralis, Achelous spinicarpus, Callinectes toxotes, Callinectes danae, Callinectes ornatus, Callinectes marginatus, Callinectes affinis, Callinectes rathbunae, Callinectes bocourti, Callinectes similis, Callinectes bellicosus, Callinectes arcuatus, Metanephrops armatus, Metanephrops mozambicus, Metanephrops japonicus, Metanephrops spp., Metanephrops binghami, Parastacus pugnax, Paranephrops zealandicus, Callinectes exasperatus, Palinurus spp., Sagmariasus verreauxi, Metanephrops rubellus, Metanephrops challengeri, Metanephrops neptunus, Metanephrops australiensis, Metanephrops arafurensis, Metanephrops boschmai, Metanephrops formosanus, Metanephrops sinensis, Lithodes ferox, Oratosquillina interrupta, Odontodactylus japonicus, Miyakella nepa, Erugosquilla woodmasoni, Clorida decorata, Dictyosquilla foveolata, Anchisquilla fasciata, Scyllarides herklotsii, Astacus astacus, Portunus hastatus, Achelous ordwayi, Carcinus maenas, Portunus inaequalis, Astacoides madagascariensis, Erimacrus isenbecki, Hemisquilla australiensis, Austrosquilla tsangi, Fallosquilla fallax, Echinosquilla guerinii, Coronis scolopendra, Chorisquilla tweediei, Chorisquilla hystrix, Chorisquilla excavate, Busquilla plantei, Alima pacifica, Alima orientalis, Alachosquilla vicina, Gonodactylellus espinosus, Gonodactylellus affinis, Kempella mikado, Hemisquilla californiensis, Haptosquilla trispinosa, Haptosquilla glyptocercus, Gonodactylus platysoma, Gonodactylaceus falcatus, Gonodactylus childi, Gonodactylellus annularis, Odontodactylus scyllarus, Odontodactylus latirostris, Odontodactylus havanensis, Odontodactylus cultrifer, Neogonodactylus oerstedii, Neogonodactylus bredini, Neogonodactylus bahiahondensis, Lysiosquillina sulcata, Squilla rugosa, Raoulserenea spp., Raoulserenea oxyrhyncha, Pseudosquillopsis marmorata, Raoulserenea komaii, Protosquilla folini, Ibacus alticrenatus, Scyllarides nodifer, Scyllarides haanii, Scyllarides brasiliensis, Taku spinosocarinatus, Jasus frontalis, Procambarus paeninsulanus, Puerulus sewelli, Panulirus polyphagus, Panulirus longipes., Panulirus interruptus, Panulirus marginatus, Ibacus peronii, Ibacus chacei, Faxonella clypeata, Fallicambarus kountzeae, Arenaeus mexicanus, Cambarus tartarus, Chionoecetes tanneri, Thenus unimaculatus, Thenus indicus, Haptosquilla hamifera, Lithodes turritus, Bouchardina robisoni, Troglocambarus maclanei, Hobbseus yalobushensis, Hobbseus prominens, Charybdis spp., Hobbseus petilus, Faxonella creaseri, Thranita danae., Monomia petrea, Neogonodactylus wennerae, Xiphonectes pseudohastatoides, Gonodactylellus viridis, Gonodactylaceus ternatensis, Belosquilla laevis, Procambarus okaloosae, Procambarus morrisi, Procambarus milleri, Procambarus mancus, Procambarus lunzi, Hobbseus cristatus, Procambarus acutissimus, Faxonius pagei, Manningia pilaensis, Pontastacus leptodactylus, Procambarus zonangulus, Procambarus youngi, Procambarus seminolae, Procambarus pycnogonopodus, Procambarus orcinus, Procambarus pallidus, Alima maxima, Scyllarides deceptor, Monomia argentata, Xiphonectes pulchricristatus, Paralithodes platypus, Lopholithodes foraminatus, Faughnia formosae, Faughnia profunda, Bathysquilla crassispinosa, Eriocheir sinensis, Harpiosquilla harpax, Callinectes sapidus, Squilla mantis, Portunus trituberculatus, Panulirus japonicus, Cancer pagurus, Chionoecetes japonicus, Scylla tranquebarica, Scylla serrata, Eriocheir japonica, Eriocheir hepuensis, Cherax destructor, Squilla empusa, Lysiosquillina maculata, Gonodactylus chiragra, Panulirus homarus, Homarus americanus, Panulirus ornatus, Oratosquilla oratoria, Panulirus stimpsoni, Charybdis japonica, Scylla paramamosain, Scylla olivacea, Cherax quadricarinatus, Cherax cainii, Paralithodes brevipes, Paralithodes camtschaticus, Scyllarides latus, Procambarus clarkii, Procambarus fallax, Homarus gammarus, Thenus orientalis, Lithodes nintokuae, Cherax cairnsensis, Cherax dispar, Cherax quinquecarinatus, Cherax robustus, Cherax monticola, Cherax glaber, Cherax holthuisi, Astacopsis gouldi, Portunus pelagicus, Paranephrops planifrons, Nephrops norvegicus, Ibacus ciliatus, Charybdis feriata, Metanephrops sibogae, Panulirus cygnus, Metanephrops thomsoni, Faxonius limosus, Squilloides leptosquilla, Cherax bicarinatus, Austropotamobius pallipes, Cherax tenuimanus, Cherax boesemani, Charybdis (Charybdis) natator, Procambarus acutus, Pacifastacus leniusculus, Munida gregaria, Panulirus versicolor, Faxonius rusticus, Portunus sanguinolentus, Procambarus alleni, Metacarcinus magister, Puerulus angulatus, Lupocycloporus gracilimanus, Monomia gladiator, Varuna yui, Panulirus argus, Munida isos, Scyllarides squammosus, Cambaroides similis, Charybdis bimaculata, Cambarus robustus, Thalamita sima, Thranita crenata, Orconectes luteus, Orconectes punctimanus, Orconectes sanbornii, Cherax spp., Cherax crassimanus, Cherax preissii, Munida spinosa, Munida asprosoma, Munida leagora, Munida alonsoi, Munida taenia, Munida gordoae, Munida zebra, Munida distiza, Munida psamathe, Munida thoe, Munida guttata, Munida stia, Munida ommata, Munida roshanei, Munida compressa, Munida clinata, Munida chydaea, Munida compacta, Munida eclepsis, Munida tyche, Munida philippinensis, Munida armilla, Munida mesembria, Munida spilota, Munida benguela, Munida endeavourae, Munida agave, Munida idyia, Munida militaris, Munida flinti, Munida congesta, Munida rubridigitalis, Munida iris, Munida microphthalma, Munida rufiantennulata, Munida pusilla, Munida remota, Munida leptosyne, Munida rosula, Munida munin, Munida valida, Munida proto, Enriquea leviantennata, Munida multilineata, Munida pagesi, Munida stomifera, Munida quadrispina, Munida tiresias, Munida psylla, Munida heteracantha, Paralomis formosa, Paralomis spinosissima, Paralomis birsteini, Paralomis hirtella, Scyllarus subarctus, Scyllarus pygmaeus, Scyllarus chacei, Scyllarus caparti, Scyllarus americanus, Episesarma palawanense, Episesarma singaporense, Austropotamobius fulcisianus orientalis, Achelous tumidulus, Achelous asper, Achelous sebae, Portunus acuminatus, Achelous tuberculatus, Achelous iridescens, Portunus xantusii, Achelous depressifrons, Achelous rufiremus, Achelous gibbesii, Portunus minimus, Achelous stanfordi, Achelous brevimanus, Portunus affinis, Achelous angustus, Achelous binoculus, Oratosquillina inornata, Oratosquillina asiatica, Oratosquillina anomala, Oratosquillina perpensa, Erugosquilla graham, Busquilla quadraticauda, Kempella stridulans, Gonodactylaceus graphurus, Gonodactylaceus randalli, Carcinus aestuarii, Menippe rumphii, Menippe nodifrons, Menippe spp., Procambarus liberorum, Procambarus toltecae, Procambarus curdi, Procambarus digueti, Procambarus nigrocinctus, Procambarus versutus, Procambarus gibbus, Cambarus pecki, Procambarus geminus, Charybdis acuta, Creaserinus fodiens, Fallicambarus jeanae, Creaserinus gordoni, Creaserinus caesius, Fallicambarus dissitus, Creaserinus danielae, Fallicambarus oryktes, Fallicambarus byersi, Creaserinus burrisi, Creaserinus gilpini, Fallicambarus harpi, Fallicambarus macneesei, Fallicambarus petilicarpus, Fallicambarus wallsi, Fallicambarus strawni, Fallicambarus devastator, Fallicambarus houstonensis, Fallicambarus hortoni, Arenaeus cribrarius, Cambarus spp., Cambarus deweesae, Cambarus striatus, Cambarus graysoni, Cambarus monongalensis, Cambarus pyronotus, Cambarus maculatus, Cambarus latimanus, Cambarus strigosus, Cambarus parrishi, Cambarus bouchardi, Cambarus fasciatus, Cambarus harti, Cambarus nerterius, Cambarus setosus, Cambarus batchi, Cambarus halli, Cambarus unestami, Cambarus reburrus, Cambarus gentry, Cambarus hubbsi, Cambarus friaufi, Cambarus obeyensis, Cambarus cracens, Cambarus asperimanus, Cambarus hobbsorum, Cambarus williami, Cambarus howardi, Cambarus obstipus, Cambarus girardianus, Cambarus cryptodytes, Cambarus sciotensis, Cambarus georgiae, Cambarus pristinus, Cambarus aculabrum, Cambarus englishi, Cambarus brachydactylus, Cambarus cumberlandensis, Cambarus dubius, Cambarus reflexus, Cambarus scotti, Cambarus longirostris, Cambarus hubrichti, Monomia lucida, Faughnia serenei, Harpiosquilla melanoura, Harpiosquilla annandalei, Cherax cuspidatus, Cherax paniaicus, Cherax lorentzi, Cherax albertisii, Cherax rotundus, Cherax leckii, Cherax murido, Cherax wasselli, Cherax parvus, Cherax pallidus, Cherax cartalacoolah, Cherax rhynchotus, Cherax pulcher, Cherax peknyi, Cherax setosus, Cherax misolicus, Cherax warsamsonicus, Cherax snowden, Cherax boschmai, Cherax nucifraga, Cherax barrette, Oratosquilla fabricii, Astacopsis tricornis, Thalamita admete, Faxonius virilis, Thranita prymna, Astacopsis franklinii, Cambaroides schrenckii, Orconectes australis, Thalamita chaptalii, Zygita longifrons, Thalamita picta, Thalamita seurati, Thranita pelsarti, Orconectes barri, Faxonius ronaldi, Faxonius neglectus, Orconectes compressus, Orconectes forceps, Orconectes pellucidus, Neoeriocheir leptognathus, Penaeus kerathurus, Penaeus marginatus, Penaeus longistylus, Penaeus plebejus, Metapenaeopsis liui, Metapenaeopsis lamellata, Metapenaeopsis acclivis, Metapenaeopsis commensalis, Atypopenaeus stenodactylus, Aristeus antillensis, Solenocera vioscai, Penaeus chinensis, Penaeus spp., Metapenaeopsis barbata, Penaeus esculentus, Heteropenaeus longimanus, Atypopenaeus dearmatus, Funchalia taaningi, Xiphopenaeus kroyeri, Trachypenaeopsis mobilispinis, Rimapenaeus similis, Parapenaeus politus, Solenocera membranacea, Alcockpenaeopsis hungerfordii, Batepenaeopsis tenella, Pandalus platyceros, Metapenaeus moyebi, Metapenaeus joyneri, Pandalus montagui, Penaeus brasiliensis, Aristeus antennatus, Heterocarpus laevigatus, Heterocarpus lepidus, Funchalia villosa, Hemipenaeus carpenter, Mesopenaeus tropicalis, Pelagopenaeus balboae, Penaeus hathor, Metapenaeopsis provocatoria, Aristeus virilis, Aristeus alcock, Penaeus aztecus, Heterocarpus abulbus, Penaeus setiferus, Cerataspis monstrosus, Pleoticus robustus, Aristaeopsis edwardsiana, Solenocera necopina, Parapenaeus cayrei, Parapenaeus fissurus, Parapenaeus investigatoris, Parapenaeus fissuroides, Parapenaeus americanus, Heterocarpus ensifer, Kishinouyepenaeopsis cornuta, Parapenaeus perezfarfantae, Parapenaeus murrayi, Parapenaeus longipes, Parapenaeus spp., Heterocarpus chani, Heterocarpus sibogae, Heterocarpus dorsalis, Metapenaeopsis andamanensis, Metapenaeopsis coniger, Macrobrachium idella, Trachysalambria brevisuturae, Trachysalambria aspera, Trachysalambria albicoma, Euphausia superba, Solenocera hextii, Hymenopenaeus equalis, Rimapenaeus constrictus, Crangon crangon, Trachypenaeus anchoralis, Megokris spp., Trachysalambria longipes, Trachysalambria starobogatovi, Trachysalambria nansei, Trachysalambria malaiana, Trachysalambria spp., Trachysalambria parvispina, Crangon uritai, Pandalus borealis, Metapenaeus monoceros, Pandalus nipponensis, Hadropenaeus lucasii, Ganjampenaeopsis uncta, Solenocera annectens, Solenocera melantho, Parapenaeopsis stylifera, Penaeus japonicus, Penaeus brevirostris, Penaeus notialis, Penaeus duorarum, Penaeus schmitti, Artemesia longinaris, Penaeus subtilis, Penaeus stylirostris, Penaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, Pandalus hypsinotus, Heterocarpus spp., Pandalus jordani, Macrobrachium bullatum, Penaeus merguiensis, Metapenaeus ensis, Acetes chinensis, Macrobrachium nipponense, Penaeus californiensis, Macrobrachium lanchesteri, Pleoticus muelleri, Metapenaeus affinis, Hymenopenaeus neptunus, Penaeus indicus, Aristaeomorpha foliacea, Solenocera spp., Mierspenaeopsis hardwickii, Penaeus latisulcatus, Penaeus semisulcatus, Penaeus isabelae, Sicyonia lancifer, Metapenaeopsis dalei, Metapenaeopsis gerardoi, Parapenaeus longirostris, Pandalus eous, Pandalus miyakei, Pandalus japonicas, Pandalus glabrus, Pandalus teraoi, Pandalus ivanovi, Pandalus coccinatus, Pandalus formosanus, Pandalus chani, Pandalus spp., Pandalus longirostris, Pandalus latirostris, Metapenaeus spp., Palaemon spp., Palaemon serratus, Macrobrachium nipponense, 15 Palaemon capensis, Palaemon sinensis, Palaemon annandalei, Palaemon gravieri, Palaemon serenus, Palaemon carinicauda, Palaemon pugio, Palaemon pandaliformis, Palaemon elegans, Palaemon longirostris, Palaemon peringueyi, Palaemon debilis, Palaemon carteri, Palaemon ritteri, Palaemon orientis, Macrobrachium gracilirostre, Palaemon vulgaris, Palaemon serrifer, Palaemon varians, Palaemon macrodactylus, Palaemon tonkinensis, Palaemon xiphias, Palaemon ivonicus, Palaemon pacificus, Palaemon atrinubes, Palaemon intermedius, Palaemon concinnus, Palaemon yuna, Palaemon antennarius, Palaemon dolospinus, Palaemon gracilis, Palaemon mundusnovus, Palaemon suttkusi, Palaemon zariquieyi, Macrobrachium australiense, Palaemon semmelinkii, Palaemon litoreus, Palaemon septemtrionalis, Palaemon guangdongensis, Palaemon hancocki, Palaemon vietnamicus, Palaemon texanus, Palaemon ortmanni, Palaemon turcorum, Palaemon kadiakensis, Macrobrachium asperulum, Macrobrachium australe, Macrobrachium olfersii, Macrobrachium jelskii, Macrobrachium villosimanus, Macrobrachium equidens, Macrobrachium potiuna, Macrobrachium malcolmsonii, Macrobrachium superbum, Macrobrachium striatum, Macrobrachium latidactylus, Macrobrachium hancocki, Macrobrachium acanthurus, Macrobrachium inflatum, Macrobrachium crenulatum, Macrobrachium carcinus, Macrobrachium americanum, Macrobrachium latimanus, Macrobrachium mammillodactylus, Macrobrachium faustinum, Macrobrachium heterochirus, Macrobrachium scabriculum, Macrobrachium digueti, Macrobrachium tenellum, Macrobrachium idae, Macrobrachium formosense, Macrobrachium dienbienphuense, Macrobrachium placidulum, Macrobrachium sintangense, Macrobrachium niphanae, Macrobrachium totonacum, Macrobrachium tuxtlaense, Macrobrachium vicconi, Macrobrachium villalobosi, Macrobrachium amazonicum, Macrobrachium canarae, Macrobrachium tratense, Macrobrachium forcipatum, Macrobrachium hirsutimanus, Macrobrachium borellii, Macrobrachium brasiliense, Macrobrachium aemulum, Macrobrachium handschini, Macrobrachium horstii, Macrobrachium ferreirai, Macrobrachium lanatum, Macrobrachium novaehollandiae, Macrobrachium tolmerum, Macrobrachium iheringi, Macrobrachium saigonense, Macrobrachium nattereri, Macrobrachium aracamuni, Macrobrachium inpa, Macrobrachium depressimanum, Macrobrachium surinamicum, Macrobrachium denticulatum, Macrobrachium pilimanus, Macrobrachium ohione, Macrobrachium hainanense, Macrobrachium lepidactyloides, Macrobrachium jaroense, Macrobrachium esculentum, Macrobrachium maculatum, Macrobrachium edentatum, Macrobrachium grandimanus, Macrobrachium malayanum, Macrobrachium meridionale, Macrobrachium neglectum, Macrobrachium platycheles, Macrobrachium naso, Macrobrachium placidum, Macrobrachium yui, Macrobrachium shokitai, Macrobrachium sundaicum, Macrobrachium rude, Macrobrachium lamarrei, Macrobrachium sankolli, Macrobrachium gangeticum, Trachysalambria palaestinensis, Euphausia pacifica, Euphausia lucens, Euphausia vallentini, Euphausia triacantha, Euphausia longirostris, Euphausia similis, Euphausia recurve, Euphausia krohni, Euphausia frigida, Euphausia gibboides, Euphausia eximia, Euphausia americana, Euphausia tenera, Euphausia pseudogibba, Euphausia hemigibba, Euphausia brevis, Hymenopenaeus debilis and Nematopalaemon tenuipes (Group 1).
More specifically, the identified species of the family of Crustacean are selected from Group 1 and are listed in Group 1A: Jasus edwardsii, Metanephrops japonicus, Astacus astacus, Eriocheir sinensis, Cancer pagurus, Chionoecetes opilio, Cherax destructor, Homarus americanus, Paralithodes camtschaticus, Procambarus clarkii, Homarus gammarus, Nephrops norvegicus, Panulirus argus, Penaeus setiferus, Aristaeopsis edwardsiana, Crangon, Pandalus borealis, Metapenaeus monoceros, Penaeus notialis, Penaeus duorarum, Penaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, Penaeus californiensis, Pleoticus muelleri, Penaeus indicus, and Monomia gladiator.
More specifically, the identified species of the family of Crustacean are selected from Group 1 and are listed in Group 1B: Cancer pagurus, Chionoecetes opilio, Homarus americanus, Paralithodes camtschaticus, Procambarus clarkii, Homarus gammarus, Nephrops norvegicus, Panulirus argus, Crangon, Pandalus borealis, Metapenaeus monoceros, Penaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Pleoticus muelleri.
Specifically, the identified species of the family of Cephalopods are selected from the following Group 2: Loligo forbesii, Nototodarus sloanii, Sepia spp., Sepia lorigera, Sepia pardex, Rossia pacifica, Berryteuthis magister, Eledone massyae, Sepia robsoni, Loligo reynaudii, Doryteuthis (Amerigo) pealeii, Doryteuthis (Amerigo) gahi, Sepiola rondeletii, Adinaefiola ligulata, Sepia smithi, Sepia elliptica, Eledone palari, Eledone moschata, Rossia palpebrosa, Gonatus madokai, Gonatus kamtschaticus, Eledone cirrhosa, Sepia elegans, Rossia bipillata, Sepiola atlantica, Lolliguncula (Lolliguncula) panamensis, Octopus maya, Illex illecebrosus, Nototodarus gouldi, Gonatopsis octopedatus, Illex coindetii, Berryteuthis anonychus, Gonatus fabricii, Lusepiola birostrata, Octopus tetricus, Uroteuthis (Photololigo) sibogae, Doryteuthis (Doryteuthis) pleii, Doryteuthis sanpaulensis, Doryteuthis (Amerigo) surinamensis, Octopus hubbsorum, Macrotritopus defilippi, Octopus insularis, Loliolus (Nipponololigo) sumatrensis, Sepia recurvirostra, Sepia madokai, Sepia kobiensis, Amphioctopus aegina, Sepia officinalis, Sepioteuthis lessoniana, Todarodes pacificus, Octopus vulgaris, Heterololigo bleekeri, Octopus sinensis, Octopus americanus, Narrowteuthis nesisi, Ommastrephes bartramii, Sepiella japonica, Uroteuthis (Photololigo) edulis, Doryteuthis (Amerigo) opalescens, Architeuthis dux, Dosidicus gigas, Sepia esculenta, Amphioctopus fangsiao, Loligo vulgaris, Sepiola spp., Octopus mimus, Octopus spp., Octopus bimaculoides, Uroteuthis (Photololigo) chinensis, Uroteuthis (Photololigo) duvaucelii, Illex argentinus, Sepia aculeata, Sepiella inermis, Sepia lycidas, Sepia latimanus, Sepia apama, Sepia pharaonis, Loliolus (Nipponololigo) beka, Alloteuthis subulata, Nototodarus hawaiiensis, Sepia orbignyana, Sepia papuensis, Rossia macrosoma, Lolliguncula (Lolliguncula) brevis, Lolliguncula (Loliolopsis) diomedeae, Afrololigo mercatoris, Octopus bimaculatus, Octopus cyanea, Callistoctopus ornatus, Enteroctopus megalocyathus, Sasakiopus salebrosus, Octopus berrima, Amphioctopus marginatus, Octopus maorum, Octopus fitchi, Amphioctopus neglectus, Loliolus (Nipponololigo) uyii, Loliolus (Nipponololigo) japonica, Bathyteuthis abyssicola, Semirossia patagonica, Cistopus taiwanicus, Sthenoteuthis oualaniensis, Watasenia scintillans, Gonatopsis okutanii, Uroteuthis (Aestuariolus) noctiluca, Sepioteuthis australis, Sepioteuthis sepioidea, Amphioctopus kagoshimensis, Amphioctopus membranaceus, Amphioctopus exannulatus, Amphioctopus rex and Sepia peterseni (Group 2).
More specifically, the identified species of the family of Cephalopods are selected from Group 2 and are listed in Group 2A: Doryteuthis (Amerigo) gahi, Eledone moschata, Octopus maya, Doryteuthis (Doryteuthis) pleii, Amphioctopus aegina, Sepia officinalis, Octopus vulgaris, Sepiella japonica, Uroteuthis (Photololigo) edulis, Doryteuthis (Amerigo) opalescens, Dosidicus gigas, Loligo vulgaris, Uroteuthis (Photololigo) chinensis, Uroteuthis (Photololigo) duvaucelii, Sepiella inermis, Sepia pharaonis, and Amphioctopus membranaceus.
More specifically, the identified species of the family of Cephalopods are selected from Group 2 and are listed in Group 2B: Doryteuthis (Amerigo) gahi, Doryteuthis (Doryteuthis) pleii, Sepia officinalis, Octopus vulgaris, Sepiella japonica, Uroteuthis (Photololigo) edulis, Dosidicus gigas, Loligo vulgaris, Uroteuthis (Photololigo) chinensis, and Uroteuthis (Photololigo) duvaucelii.
Specifically, the identified species of the family of Gastropoda are selected from the following Group 3: Helix pomatia, Achatina fulica, Helix aspersa, Helix aspersa maxima, Helix thessalica, Helix lucorum, Helix nicaeensis, Achatina reticulata, Helix aperta, Helix albescens, Tyrrhenaria ceratina, Helix vladika, Helix spp., Pleurodonte discolor, Pleurodonte lychnuchus, Erctella mazzullii, Erctella cephalaeditana, Pleurodonte formosa, Helix christophi, Helix nordmanni, Pleurodonte nucleola, Pleurodonte parilis, Gonostomopsis auridens, Caracolus caracollus, Lacteoluna selenina, Cernuella cisalpine, Cochlicella acuta, Disculella maderensis, Dialeuca nemoraloides, Monadenia fidelis, Cepaea nemoralis, Sphincterochila candidissima, Microphysula ingersolli, Helicodonta obvoluta and Cernuella virgata (Group 3).
More specifically, the identified species of the family of Gastropoda are selected from Group 3 and are listed in Group 3A: Helix pomatia, Achatina fulica, Helix aspersa, Helix aspersa maxima, Helix lucorum, and Achatina reticulata.
More specifically, the identified species of the family of Gastropoda are selected from Group 3 and are listed in Group 3B: Helix pomatia and Achatina reticulata.
Specifically, the identified species of the family of Veneridae are selected from the following Group 4: Tridacna mbalavuana, Siliqua alta, Megangulus zyonoensis, Megangulus venulosus, Donax faba, Donax cuneatus, Donax kiusiuensis, Mactra quadrangularis, Ensis ensis, Chamelea gallina, Spisula subtruncata, Polititapes rhomboides, Callista chione, Venerupis corrugata, Polititapes aureus, Venus crebrisulca, Mercenaria campechiensis, Antigona lamellaris, Ameghinomya antiqua, Ameghinomya spp., Callista erycina, Venerupis aspera, Paphia philippiana, Venus casina, Ensis spp., Mactra stultorum, Ensis macha, Siliqua minima, Ensis leei, Polititapes durus, Cerastoderma glaucum, Tridacna spp., Donax longissimus, Solen 5 vaginoides, Venus verrucosa, Ezocallista brevisiphonata, Procardium indicum, Cardium maxicostatum, Cardium costatum, Acanthocardia paucicostata, Acanthocardia echinata, Acanthocardia aculeata, Solen spp., Ruditapes philippinarum, Corculum cardissa, Spisula solida, Scrobicularia plana, Mactra spp., Chamelea striatula, Ensis siliqua, Serripes groenlandicus, Tridacna elongatissima, Tridacna rosewateri, Meretrix lamarckii, Meretrix lusoria, Paphia euglypta, Meretrix spp., Acanthocardia tuberculata, Tridacna maxima, Lutraria rhynchaena, Meretrix lyrata, Arctica islandica, Solen strictus, Paratapes undulatus, Paratapes textilis, Paphia amabilis, Solen grandis, Lutraria maxima, Donax vittatus, Donax variegatus, Donax trunculus, Donax semistriatus, Ruditapes decussatus, Cerastoderma edule, Tridacna squamosa, Mactra chinensis and Mercenaria mercenaria (Group 4).
More specifically, the identified species of the family of Veneridae are selected from Group 4 and are listed in Group 4A: Ensis ensis, Chamelea gallina, Callista chione, Venus verrucosa, Ruditapes philippinarum, Ensis siliqua, Meretrix lyrata, Donax trunculus, and Cerastoderma edule.
More specifically, the identified species of the family of Veneridae are selected from Group 4 and are listed in Group 4B: Ensis ensis, Callista chione, Ruditapes philippinarum, Meretrix lyrata, and Cerastoderma edule.
Specifically, the identified species of the family of Ostreidae are selected from the following Group 5: Magallana bilineata, Magallana gigas, Crassostrea virginica, Magallana spp., Magallana angulata, Magallana sikamea, Magallana ariakensis, Ostrea denselamellosa, Magallana nippona, Ostrea edulis, Crassostrea spp., Crassostrea tulipa, Ostrea angasi, Magallana belcheri, Crassostrea rhizophorae, Talonostrea talonata, Crassostrea corteziensis, Ostrea spp., Ostrea chilensis, Ostrea algoensis, Ostrea megodon, Saccostrea cuccullata, Saccostrea palmula, Saccostrea malabonensis, Saccostrea scyphophilla, Saccostrea kegaki, and Saccostrea spp. (Group 5).
More specifically, the identified species of the family of Ostreidae are selected from Group 5 and are listed in Group 5A: Magallana gigas, Crassostrea virginica, and Ostrea edulis.
Specifically, the identified species of the family of Pectinidae are selected from the following Group 6: Euvola spp., Mimachlamys crassicostata, Gloripallium pallium, Flexopecten glaber, Aequipecten opercularis, Nodipecten nodosus, Scaeochlamys livida, Pecten spp., Talochlamys multistriata, Patinopecten caurinus, Chlamys behringiana, Placopecten septemradiatus, Pecten maximus, Zygochlamys delicatula, Chlamys hastata, Ylistrum japonicum, Talochlamys gemmulata, Zygochlamys patagonica, Argopecten purpuratus, Argopecten irradians, Azumapecten farreri, Mizuhopecten yessoensi, Placopecten magellanicus, Chlamys islandica, Argopecten ventricosus, Mimachlamys varia, Amusium pleuronectes, Mimachlamys sanguinea, Talochlamys dichroa, Mimachlamys gloriosa, Mimachlamys cloacata, Mimachlamys asperrima, Annachlamys striatula, Decatopecten radula, Bractechlamys vexillum, Aequipecten glyptus, Scaeochlamys lemniscata, Chlamys rubida, Karnekampia sulcata, Crassadoma gigantea, and Ylistrum balloti (Group 6).
More specifically, the identified species of the family of Pectinidae are selected from Group 6 and are listed in Group 6A: Aequipecten opercularis, Pecten maximus, Pecten jacobaeus, Zygochlamys patagonica, Argopecten purpuratus, Mizuhopecten yessoensi, and Placopecten magellanicus.
Specifically, the identified species of the family of Mytilidae are selected from the following Group 7: Mytilus spp., Perna perna, Mytilus unguiculatus, Perna viridis, Mytilus californianus, Mytilus trossulus, Mytilus galloprovincialis, Mytilus edulis and Perna canaliculus (Group 7).
More specifically, the identified species of the family of Mytilidae are selected from Group 7 and are listed in Group 7A: Perna perna Mytilus californianus, Mytilus trossulus, Mytilus galloprovincialis, Mytilus edulis and Perna canaliculus.
More specifically, the identified species of the family of Mytilidae are selected from Group 7 and are listed in Group 7B: Mytilus galloprovincialis, Mytilus edulis and Perna canaliculus.
Further provided herein is a kit for identifying seafood species in a sample, comprising one or more primer sets selected from the group of
Further provided herein is a library of primer sequences comprising any one of SEQ ID NOs: 1 to 25, or any combinations of SEQ ID NOs: 1 to 25.
Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks, such as Sambrook et al, “Molecular Cloning: A Laboratory Manual” (4th Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (2012); Krebs et al., “Lewin's Genes XI”, Jones & Bartlett Learning, (2017); Berg et al, “Stryer Biochemie” Springer Verlag, 2018; and Murphy & Weaver, “Janeway's Immunobiology” (9th Ed., or more recent editions), Taylor & Francis Inc, 2017.
The subject matter of the claims specifically refers to artificial products or methods employing or producing such artificial products, which may be variants of native (wild-type) products. Though there can be a certain degree of sequence identity to the native structure, it is well understood that the materials, methods and uses of the invention, e.g., specifically referring to isolated nucleic acid sequences, amino acid sequences, expression constructs, transformed host cells and modified proteins and enzymes, are “man-made” or synthetic, and are therefore not considered as a result of “laws of nature”.
The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.
The term “about” as used herein refers to the same value or a value differing by +/−5% of the given value.
As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.
The method of the present invention relates to the field of DNA barcoding. Compared with existing morphological identification methods, the method of the present invention is not affected by the experience of inspectors or the morphological changes after processing, which greatly improves the feasibility of sample detection. Compared with the existing DNA barcoding techniques, the present invention solves the problem of false negative results in highly processed foods caused by the difficulty in amplifying DNA of bivalve organisms by the existing DNA barcodes. Therefore, the DNA barcode and its application method established by the present invention are important supplements to the existing DNA barcoding techniques, and can be used to simultaneously identify a plurality of seafood species of the family of Crustacean, Cephalopods, Gastropoda, Veneridae, Ostreidae, Pectinidae, and Mytilidae even if present in very low amounts.
The DNA barcoding identification technique is mainly used for species identification by using relatively short DNA fragments in the organism with sufficient variation that can represent and map this species by means of PCR amplification, sequencing and alignment, etc. DNA barcoding aims at detecting a broad range of species by using universal primer systems. DNA metabarcoding allows the identification of multiple species in food samples in one and the same sequencing run.
DNA barcodes commonly contain conserved regions at both ends, serving as binding sites for universal primers, and a variable part in between the primer binding sites, for differentiation between the species of interest. Due to its high copy number and robustness, mitochondrial DNA (mtDNA) is preferred over genomic DNA. The mtDNA regions most commonly used for species identification are cytochrome c oxidase subunit I (COI), cytochrome b (cyt b), and 16S ribosomal DNA (16S rDNA).
According to the invention, seafood species are identified based on a barcoding method. The term “seafood species” refers to species of the following families: Crustacean, Cephalopods, Gastropoda, Veneridae, Ostreidae, Pectinidae, and Mytilidae. In biological classification, the taxonomic rank is the relative level of a group of organisms in a taxonomic hierarchy. As used herein, the following taxonomic ranks are referred to as according to their relative level of a group of organisms in descending taxonomic hierarchy: family, genus, species. As an example, the species Pecten jacobaeus and Placopecten magellanicus belong to the genus Pecten and the genus Pecten belongs to the family of Pectinidae.
According to a specific embodiment, the seafood species identified according to the invention are the species described herein selected from the group of families of Crustacean, Cephalopods, Gastropoda (snails), Veneridae (venus clams), Ostreidae (oysters), Pectinidae (scallops), and Mytilidae (mussels).
The term “sample” refers to samples which may be taken from different seafood, foodstuff, different origin of the foodstuff and various processing degrees of the food. For example, the sample is taken from sauces, soups, seafood mix, chips, pastes, seafood in cans e.g. mussels in various sauces, raw or frozen seafood, or raw or frozen seafood ingredients.
According to one embodiment of the invention, DNA is isolated from a sample. DNA can be isolated from a sample e.g., by using special kits for DNA isolation/extraction or by using the CTAB (ionic detergent cetyltrimethylammonium bromide)-method. The method for isolating DNA according to the invention enables the isolation of DNA from different samples, specifically from food samples.
According to one embodiment of the invention, the amplification of DNA fragments of isolated DNA is performed by PCR (polymerase chain reaction). Variations of PCR may be used, e.g. real time PCR with intercalating dyes.
In another embodiment, in order to improve the PCR amplification according to the invention, concentrations of compounds commonly used in PCR technology may be adapted. For example, the concentration of magnesium chloride, commonly used in PCR amplification assays, may be increased or decreased depending on the specific setup.
In yet another embodiment, PCR methods rely commonly on thermal cycling, wherein the DNA is replicated by repeated cycles of heating and cooling permitting different temperature-dependent reactions. In general, one PCR-cycle comprises the steps of denaturation, annealing, and extension. Accordingly, the PCR method according to the invention comprises 25-30 cycles for the amplification of DNA fragments. Specifically, the PCR method according to the invention comprises 25, 26, 27, 28, 29, or 30 cycles.
In yet another specific embodiment, the PCR method according to the invention comprises an annealing temperature of 60-65° C., specifically 60, 61, 62, 63, 64, or 65° C. More specifically, the annealing temperature is 62° C.
According to one embodiment, in the PCR assay of the invention, DNA fragments of different seafood species are amplified in one single PCR assay due to the usage of multiple primer pairs. Thereby, a primer pair is consisting of one forward and one reverse primer. Multiplex PCR is enabled since multiple primer pairs are used within a single PCR mixture to produce amplicons of different DNA sequences. Thereby, several different DNA sequences can be amplified simultaneously. By amplifying multiple different DNA fragments at once, additional information is gained from a single test-run.
According to one embodiment of the invention, fragments of the 16S rDNA of the mtDNA region are amplified from isolated DNA samples. Thus, the mtDNA region used for seafood species identification is the 16S rDNA.
In a specific embodiment, the 16S rDNA fragments comprise 130 bp to 220 bp. The length of 16S rDNA fragments vary among the seafood species to be identified. The 16S rDNA fragments comprise 190 bp to 220 bp, 150 bp to 160 bp, or 130 bp to 150 bp. Specifically, for species of the family of Crustacean, the 16S rDNA fragments comprise 198-220 bp. Specifically, for species of the family of Cephalopods, the 16S rDNA fragments comprise 194-220 bp. Specifically, for species of the family of Gastropoda, the 16S rDNA fragments comprise 154-157 bp. Specifically, for species of the family of Veneridae, the 16S rDNA fragments comprise 128-149 bp. Specifically, for species of the families of Ostreidae, Pectinidae, and Mytilidae, the 16S rDNA fragments comprise 133-148 bp.
According to the invention, a primer pair consisting of one forward primer and one reverse primer binds to a conserved region at both ends of the 16S rDNA fragment which is targeted to be amplified by PCR. In order to identify the seafood species according to the invention, a PCR product has to be obtained for the species to be identified. Due to high sequence variability between closely related seafood species of certain families, more than one primer pair is needed to obtain a PCR product for all the species of the family.
Specifically, the term “primer set” as used herein refers to the set of all the primer pairs which are needed for the identification of seafood species of a specific family, wherein the families are the following: Crustacean, Cephalopods, Gastropoda, Veneridae, Ostreidae, Pectinidae, and Mytilidae. As an example, three primer sets are needed for the identification of seafood species belonging the three families, Pectinidae, Ostreidae, and Mytilidae and each primer set is specific for one of the families.
More specifically, depending on the specific family, the primer set comprises one primer pair consisting of one forward primer and one reverse primer or comprises more than one primer pair. In the case that the primer set comprises more than one primer pair, each of the primer sequences of the primer set can be used to form multiple primer pairs. For example, if one primer set comprises two forward primer sequences and one reverse primer sequence, two primer pairs are formed. These two primer pairs are formed by selecting one forward primer from the two forward primer sequences and combining the forward primer sequence with the reverse primer sequence.
The herein described primers enable a simultaneous use in one single PCR reaction because of their specific characteristics such as their sequence length. For a simultaneous PCR reaction using different primers and primer pairs, it is important that all primers are suitable for the selected PCR conditions. For example, slight variations of the sequence of a primer (or primer pair) can result in an altered melting temperature which can ultimately lead to an unsuccessful PCR reaction if the annealing temperature of the PCR reaction is not adjusted. For example, using other primers known in the art, such as the primers disclosed in JP 2010004890, could possibly result in no PCR products since such primers are possibly not compatible with the primers described herein concerning e.g., their length, melting temperature or during the following sequencing.
According to one embodiment of the invention, the primers sequences of the primer sets specific for the identification of seafood species of the families Crustacean, Cephalopods, Gastropoda, Veneridae, Ostreidae, Pectinidae, and Mytilidae are given in Table 1. The forward primer SEQ ID NOs: 1 to 14 and reverse primer SEQ ID NOs: 15 to 25 are given in 5′-3′ direction. The reverse complement of the primer sequences can also be used in the method provided herein.
Specifically, primer set (ps) 1 is used for the identification of species of the family of Crustacean. In ps 1, one or more, specifically one or two primer pairs are formed by selecting a forward primer from forward primer sequences SEQ ID NOs: 1 and 2 and combining the selected forward primer with the reverse primer given in reverse primer sequence SEQ ID NO: 15. The primer set for the identification of Crustacean comprises SEQ ID NOs: 1, 2, and 15.
Specifically, ps 2 is used for the identification of species of the family of Cephalopods. In ps 2, one or more, specifically one, two, or three primer pairs are formed by selecting a forward primer from SEQ ID NOs: 3 to 5 and combining the selected forward primer with the reverse primer given in reverse primer sequence SEQ ID NO: 16. The primer set for the identification of Cephalopods comprises SEQ ID NOs: 3, 4, 5, and 16.
Specifically, ps 3 is used for the identification of species of the family of Gastropoda. In ps 3, one or more, specifically one or two primer pairs are formed by forward primer from SEQ ID NO: 6 and combining the forward primer with one reverse primer selected from reverse primer sequences SEQ ID NOs: 17 to 18. The primer set for the identification of Gastropoda comprises SEQ ID NOs: 6, 17, and 18.
Specifically, ps 4 is used for the identification of species of the family of Veneridae. In ps 4, one or more, specifically one, two, three, four, or five primer pairs are formed by selecting a forward primer from SEQ ID NOs: 7 to 11 and combining the selected forward primer with one reverse primer selected from reverse primer sequences SEQ ID NOs: 19 to 21. The primer set for the identification of Veneridae comprises SEQ ID NOs: 7, 8, 9, 10, 11, 19, 20, and 21.
Specifically, ps 5 is used for the identification of species of the family of Ostreidae. In ps 5, one primer pair is formed by the forward primer SEQ ID NO: 12 and reverse primer sequence SEQ ID NO: 22. The primer set for the identification of Ostreidae comprises SEQ ID NOs: 12 and 22.
Specifically, ps 6 is used for the identification of species of the family of Pectinidae. In ps 6, one primer pair is formed by the forward primer SEQ ID NO: 13 and reverse primer sequence SEQ ID NO: 23. The primer set for the identification of Pectinidae comprises SEQ ID NOs: 13 and 23.
Specifically, ps 7 is used for the identification of species of the family of Mytilidae. In ps 7, one or more, specifically one or two primer pairs are formed by the forward primer SEQ ID NO: 14 and combining the forward primer with one reverse primer sequence selected from reverse primer sequences SEQ ID NOs: 24 to 25. The primer set for the identification of Mytilidae comprises SEQ ID NOs: 14, 24, and 25.
Specifically, the amplification of DNA fragments according to the invention is performed with at least 1, 2, 3, 4, 5, 6, or 7 primer sets.
In one embodiment, the concentrations of the primers may be adapted to the specific combination of forward and reverse primers in a primer set for a single species. For example, if the PCR amplification of Mytilidae is performed with two primer pairs, namely primer pair 1 being SEQ ID NO: 14 and SEQ ID NO: 24, and primer pair 2 being SEQ ID NO: 14 and SEQ ID NO: 25, then the concentration in the PCR assay of SEQ ID NO: 14 may be twice as high as the concentration of each SEQ ID NO: 24 and SEQ ID NO: 25.
According to the invention, the amplified sequences are sequenced in order to determine the nucleotide sequence of the amplified 16S rDNA fragment. Specifically, Sanger sequencing or next generation sequencing (NGS) is applied as sequencing technology.
According to the invention, the sequenced DNA fragments are identified based on a comparison with reference sequences of the respective species. Specifically, the variable part in between the primer binding sites is used for differentiation between the species of interest. Methods for sequence comparison are commonly known in the art. Specifically, BLASTn provided by NCBI (National Center for Biotechnology Information) can be used for sequence comparison of a sequenced DNA fragment with a reference sequence database in order to identify the seafood species in the sample. More specifically, the sequences DNA fragments obtained by the method of the invention are compared to reference sequences comprising the 16S rDNA sequence of the targeted seafood species.
According to a specific embodiment, the reference sequences of the invention are selected from any one of SEQ ID NOs: 28 to 1153. More specifically, the sequenced DNA fragments are identified based on a comparison with a database or a library comprising reference sequences of the species identified according to the invention. In this case, the library of reference sequences comprises one or more of SEQ ID NOs: 28 to 1153, or all of SEQ ID NOs: 28 to 1153, or any combinations thereof.
According to one embodiment of the invention, a library of primer sequences is provided herein comprising SEQ ID NOs: 1 to 25 and/or the reverse complement sequences thereof. The sequences of said primer sequences (SEQ ID NOs: 1 to 25) are given in the Table 1. The primer library of the invention may comprise all primers listed in Table 1. Alternatively, the primer library comprises primers of primer set 1, 2, 3, 4, 5, 6, and/or 7.
As used herein, the term “Fwd” refers to a forward and the term “Rev” refers to a reverse primer. Alternatively, also the term “For” may be used as abbreviation for a forward primer.
The nomenclature of the nucleotide sequences of the invention follows the general guidelines of the IUPAC nucleotide code. Specifically, nucleotide code A refers to adenine, C refers to cytosine, G refers to guanine, T refers to thymine, and W refers to A or T.
According to a specific embodiment, if a primer sequence comprises a “W” at a specific position in the nucleotide sequence, said primer is used as a mixture of two sequences, wherein one primer has an adenine at the specific position and the other primer has thymine at the specific position.
Crustacean
Crustacean
Crustacean
Cephalopods
Cephalopods
Cephalopods
Cephalopods
Gastropoda
Gastropoda
Gastropoda
Veneridae
Veneridae
Veneridae
Veneridae
Veneridae
Veneridae
Veneridae
Veneridae
Ostreidae
Ostreidae
Pectinidae
Pectinidae
Mytilidae
Mytilidae
Mytilidae
According to one embodiment of the invention, a kit for identifying seafood species in a sample is provided, wherein the kit comprises the primer library of the invention. Specifically, the kit comprises primer sequences of one or more of the primer sets selected from primer set 1, primer set 2, primer set 3, primer set 4, primer set 5, primer set 6, and primer set 7. More specifically, the kit comprises at least 1, 2, 3, 4, 5, 6, or 7 of the primer sets according to the invention. The kit may also comprise the reverse complement sequences of the primer sequences.
Said kit may further comprise PCR components, buffers, reagents and/or an instruction manual. Specifically, the kit may comprise as PCR components a polymerase, a master mix, and/or magnesium chloride.
The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from scope of the invention. Accordingly, it should be understood that the examples are illustrative only and are not limiting upon the scope of the invention.
In Example 1, the identification of seafood species of the family of Pectinidae, Ostreidae, and Mytilidae (together known as “bivalves”) in raw and processed food products is described. The method was developed on the Illumina MiSeq® and iSeq® platforms.
86 commercial food products were collected from regional supermarkets, fish markets, and delicacy shops (Table 2). Table 2 shows the declaration, origin and processing condition of the 86 commercial food products. Samples were either fresh, deep-frozen, or in processed condition. Each sample was given a specific ID number, with the letter “0” referring to oysters, “S” to scallops, “M” to mussels, and “Mi” to mixed-species seafood. Samples were stored at −20° C. until DNA extraction.
Crassostrea gigas
Ostrea edulis
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Crassostrea gigas
Mytilus edulis
Mytilus
galloprovincialis
Perna canaliculus
Mytilus spp.
Mytilus edulis
Mytilus edulis
Mytilus chilensis
Mytilus spp.
Mytilus edulis
Mytilus
galloprovincialis
Mytilus chilensis
Mytilus chilensis
Mytilus
galloprovincialis
Mytilus
galloprovincialis
Mytilus chilensis
Mytilus chilensis
Mytilus chilensis
Mytilus
galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus edulis
Placopecten
magellanicus
Mizuhopecten
yessoensis
Pecten maximus
Pecten spp.
Placopecten
magellanicus
Pecten jacobaeus
Zygochlamys
patagonica
Patinopecten
yessoensis
Placopecten
magellanicus
Argopecten
purpuratus
Patinopecten
yessoensis
Pecten sp.
Placopecten
magellanicus
Aequipecten
opercularis
Placopecten
magellanicus
Patinopecten
yessoensis
Mytilus chilensis
Mytilus chilensis,
Mytilus edulis
Mytilus chilensis
Mytilus spp.
Mytilus
galloprovincialis
Mytilus chilensis
Mytilus chilensis
Mytilus edulis,
Mytilus chilensis
Mytilus chilensis
Mytilus chilensis
Mytilus chilensis
Zygochlamys
patagonica,
Chlamys
opercularis
Mytilus chilensis
Eleven out of the 86 samples (“reference samples”), comprising three mussel, six scallop, and two oyster species, were used for method development. Table 3 shows the bivalve species used for development of the DNA metabarcoding method. Identity of bivalve species in these reference samples (samples M12, M13 and M27 for mussels; samples S42, S46, S47, S49, S50, and S55 for scallops; samples 02 and 03 for oysters; Table 2) was verified by subjecting DNA extracts to Sanger sequencing (Microsynth, Balgach, Switzerland) and matching the sequences against the public databases provided by the National Center for Biotechnology Information (NCBI).
Mytilus edulis
Mytilus galloprovincialis
Perna canaliculus
Placopecten magellanicus
Mizuhopecten yessoensis
Pecten jacobaeus
Zygochlamys patagonica
Argopecten purpuratus
Aequipecten opercularis
Magallana gigas
Ostrea edulis
Raw material was cut into smaller pieces or homogenized. To 2.0 gram of each sample, 10 mL of a hexadecyltrimethylammonium bromide (CTAB) buffer was added. After addition of 80 μL proteinase K, the mixture was incubated on an Intelli-Mixer™ RM2 (LTF Labortechnik) overnight at 50° C.
For DNA isolation, a commercial kit (Maxwell® 16 FFS Nucleic Acid Extraction System Custom-Kit, Promega, Madison, USA) was used according to the manufacturer's instruction. DNA concentration was determined fluorometrically (Qubit® 2.0 fluorometer, Thermo Fisher Scientific, Oregon, USA). For higher concentrations, the Qubit® dsDNA broad range assay kit (2 to 1000 ng) and for lower concentrations, the Qubit® dsDNA high sensitivity assay kit (0.2 to 100 ng) was used. DNA purity was assessed from the ratio of the absorbance at 260 nm and 280 nm (QlAxpert spectrophotometer, software version 2.2.0.21, Qiagen, Hilden, Germany). DNA extracts were stored at −20° C. until further use.
Ternary DNA extract mixtures were prepared by mixing DNA extracts (DNA concentration 5 ng/mL) from Pecten spp., Magallana gigas and Mytilus galloprovincialis, representing the three bivalve species Pectinidae, Ostreidae, and Mytilidae, respectively. Individual DNA extracts were mixed in a ratio of 98.0:1.5:0.5 (v/v/v).
In addition, DNA extract mixtures consisting of DNA from species belonging to one bivalve species were prepared. In these mixtures, DNA from one species was present as the main component, DNA from the other species as minor components (1.0% each). Since only two oyster species were available, the DNA extract mixture representing the bivalve species Ostreidae contained the closely related scallop (Placopecten magellanicus) as major component (98.0%) and DNA from the two oyster species as minor components (1.0% each).
In addition to mixtures consisting of DNA from bivalve species only, a DNA extract mixture containing another mollusc species was prepared. DNA extract from a squid species (Sepiella inermis) was chosen as the main component (97.0%) and DNA from the bivalve species Placopecten magellanicus, Ostrea edulis and Perna canaliculus were present as minor components (1.0% each).
A 150 bp fragment of the mitochondrial 16S rDNA gene was used as DNA barcode. Reference sequences for commonly consumed bivalve species and some exotic seafood species, that are permitted for consumption in Austria (“Codex Alimentarius Austriacus” chapter B35, (see Bundesministerium für Arbeit, Soziales, Gesundheit und Konsumentenschutz, Codexkapitel/B 35/Fische, Krebse, Weichtiere und daraus hergestellte Erzeugnisse: BMGF-75210/0026-II/B/13/2017, 2007), were downloaded from the NCBI databases by using CLC Genomics Workbench 10.1.1 (Qiagen). If available, complete reference sequences from the RefSeq database were preferentially downloaded due to their reliability. In case complete reference sequences were not available, all DNA sequences of the mitochondrial 16S rDNA available for one and the same species, submitted by individual scientists, were aligned and checked for similarity and unidentified nucleotides. Subsequently, the DNA sequence with the highest quality (e.g. without unknown nucleotides, full-length of the DNA barcode) was chosen as a reference sequence.
The reference sequences according to the invention are given in Table 4. This list of sequences comprises all reference sequences used for the identification of all seafood species of the invention and thus, does not only comprise the sequences for example 1 but for the seafood species identified according to the invention.
Mytilus
chilensis
Perna
perna
Mytilus
unguiculatus
Perna
viridis
Mytilus
californianus
Mytilus
trossulus
Mytilus
galloprovincialis
Mytilusedulis
Perna
canaliculus
Mytilus
platensis
Magallana
bilineata
Magallana
gigas
Crassostrea
virginica
Magallana
hongkongensis
Magallana
angulata
Magallan
asikamea
Magallana
ariakensis
Ostrea
denselamellosa
Magallana
nippona
Ostreaedulis
Ostrea
lurida
Crassostrea
tulipa
Ostrea
angasi
Magallana
belcheri
Crassostrea
rhizophorae
Crassostrea
brasiliana
Talonostrea
talonata
Crassostrea
corteziensis
Magallana
rivularis
Ostrea
angelica
Ostrea
permollis
Ostrea
chilensis
Ostrea
algoensis
Ostrea
puelchana
Ostrea
megodon
Saccostrea
cuccullata
Saccostrea
palmula
Saccostrea
malabonensis
Saccostre
ascyphophilla
Saccostrea
glomerata
Saccostrea
kegaki
Saccostre
aechinata
Saccostrea
mytiloides
Euvola
vogdesi
Mimachlamys
crassicostata
Gloripallium
pallium
Flexopecten
glaber
Pecten
jacobaeus
Pecten
novaezelandiae
Euvola
raveneli
Aequipecten
opercularis
Euvola
perula
Nodipecten
nodosus
Scaeochlamys
livida
Pecten
keppelianus
Talochlamys
multistriata
Patinopecten
caurinus
Chlamys
behringiana
Placopecten
septemradiatus
Pecten
maximus
Pecten
albicans
Zygochlamys
delicatula
Chlamys
hastata
Ylistrum
japonicum
Pecten
fumatus
Talochlamys
gemmulata
Zygochlamys
patagonica
Argopecten
purpuratus
Argopecten
irradians
Azumapecten
farreri
Mizuhopecten
yessoensi
Placopecten
magellanicus
Euvola
ziczac
Pecten
sulcico
status
Chlamys
islandica
Argopecten
ventricosus
Mimachlamys
varia
Amusium
pleuronectes
Mimachla
myssanguinea
Talochlamys
dichroa
Mimachla
mys
gloriosa
Mimachlamys
cloacata
Mimachlamys
asperrima
Annachla
mysstriatula
Decatopecten
radula
Bractechlamys
vexillum
Aequipecten
glyptus
Scaeochlamys
lemniscata
Chlamys
rubida
Karneka
mpiasulcata
Crassadoma
gigantea
Ylistrum
balloti
Tridacna
mbalavua
na
Siliqua
alta
Megangulus
zyonoensis
Megangulus
venulosus
Donax
faba
Donax
cuneatus
Donax
kiusiuensis
Mactra
quadrang
ularis
Ensisensis
Chamelea
gallina
Spisula
subtruncata
Callista
chione
Polititapes
rhomboides
Venerupis
corrugata
Polititapes
aureus
Venus
crebrisulca
Mercenaria
campechiensis
Antigona
lamellaris
Ameghinomya
antiqua
Callistaerycina
Venerupis
aspera
Paphia
philippiana
Venus
casina
Ensis
terranovensis
Mactra
stultorum
Ensis
macha
Siliqua
minima
Ensis
leei
Polititapes
durus
Cerastoderma
glaucum
Donax
longissimus
Solen
vaginoides
Venus
verrucosa
Ezocallista
brevisiphonata
Procardium
indicum
Cardium
maxicostatum
Cardium
costatum
Acanthocardia
paucicostata
Acanthoc
ardiaechinata
Acanthocardia
aculeata
Ruditapes
philippinarum
Corculum
cardissa
Spisula
solida
Scrobicularia
plana
Mactra
violacea
Chamelea
striatula
Ensissiliqua
Serripes
groenlandicus
Tridacna
elongatissima
Tridacna
rosewateri
Meretrix
lamarckii
Meretrix
lusoria
Paphiaeuglypta
Meretrix
meretrix
Meretrix
petechialis
Acanthocardia
tuberculata
Tridacna
maxima
Lutraria
rhynchaena
Meretrix
lyrata
Arctica
islandica
Solen
strictus
Paratapes
undulatus
Paratapes
textilis
Paphia
amabilis
Solen
grandis
Lutraria
maxima
Donax
vittatus
Donax
variegatus
Donax
trunculus
Donax
semistriatus
Ruditapes
decussatus
Cerastoderma
edule
Tridacna
squamosa
Ruditapes
philippinarum
Mactra
chinensis
Tridacna
derasa
Mercenaria
mercenaria
Tridacna
gigas
Mactra
antiquata
Helix
pomatia
Achatina
fulica
Helix
aspersa
Helix
thessalica
Helix
pomatia
Helix
lucorum
Helix
nicaeensis
Achatina
reticulata
Helix
aspersa
maxima
Helix
aperta
Helix
albescens
Tyrrhenaria
ceratina
Helix
vladika
Pleurodonte
discolor
Pleurodonte
lychnuchus
Erctella
mazzullii
Erctella
cephalae
ditana
Pleurodonte
formosa
Helix
christophi
Helix
nordmanni
Pleurodonte
nucleola
Pleurodonte
parilis
Gonostomopsis
auridens
Caracolus
caracollus
Lacteolun
aselenina
Cernuella
cisalpine
Cochlicella
acuta
Disculella
maderensis
Dialeuca
nemoraloides
Monadenia
fidelis
Cepaea
nemoralis
Sphincterochila
candidissima
Microphysula
ingersolli
Helicodonta
obvoluta
Cernuella
virgata
Loligo
forbesii
Nototodarus
sloanii
Sepia
lorigera
Sepia
pardex
Sepia
kobiensis
Rossia
pacifica
Berryteuthis
magister
Eledone
massyae
Sepia
robsoni
Loligo
reynaudii
Doryteuthis
(Amerigo)
pealeii
Doryteuthis
(Amerigo)
gahi
Sepiola
rondeletii
Sepiola
robusta
Sepiola
intermedia
Adinaefiola
ligulata
Sepia
smithi
Sepia
elliptica
Eledone
palari
Eledone
moschata
Sepiola
affinis
Rossia
palpebrosa
Gonatus
madokai
Gonatus
kamtschaticus
Eledone
cirrhosa
Sepia
elegans
Rossia
bipillata
Sepiola
atlantica
Lolliguncula
(Lolliguncula)
panamensis
Octopus
maya
Illex
illecebrosus
Nototodarus
gouldi
Gonatopsis
octopedatus
Illex
coindetii
Berryteuthis
anonychus
Gonatus
fabricii
Lusepiola
birostrata
Octopus
tetricus
Uroteuthis
(Photololigo)
sibogae
Doryteuthis
(Doryteuthis)
pleii
Doryteuthis
sanpaulensis
Doryteuthis
(Amerigo)
surinamensis
Octopus
hubbsorum
Macrotritopus
defilippi
Octopus
insularis
Loliolus
(Nipponol
oligo)
sumatrensis
Sepia
stellifera
Sepia
recurvirostra
Sepia
madokai
Sepia
kobiensis
Amphioctopus
aegina
Sepia
officinalis
Sepioteuthis
lessoniana
Todarodes
pacificus
Octopus
vulgaris
Heterololigo
bleekeri
Octopus
sinensis
Octopus
americanus
Narrowteuthis
nesisi
Ommastrephes
bartramii
Sepiella
japonica
Uroteuthis
(Photololigo)
edulis
Doryteuthis
(Amerigo)
opalescens
Architeuthis
dux
Dosidicus
gigas
Sepiae
sculenta
Amphioctopus
fangsiao
Loligo
vulgaris
Octopus
mimus
Octopus
conispadiceus
Octopus
bimaculoides
Uroteuthis
(Photololigo)
chinensis
Uroteuthis
(Photololigo)
duvaucelii
Illex
argentinus
Sepia
aculeata
Sepiella
inermis
Sepia
lycidas
Sepia
latimanus
Sepia
apama
Sepia
pharaonis
Loliolus
(Nipponol
oligo)
beka
Alloteuthis
subulata
Nototodarus
hawaiiensis
Sepia
orbignyana
Sepia
papuensis
Rossia
macrosoma
Lolliguncula
(Lolliguncula)
brevis
Lolliguncula
(Loliolopsis)
diomedeae
Afrololigo
mercatoris
Octopus
minor
Octopus
bimaculatus
Octopus
variabilis
Octopus
cyanea
Callistoctopus
ornatus
Enteroctopus
megalocyathus
Enteroctopus
dofleini
Sasakiopus
salebrosus
Octopus
berrima
Amphioctopus
marginatus
Octopus
maorum
Octopus
fitchi
Amphioctopus
neglectus
Loliolus
(Nipponol
oligo) uyii
Loliolus
(Nipponol
oligo)
japonica
Bathyteuthis
abyssicola
Semirossia
patagonica
Cistopus
taiwanicus
Sthenoteuthis
oualaniensis
Wataseni
ascintillans
Gonatopsis
okutanii
Uroteuthis
(Aestuariolus)
noctiluca
Sepioteuthis
australis
Sepioteut
hissepioidea
Amphioctopus
kagoshimensis
Amphioctopus
membranaceus
Amphioctopus
exannulatus
Amphioctopus
rex
Sepia
peterseni
Varuna
litterata
Hemisquil
laensigera
Gonodact
ylussmithii
Pullosquilla
thomassini
Chorisquilla
trigibbosa
Chionoecetes
opilio
Chionoecetes
opilio
Telmessus
acutidens
Lithodes
aequispinus
Panulirus
echinatus
Jasus
paulensis
Jasus
caveorum
Parastacus
pilimanus
Parastacus
brasiliensis
Parastacus
defossus
Parastacus
nicoleti
Gonodactylus
graphurus
Jasus
lalandii
Jasus
paulensis
Lopholithodes
mandtii
Lithodes
santolla
Lithodes
maja
Jasus
edwardsii
Panulirus
regius
Panulirus
pascuensis
Panulirus
laevicauda
Panulirus
gracilis
Panulirus
guttatus
Panulirus
inflatus
Panulirus
femoristriga
Chionoecetes
bairdi
Paralomis
granulosa
Scyllarus
arctus
Palinurus
elephas
Episesarma
mederi
Austropotamobius
torrentium
Cycloachelous
granulatus
Eriocheir
recta
Cervimunida
johni
Achelous
floridanus
Portunus
sayi
Portunus
anceps
Palinurus
gilchristi
Palinurus
mauritanicus
Palinurus
charlestoni
Pseudosquilla
ciliata
Pleuroncodes
monodon
Portunus
ventralis
Achelous
spinicarpus
Callinectes
toxotes
Callinectes
danae
Callinectes
ornatus
Callinectes
marginatus
Callinectes
affinis
Callinectes
rathbunae
Callinectes
bocourti
Callinectes
similis
Callinectes
bellicosus
Callinectes
arcuatus
Metanephrops
armatus
Metanephrops
mozambicus
Metanephrops
japonicus
Metanephrops
andamanicus
Metanephrops
velutinus
Metanephrops
sagamiensis
Metanephrops
binghami
Parastacus
pugnax
Paranephrops
zealandicus
Callinectes
exasperatus
Palinurus
barbarae
Sagmariasus
verreauxi
Metanephrops
rubellus
Metanephrops
challengeri
Metanephrops
neptunus
Metanephrops
australiensis
Metanephrops
arafurensis
Metanephrops
boschmai
Metanephrops
formosanus
Metanephrops
sinensis
Lithodes
ferox
Oratosquillina
interrupta
Odontodactylus
japonicus
Miyakella
nepa
Erugosquilla
woodmasoni
Clorida
decorata
Dictyosquilla
foveolata
Anchisquilla
fasciata
Scyllarides
herklotsii
Palinurus
delagoae
Astacus
astacus
Portunus
hastatus
Achelous
ordwayi
Carcinus
maenas
Portunus
inaequalis
Astacoides
madagas
cariensis
Erimacrus
isenbecki
Hemisquilla
australiensis
Austrosquilla
tsangi
Lithodes
confundens
Fallosquilla
fallax
Echinosquilla
guerinii
Coronis
scolopendra
Chorisquilla
tweediei
Chorisquil
la
hystrix
Chorisquil
laexcavata
Busquilla
plantei
Alima
pacifica
Alima
orientalis
Alachosquilla
vicina
Gonodact
ylellusespinosus
Gonodactylellus
affinis
Kempella
mikado
Hemisquilla
californiensis
Haptosquilla
trispinosa
Haptosquilla
glyptocercus
Gonodactylus
platysoma
Gonodactylaceus
falcatus
Gonodactylus
childi
Gonodactylellus
annularis
Odontoda
ctylusscyllarus
Odontodactylus
latirostris
Odontodactylus
havanensis
Odontodactylus
cultrifer
Neogonodactylus
oerstedii
Neogonodactylus
bredini
Neogonodactylus
bahiahondensis
Lysiosquil
linasulcata
Squilla
rugosa
Raoulserenea
hieroglyphica
Raoulserenea
oxyrhyncha
Pseudosquillopsis
marmorata
Raoulserenea
ornata
Raoulserenea
komaii
Protosquilla
folini
Ibacus
alticrenatus
Scyllarides
nodifer
Scyllarides
haanii
Scyllarides
brasiliensis
Takuspinosoc
arinatus
Jasus
frontalis
Menippe
mercenaria
Procambarus
paeninsul
anus
Puerulus
sewelli
Panulirus
polyphagus
Panulirus
longipes
Panulirus
penicillatus
Panulirus
interruptus
Panulirus
marginatus
Ibacus
peronii
Ibacus
chacei
Charybdis
hellerii
Faxonella
clypeata
Fallicambarus
kountzeae
Arenaeus
mexicanus
Cambarus
tartarus
Chionoecetes
tanneri
Thenus
unimaculatus
Thenus
indicus
Haptosquilla
hamifera
Lithodes
turritus
Bouchardina
robisoni
Troglocambarus
maclanei
Hobbseus
yalobushensis
Hobbseus
prominens
Charybdis
lucifera
Hobbseus
petilus
Faxonella
creaseri
Thranita
danae
Monomia
petrea
Neogonodactylus
wennerae
Xiphonectes
pseudohastatoides
Gonodactylellus
viridis
Gonodactylaceus
ternatensis
Belosquilla
laevis
Procambarus
okaloosae
Procambarus
morrisi
Procambarus
milleri
Procambarus
mancus
Procambarus
lunzi
Hobbseus
cristatus
Procambarus
acutissimus
Faxonius
pagei
Manningia
pilaensis
Pontastacus
leptodactylus
Procambarus
zonangulus
Procambarus
youngi
Procambarus
seminolae
Procambarus
pycnogonopodus
Procambarus
orcinus
Procambarus
pallidus
Alima
maxima
Scyllarides
deceptor
Monomia
argentata
Xiphonectes
pulchricristatus
Paralithodes
platypus
Lopholithodes
foraminatus
Faughnia
formosae
Faughnia
profunda
Bathysquilla
crassispinosa
Eriocheirsinensis
Harpiosquilla
harpax
Callinectes
sapidus
Squilla
mantis
Portunus
trituberculatus
Panulirus
japonicus
Cancer
pagurus
Chionoecetes
japonicus
Scylla
tranquebarica
Scylla
serrata
Eriocheir
hepuensis
Eriocheir
japonica
Cherax
destructor
Squilla
empusa
Lysiosquillina
maculata
Gonodactylus
chiragra
Panulirus
homarus
Homarus
americanus
Panulirus
ornatus
Oratosquilla
oratoria
Panulirus
stimpsoni
Charybdis
japonica
Scylla
paramamosain
Scylla
olivacea
Cherax
quadricarinatus
Cherax
cainii
Paralithodes
brevipes
Paralithodes
camtschaticus
Scyllarides
latus
Procambarus
clarkii
Procambarus
fallax
Homarus
gammarus
Thenus
orientalis
Lithodes
nintokuae
Cherax
cairnsensis
Cherax
dispar
Cherax
quinquec
arinatus
Cherax
robustus
Cherax
monticola
Cherax
glaber
Cherax
holthuisi
Astacopsis
gouldi
Portunus
pelagicus
Paranephrops
planifrons
Nephrops
norvegicus
Ibacus
ciliatus
Charybdis
feriata
Metaneph
ropssibogae
Panulirus
cygnus
Metanephrops
thomsoni
Faxonius
limosus
Squilloides
leptosquilla
Cherax
bicarinatus
Austropot
amobius
pallipes
Cherax
tenuimanus
Cherax
boesemani
Charybdis
(Charybdis)
natator
Procambarus
acutus
Pacifastacus
leniusculus
Munida
gregaria
Panulirus
versicolor
Faxonius
rusticus
Portunus
sanguinolentus
Procambarus
alleni
Metacarcinus
magister
Puerulus
angulatus
Lupocycloporus
gracilimanus
Monomia
gladiator
Varuna
yui
Panulirus
argus
Munida
isos
Scyllarides
squammosus
Cambaroi
dessimilis
Charybdis
bimaculata
Cambarus
robustus
Thalamitasima
Thranita
crenata
Orconectes
luteus
Orconectes
punctimanus
Orconectes
sanbornii
Cherax
crassimanus
Cherax
preissii
Munida
spinosa
Munida
asprosoma
Munida
leagora
Munida
alonsoi
Munida
taenia
Munida
gordoae
Munida
zebra
Munida
distiza
Munida
psamathe
Munida
thoe
Munida
guttata
Munidastia
Munida
ommata
Munida
roshanei
Munida
compressa
Munida
clinata
Munida
chydaea
Munida
compacta
Munidaeclepsis
Munida
tyche
Munida
philippinensis
Munida
armilla
Munida
mesembria
Munida
spilota
Munida
benguela
Munida
endeavourae
Munida
agave
Munida
idyia
Munida
militaris
Munida
flinti
Munida
congesta
Munida
rubridigitalis
Munida
iris
Munida
microphth
alma
Munida
rufiantenn
ulata
Munidapusilla
Munida
remota
Munida
leptosyne
Munida
rosula
Munida
munin
Munida
valida
Munida
proto
Enriquea
leviantennata
Munida
multilineata
Munida
pagesi
Munida
stomifera
Munida
quadrispina
Munida
tiresias
Munida
psylla
Munida
heteracantha
Paralomis
formosa
Paralomis
spinosissima
Paralomis
birsteini
Paralomis
hirtella
Scyllarus
subarctus
Scyllarus
pygmaeus
Scyllarus
chacei
Scyllarus
caparti
Scyllarus
americanus
Episesarma
palawanense
Episesar
masingaporense
Austropotamobius
fulcisianus
orientalis
Achelous
tumidulus
Achelous
asper
Achelous
sebae
Portunus
acuminatus
Achelous
tuberculatus
Achelous
iridescens
Portunus
xantusii
Achelous
depressifrons
Achelous
rufiremus
Achelous
gibbesii
Portunus
minimus
Achelous
stanfordi
Achelous
brevimanus
Portunus
affinis
Achelous
angustus
Achelous
binoculus
Oratosquillina
inornata
Oratosquillina
asiatica
Oratosquillina
anomala
Oratosquillina
perpensa
Erugosquilla
graham
Busquilla
quadraticauda
Kempella
stridulans
Gonodactylaceus
graphurus
Gonodactylaceus
randalli
Carcinus
aestuarii
Menippe
rumphii
Menippe
nodifrons
Menippe
adina
Procambarus
liberorum
Procambarus
toltecae
Procambarus
curdi
Procambarus
digueti
Procambarus
nigrocinctus
Procambarus
versutus
Procambarus
gibbus
Cambarus
pecki
Procambarus
geminus
Charybdis
acuta
Creaserinus
fodiens
Fallicambarus
jeanae
Creaserinus
gordoni
Creaserinus
caesius
Fallicambarus
dissitus
Creaserinus
danielae
Fallicambarus
oryktes
Fallicambarus
byersi
Creaserinus
burrisi
Creaserinus
gilpini
Fallicambarus
harpi
Fallicambarus
macneesei
Fallicambarus
petilicarpus
Fallicambarus
wallsi
Fallicambarus
strawni
Fallicambarus
devastator
Fallicambarus
houstonensis
Fallicambarus
hortoni
Arenaeus
cribrarius
Cambarus
tenebrosus
Cambarus
deweesae
Cambarus
striatus
Cambarus
graysoni
Cambarus
monongalensis
Cambarus
pyronotus
Cambarus
maculatue
Cambarus
coosawat
tae
Cambarus
latimanus
Cambarus
strigosus
Cambarus
parrishi
Cambarus
bouchardi
Cambarus
fasciatus
Cambarus
harti
Cambarus
nerterius
Cambarus
setosus
Cambarus
batchi
Cambarus
halli
Cambarus
crinipes
Cambarus
unestami
Cambarus
reburrus
Cambarus
gentry
Cambarus
hubbsi
Cambarus
friaufi
Cambarus
obeyensis
Cambarus
cracens
Cambarus
asperimanus
Cambarus
hobbsorum
Cambarus
williami
Cambarus
howardi
Cambarus
obstipus
Cambarus
girardianus
Cambarus
cryptodytes
Cambarus
speciosus
Cambarus
sciotensis
Cambarus
georgiae
Cambarus
pristinus
Cambarus
aculabrum
Cambarus
englishi
Cambarus
brachydactylus
Cambarus
cumberlandensis
Cambarus
dubius
Cambarus
reflexus
Cambarus
rusticiformis
Cambarus
scotti
Cambarus
coosae
Cambarus
distans
Cambarus
longirostris
Cambarus
hubrichti
Monomia
lucida
Faughniaserenei
Harpiosquilla
melanoura
Harpiosquilla
annandalei
Cherax
communis
Cherax
cuspidatus
Cherax
paniaicus
Cherax
lorentzi
Cherax
albertisii
Cherax
rotundus
Cherax
leckii
Cherax
murido
Cherax
wasselli
Cherax
parvus
Cherax
pallidus
Cherax
cartalacoolah
Cherax
longipes
Cherax
rhynchotus
Cherax
pulcher
Cherax
peknyi
Cheraxsetosus
Cherax
misolicus
Cherax
warsamsonicus
Cheraxsolus
Cheraxsnowden
Cherax
buitendijkae
Cherax
boschmai
Cherax
nucifraga
Cherax
barrette
Oratosquilla
fabricii
Astacopsis
tricornis
Thalamita
admete
Faxonius
virilis
Thranita
prymna
Astacopsis
franklinii
Cambaroi
desschrenckii
Orconectes
australis
Thalamita
chaptalii
Zygita
longifrons
Thalamita
picta
Thalamitaseurati
Thranita
pelsarti
Orconectes
barri
Faxonius
ronaldi
Faxonius
neglectus
Orconectes
compressus
Orconectes
forceps
Orconectes
pellucidus
Neoeriocheir
leptognathus
Penaeus
kerathurus
Penaeus
marginatus
Penaeus
longistylus
Penaeus
plebejus
Metapenaeopsis
liui
Metapenaeopsis
lamellata
Metapenaeopsis
acclivis
Metapenaeopsis
commensalis
Atypopen
aeusstenodactylus
Aristeus
antillensis
Solenocera
vioscai
Trachysal
ambria
curvirostris
Penaeus
chinensis
Penaeus
canaliculatus
Metapenaeopsis
barbata
Penaeusesculentus
Heteropenaeus
longimanus
Atypopenaeus
dearmatus
Funchalia
taaningi
Xiphopenaeus
kroyeri
Trachypenaeopsis
mobilispinis
Rimapen
aeussimilis
Megokris
granulosus
Parapenaeus
politus
Solenocera
membranacea
Solenocera
koelbeli
Alcockpenaeopsis
hungerfordii
Batepenaeopsis
tenella
Pandalus
platyceros
Metapenaeus
moyebi
Metapenaeus
joyneri
Pandalus
montagui
Heterocarpus
parvispin
a
Penaeus
brasiliensis
Aristeus
antennatus
Heterocarpus
laevigatus
Heterocarpus
lepidus
Heterocarpus
gibbosus
Funchalia
villosa
Hemipenaeus
carpenter
Mesopenaeus
tropicalis
Pelagope
naeus
balboae
Penaeus
hathor
Metapenaeopsis
provocatoria
Aristeus
virilis
Aristeus
alcock
Penaeus
aztecus
Heterocarpus
abulbus
Penaeussetiferus
Cerataspis
monstrosus
Pleoticus
robustus
Aristaeopsis
edwardsiana
Solenoce
ra
necopina
Parapenaeus
indicus
Parapenaeus
cayrei
Parapenaeus
fissurus
Parapenaeus
investigatoris
Parapenaeus
fissuroides
Parapenaeus
australiensis
Parapenaeus
americanus
Parapenaeus
ruberoculatus
Heterocarpus
ensifer
Kishinouyepenaeopsis
cornuta
Parapenaeus
sextuberculatus
Parapenaeus
perezfarf
antae
Parapenaeus
murrayi
Parapenaeus
longipes
Parapenaeus
lanceolatus
Parapenaeus
kensleyi
Heterocarpus
chani
Heterocarpus
woodmasoni
Heterocarpus
sibogae
Heterocarpus
dorsalis
Metapenaeopsis
andamanensis
Heterocarpus
corona
Metapenaeopsis
coniger
Macrobrachium
idella
Trachysal
ambria
brevisutur
ae
Trachysal
ambria
aspera
Trachysal
ambria
albicoma
Euphausia
superba
Solenocera
hextii
Hymenopenaeus
equalis
Rimapenaeus
constrictus
Crangon
crangon
Trachypenaeus
anchoralis
Megokris
pescadoreensis
Trachysal
ambria
longipes
Trachysal
ambriastaro
bogatovi
Trachysal
ambria
nansei
Trachysal
ambria
malaiana
Trachysal
ambria
dentata
Trachysal
ambria
parvispina
Crangon
uritai
Pandalus
borealis
Metapenaeus
monoceros
Pandalus
nipponensis
Hadropenaeus
lucasii
Ganjampenaeopsis
uncta
Solenocera
annectens
Solenocera
melantho
parapenaeopsis
stylifera
Penaeus
japonicus
Penaeus
brevirostris
Penaeus
notialis
Penaeus
duorarum
Penaeusschmitti
Artemesia
longinaris
Penaeus
subtilis
Penaeus
stylirostris
Penaeus
vannamei
Macrobrachium
rosenbergii
Penaeus
monodon
Pandalus
hypsinotus
Ganjam
penaeopsis
uncta
Pandalus
jordani
Macrobrachium
bullatum
Penaeus
penicillatus
Penaeus
merguiensis
Metapenaeus
ensis
Acetes
chinensis
Macrobrachium
nipponense
Penaeus
californiensis
Macrobrachium
lanchesteri
Pleoticus
muelleri
Metapenaeus
affinis
Hymenopenaeus
neptunus
Penaeus
indicus
Aristaeomorpha
foliacea
Solenocera
crassicornis
Mierspen
aeopsis
hardwickii
Penaeus
latisulcatus
Penaeus
semisulcatus
Penaeussilasi
Penaeus
isabelae
Sicyonia
lancifer
Metapenaeopsis
dalei
Metapenaeopsis
gerardoi
Parapenaeus
longirostris
Pandaluseous
Pandalus
miyakei
Pandalus
japonicas
Pandalus
glabrus
Pandalus
teraoi
Pandalus
ivanovi
Pandalus
coccinatus
Pandalus
formosanus
Pandalus
princeps
Pandalus
chani
Pandalus
houyuu
Pandalus
capillus
Pandalus
longirostris
Pandalus
ochotensis
Pandalus
latirostris
Metapenaeus
brevicornis
Metapenaeus
dobsoni
Heterocarpus
hayashii
Heterocarpus
fascirostratus
Palaemon
adspersus
Palaemon
serratus
Palaemon
sinensis
Palaemon
capensis
Palaemon
annandalei
Palaemon
gravieri
Palaemon
serenus
Palaemon
carinicauda
Palaemon
pugio
Palaemon
pandaliformis
Palaemon
elegans
Palaemon
longirostris
Palaemon
peringuey
Palaemon
debilis
Palaemon
carteri
Palaemon
ritteri
Palaemon
orientis
Macrobrachium
gracilirostre
Palaemon
vulgaris
Palaemon
serrifer
Palaemon
varians
Palaemon
macrodactylus
Palaemon
tonkinensis
Palaemon
xiphias
Palaemon
ivonicus
Palaemon
pacificus
Palaemon
atrinubes
Palaemon
intermedius
Palaemon
concinnus
Palaemon
yuna
Palaemon
antennarius
Palaemon
dolospinus
Palaemon
gracilis
Palaemon
mundusn
ovus
Palaemon
suttkusi
Palaemon
zariquieyi
Macrobrachium
australiense
Palaemon
semmelin
kii
Palaemon
litoreus
Palaemon
septemtri
onalis
Palaemon
guangdon
gensis
Palaemon
hancocki
Palaemon
vietnamicus
Palaemon
texanus
Palaemon
ortmanni
Palaemon
turcorum
Palaemon
kadiakensis
Macrobrachium
asperulum
Macrobrachium
australe
Macrobrachium
olfersii
Macrobrachium
jelskii
Macrobrachium
villosimanus
Macrobrachium
equidens
Macrobrachium
potiuna
Macrobrachium
malcolmsonii
Macrobrachium
superbum
Macrobrachium
striatum
Macrobrachium
latidactylus
Macrobrachium
hancocki
Macrobrachium
acanthurus
Macrobrachium
inflatum
Macrobrachium
crenulatum
carcinus
Macrobrachium
americanum
Macrobrachium
latimanus
Macrobrachium
mammillo
dactylus
Macrobrachium
faustinum
Macrobrachium
heterochirus
Macrobrachium
scabriculum
Macrobrachium
digueti
Macrobrachium
tenellum
Macrobrachium
idae
Macrobrachium
japonicum
Macrobrachium
formosense
Macrobrachium
dienbienp
huense
Macrobrachium
placidulum
Macrobrachium
sintangense
Macrobrachium
niphanae
Macrobrachium
totonacum
Macrobrachium
tuxtlaense
Macrobrachium
vicconi
Macrobrachium
villalobosi
Macrobrachium
amazonicum
Macrobrachium
canarae
Macrobrachium
tratense
Macrobrachium
forcipatum
Macrobrachium
hirsutimanus
Macrobrachium
borellii
Macrobrachium
brasiliense
Macrobrachium
aemulum
Macrobrachium
handschini
Macrobrachium
horstii
Macrobrachium
ferreirai
Macrobrachium
lanatum
Macrobrachium
novaeholl
andiae
Macrobrachium
tolmerum
Macrobrachium
iheringi
Macrobrachium
saigonense
Macrobrachium
nattereri
Macrobrachium
aracamuni
Macrobrachium
inpa
Macrobrachium
depressimanum
Macrobrachium
surinamicum
Macrobrachium
denticulatum
Macrobrachium
pilimanus
Macrobrachium
ohione
Macrobrachium
hainanense
Macrobrachium
lepidactyloides
Macrobrachium
jaroense
Macrobrachium
esculentum
Macrobrachium
maculatum
Macrobrachium
edentatum
Macrobrachium
grandimanus
Macrobrachium
malayanum
Macrobrachium
meridionale
Macrobrachium
neglectum
Macrobrachium
platycheles
Macrobrachium
naso
Macrobrachium
placidum
Macrobrachium
yui
Macrobrachium
shokitai
Macrobrachium
sundaicum
Macrobrachium
rude
Macrobrachium
lamarrei
Macrobrachium
sankolli
Macrobrachium
gangeticum
Trachysal
ambria
palaestinensis
Euphausia
pacifica
Euphausia
lucens
Euphausia
vallentini
Euphausia
triacantha
Euphausia
longirostris
Euphausia
similis
Euphausia
recurve
Euphausia
krohni
Euphausia
frigida
Euphausia
gibboides
Euphausia
eximia
Euphausia
americana
Euphausia
tenera
Euphausia
pseudogibba
Euphausia
hemigibba
Euphausia
brevis
Hymenopenaeus
debilis
Nematopalaemon
tenuipes
Primers were designed manually on a multiple DNA sequence alignment of the mitochondrial 16S rDNA of approximately 90 bivalve species using the CLC Genomics Workbench 10.1.1. The designed primers were checked for their physical and structural properties (e.g. formation of dimers, secondary structure, annealing temperature) using Oligo Calc, the OligoAnalyzer Tool provided by Integrated DNA Technologies (IDT) and the online product descriptions from TIB Molbiol (Berlin, Germany). The primers, listed in Table 5, were synthesized by TIB Molbiol. Table 5 also shows the Illumina overhang adapter sequences which were linked to the target-specific primers.
All in-house designed primers were tested in real-time PCR with DNA extracted from the eleven reference samples, comprising three mussel, six scallop, and two oyster species. During optimization, the following PCR conditions/parameters were kept constant and applied as published previously: DNA input amount of 12.5 ng, ‘ready-to-use’ HotStarTaq Master Mix Kit, annealing temperature (62° C.), 25 cycles (Dobrovolny, S. et al., 2019). Only one variable, the addition of magnesium chloride solution, was modified (addition of 1.5 mM or 3 mM MgCl2). Real-time PCR reactions were carried out using a fluorescent intercalating dye (EvaGreen® (20× in water)) in strip tubes or in 96-well plates, depending on the thermocycler used, the Rotor-Gene Q (Qiagen) or the LightCycler® 480 System (Roche, Penzberg, Germany), respectively. The total volume of the PCR reactions was 25 μL, consisting of 22.5 μL reaction mix and 2.5 μL of template DNA (diluted DNA samples (5 ng/μL)) or water as negative control. In the reaction mix, the HotStarTaq Master Mix Kit (Qiagen) was used at a final concentration of 1× and the final concentration of primers was 0.2 μM, expect the forward primer for mussels (0.4 μM). PCR cycling conditions were 15 min initial denaturation at 95° C., 25 cycles at 95° C., 62° C. and 72° C. for 30 s each, and a final elongation for 10 min at 72° C. The primer pairs for mussels, scallop and oysters with and without Illumina overhang adapter sequences were first used in singleplex PCR assays. Then, the seven primers (three forward and four reverse primers) listed in Table 5 were combined in a triplex assay. The identity of the PCR products was confirmed by melting curve analysis and/or agarose gel electrophoresis.
In general, samples were sequenced by using either the MiSeq® or the iSeq® platform (Illumina, San Diego, California, USA). DNA extracts were diluted to a DNA concentration of 5 ng/μL. Extracts with a DNA concentration <5 ng/μL were used undiluted.
DNA library preparation was performed according to Dobrovolny, S. et al. (2019) with minor modifications (excess of magnesium chloride, final concentration 3 mM; average library size: 278 bp; diluted libraries of the iSeq© system were denatured automatically on the instrument).
For the MiSeq© and iSeq® platform, the DNA library was adjusted to 4 nM and 1 nM, respectively, with 10 mM Tris-HCL, pH 8.6. After pooling individual DNA libraries (5 μL MiSeq®, 7 μL iSeq®), the DNA concentration was determined using Qubit© 2.0 fluorimeter.
All sequencing runs were performed using either the MiSeq® Reagent Kit v2 (300-cycles) or the iSeq® 100 i1 Reagent v2 (300-cycles) with a final loading concentration of 8 μM. The pooled DNA libraries contained a 5% PhiX spike-in.
Reference samples were sequenced in six replicates (three sequencing runs, two replicates per run), while DNA extract mixtures were sequenced in nine replicates (three sequencing runs, three replicates per run). Commercial food products (O5-Mi86, above the double line in Table 9) were sequenced in one replicate (three sequencing runs, one replicate per run) and food products (O1-S61, below the double line in Table 9) were sequenced at least once by using either the MiSeq® or the iSeq® platform.
After paired-end sequencing, the resulting FastQ files, generated by the instrument control software, were used as input for data analysis. The sequencing output in FastQ format was then processed with an analysis pipeline as described previously by using Galaxy (Version 19.01) (Dobrovolny, S. et al., 2019). The published amplicon analysis workflow was modified as follows: the target-specific primers were trimmed from both ends using the tool Cutadapt and reads were not clustered into Operational Taxonomic Units (OTUs) (Martin, M., 2011). Completely identical sequences were collapsed into a single representative sequence with the tool Dereplicate to minimize the number of reads, and then compared against a customized database for bivalves using BLASTn (Edgar, R. C., 2010).
The aim was to develop a DNA metabarcoding method allowing the differentiation between species belonging to the bivalves Pectinidae, Ostreidae, and Mytilidae. To be applicable in routine analysis, the method should allow identifying the economically most important bivalve species in raw and highly processed food products.
Three primer sets were designed, one for each of the three bivalves, Pectinidae, Ostreidae, and Mytilidae. Primer pairs consisting of one forward and one reverse primer allowed amplifying the DNA barcode region in scallop and oyster species (Table 5). However, in case of mussels, a primer set consisting of one forward primer and two reverse primers (Table 5) was necessary to obtain a PCR product for the mussel species listed in Table 3. With the three primer sets, PCR products differing in at least one base should be obtained for all bivalve species of interest.
Further sequence alignments indicated that the DNA barcode region selected does not allow distinguishing between all species of the following genera: Chlamys spp., Euvola spp., Pecten spp., Crassostrea spp., Magallana spp., Ostrea spp. and Saccostrea spp. These species cannot be distinguished: Chlamys rubida and Chlamys behringiana; Pecten albicans, Pecten fumatus, Pecten jacobaeus, Pecten keppelianus, Pecten novaezelandiae, Pecten sucicostatus, Crassostrea hongkongensis and Crassostrea rivularis; Ostrea angelica and Ostrea lurida; as well as Ostrea permollis and Ostrea puelchana; and Saccostrea echinata, Saccostrea glomerata and Saccostrea mytiloides. In addition, two mussel species, Mytilus platensis and Mytilus chilensis, can also not be distinguished (for Mytilus platensis only one DNA sequence entry was in the public databases provided by NCBI). However, differentiation at the genus level (Chlamys spp., Pecten spp., Crassostrea spp. Ostrea spp., Mytilus spp.) is sufficient according to the “Codex Alimentarius Austriacus” chapter B35 (see BMGF-75210/0026-II/B/13/2017, 2007).
When the primers were tested in singleplex PCR assays, for each of the reference samples a PCR product of about 150 bp in length was obtained by increasing the concentration of the forward primer for mussels to 0.4 μM and keeping the concentration of the other six primers at 0.2 μM. In addition, it was tested whether the seven primers could be combined to a triplex system. PCR products for the bivalve species of interest were obtained in one and the same vial by increasing the magnesium chloride concentration to a final concentration of 3 mM. Thus, it was achieved to perform the triplex PCR assay.
Library preparation, pooling of 5 μL or 7 μL per normalized DNA library and the sequencing process were performed as described previously (Dobrovolny, S. et al., 2019). Sequencing runs were performed in triplicates and the average run metrics were as follows: cluster density (969 K/mm2) on the flow cell, cluster passing filter (70.22%) as well as the Q-scores (Q30) for read1 and read2 were 92.6% and 89.28%, respectively. 5.02% of the total reads were identified as PhiX control sequences with an error rate of 1.49%.
Analysis of DNA Extracts from Reference Samples
PCR products were obtained for each of the reference samples, comprising three mussel samples (M12, M13 and M27, Table 2), six scallop samples (S42, S46, S47, S49, S50, and S55, Table 2) and two oyster samples (O2 and O3, Table 2). Sequencing results for reference samples are summarized in Table 6.
Table 6 shows results for DNA extracts from reference samples. The numbers are mean values (n=6, three sequencing runs, 2 replicates per run). The table shows the mean values of the total number of raw reads, the total number of reads that passed the analysis pipeline in Galaxy as well as the total number and percentage of reads that were assigned correctly to the eleven species (based on 6 replicates).
No significant differences were observed in the total number of reads (before data analysis process) between these species, except Mytilus galloprovincialis (162843), Perna canaliculus (169631), and Mytilus edulis (134500). With the exception of Perna canaliculus, >70% of the reads passed the amplicon analysis workflow. All three mussel species, six scallop species and two oyster species could be identified with this workflow at high rate (>97.5%), except Mytilus edulis.
Ostrea edulis
Ostrea edulis
Crassostrea
Magallana
gigas
gigas
Mytilus
Mytilus
galloprovincialis
galloprovincialis
Perna
Perna
canaliculus
canaliculus
Mytilus edulis
Mytilus edulis
Mizuhopecten
Mizuhopecten
yessoensis
yessoensis
Pecten
Pecten spp.
jacobaeus
Zygochlamys
Zygochlamys
patagonica
patagonica
Placopecten
Placopecten
magellanicus
magellanicus
Argopecten
Argopecten
purpuratus
purpuratus
Aequipecten
Aequipecten
opercularis
opercularis
Six ternary DNA extract mixtures were analysed containing the DNA of the three bivalve species Pectinidae, Ostreidae, and Mytilidae in ratios of 98.0:1.5:0.5 (v/v/v). The composition of the DNA extract mixtures and the results obtained by DNA metabarcoding are summarized in Table 7. Table 7 shows the results for ternary DNA extract mixtures representing the three bivalve species of interest. DNA extracts (5 ng/μL) were mixed in a ratio of 98.0:1.5:0.5 (v/v/v). Numbers are mean values (n=9, three sequencing runs, 3 replicates per run). The total number of raw reads ranged from 80856 to 147443 and the reads that passed the workflow were in the range from 65961 to 147196. For the main components (98.0%), the number of reads assigned correctly ranged from 62434 to 140147. In addition, both minor components (1.5% and 0.5%) could be identified. The number of reads assigned correctly was in the range from 1710 to 4356 and 555 to 1478, respectively.
Crassostrea
Mytilus
Pecten spp.
gigas
galloprovincialis
Crassostrea
Pecten spp.
Mytilus
gigas
galloprovincialis
Pecten spp.
Crassostrea
Mytilus
gigas
galloprovincialis
Pecten spp.
Mytilus
Crassostrea
galloprovincialis
gigas
Mytilus
Pecten spp.
Crassostrea
galloprovincialis
gigas
Mytilus
Crassostrea
Pecten spp.
galloprovincialis
gigas
In addition, three DNA extract mixtures containing DNA from species belonging to one bivalve species were analysed (Table 8). Table 8 shows the results for DNA extract mixtures representing one bivalve species. DNA from minor components was present in a proportion of 1% each. In addition, results for a DNA extract mixture containing DNA from a squid species (Sepiella inermis) as main component (97.0%) and DNA from three bivalve species (1% each) is shown. Numbers are mean values (n=9, three sequencing runs, 3 replicates per run). The mixtures contained DNA from a scallop or mussel species, respectively. DNA from other bivalve species was present in a proportion of 1.0% each. Both species being present as main components, Placopecten magellanicus and Perna canaliculus, could be identified, with the number of reads assigned correctly ranging from 58156 to 77483. However, quite different numbers of reads were correctly assigned to the minor components, ranging from 626 (Mizuhopecten yessoensis) to 50391 (Mytilus galloprovincialis).
A further DNA extract mixture was analysed containing DNA from the squid species Sepiella inermis as main component (97.0%) and DNA from the bivalve species Placopecten magellanicus, Ostrea edulis and Perna canaliculus as minor components (1.0% each). As expected, in this mixture, the main component could not be detected because the primers are not suitable for amplification of the target region for Sepiella inermis. 31424, 28162, and 806 reads, respectively, were assigned correctly to the three bivalve species.
Placopecten
magellanicus
Mizuhopecten
yessoensis
Pecten spp.
Zygochlamys
patagonica
Argopecten
purpuratus
Aequipecten
opercularis
Placopecten
magellanicus
Magallana gigas
Ostrea edulis
Perna
canaliculus
Mytilus
galloprovincialis
Mytilus edulis
Sepiella inermis
Placopecten
magellanicus
Ostrea edulis
Perna canaliculus
In order to investigate the applicability of the DNA metabarcoding method to foodstuffs, DNA extracts from 75 commercial food products were analysed. According to declaration, eight samples (O1 and O4-O10) contained oyster species, 27 samples (M11, M14-M26, and M28-M40) mussel species, 15 samples (S41, S43-45, S48, S51-S55, and S56-S61) scallop species and 25 samples (Mi62-Mi86) were mixed-species seafood products (Table 9). Table 9 shows the results obtained for commercial seafood samples. Samples listed above the double line were sequenced with the MiSeq® (three sequencing runs, 1 replicate per run, numbers are mean values); samples listed below the double line were sequenced either with the MiSeq® or the iSeq®. The ingredient list of 30 out of 75 food products did not give any information on the bivalve species. 39 samples were declared to contain “Crassostrea gigas”, “Mytilus galloprovincialis”, “Mytilus chilensis”, “Mytilus edulis”, “Zygochlamys patagonica”, “Chlamys opercularis”, “Placopecten magellanicus”, “Pecten maximus”, or “Patinopecten yessoensis”. The remaining samples (n=6) were labelled with “Mytilus spp.” and “Pecten spp.”.
Three oyster species (Saccostrea malabonensis, Magallana bilineata, Magallana gigas), three mussel species (Mytilus galloprovincialis, Mytilus edulis, Perna canaliculus), and three scallop species (Aequipecten opercularis, Placopecten magellanicus, Pecten spp.) were detected in food products (O4, O8, M17, M19, M23, M25, M26, M28, M31, M32, M35, M38-M40, S51, S56, S58-S60, Mi63, Mi65, Mi70, Mi71, Mi73-Mi76, Mi81, Mi83, Mi85, and Mi86) although they were not declared on the label. In four (O1 and O5-O7) out of eight oyster products declared to contain “Crassostrea gigas”, this species was identified.
In 21 products (M11, M16, M18, M21, M24, M33-M35, M37, M39, M40, Mi62, Mi64, Mi66, Mi69, Mi72, Mi77-Mi80, and Mi84), the mussel species Mytilus galloprovincialis was detected, indicating that it is one of the most commonly used bivalve species. In addition to Mytilus galloprovincialis, Mytilus edulis was identified (percentage of reads assigned correctly >1%) in 13 products (M24, M33, M34, M39, Mi62, Mi64, Mi66, Mi69, Mi72, Mi78-Mi80, and Mi84). However, in none of the products declared to contain Mytilus chilensis, Mytilus chilensis was detected. In four products, Mytilus edulis could not be detected although it was declared on the label.
Placopecten magellanicus and Patinopecten yessoensis were listed as ingredients in samples S41, S45, S54, and S57 and samples S48, S52, and S61, respectively. The results confirmed the presence of these two species, except for sample S57. In sample S43, declared to contain Pecten maximus, the species Mizuhopecten yessoensis was detected. In sample S44 and S53, declared as Pecten spp., the species Mizuhopecten yessoensis was also analysed.
Thus, the method of the invention allows to specifically control food declaration.
Crassostrea gigas
Magallana gigas
Crassostrea gigas
Magallana gigas
Crassostrea gigas
Magallana gigas
Saccostrea malabonensis
Magallana bilineata
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Aequipecten opercularis
Mytilus galloprovincialis
Aequipecten opercularis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Mytilus chilensis,
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus spp.
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis,
Mytilus galloprovincialis
Mytilus chilensis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Zygochlamys patagonica,
Zygochlamys patagonica
Chlamys opercularis
Pecten spp.
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Crassostrea gigas
Magallana gigas
Magallana gigas
Mytilus edulis
Mytilus galloprovincialis
Mytilus spp.
Mytilus galloprovincialis
Mytilus spp.
Mytilus edulis
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
not declared
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus spp.
Mytilus galloprovincialis
Mytilus edulis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus galloprovincialis
Mytilus edulis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Pecten spp.
Mytilus galloprovincialis
Perna canaliculus
Mytilus chilensis
Mytilus galloprovincialis
Mytilus chilensis
Mytilus galloprovincialis
Mytilus edulis
Mytilus edulis
Mytilus galloprovincialis
Placopecten magellanicus
Placopecten magellanicus
Pecten maximus
Mizuhopecten yessoensis
Pecten spp.
Mizuhopecten yessoensis
Placopecten magellanicus
Placopecten magellanicus
Patinopecten yessoensis
Mizuhopecten yessoensis
Placopecten magellanicus
Patinopecten yessoensis
Mizuhopecten yessoensis
Pecten spp.
Mizuhopecten yessoensis
Placopecten magellanicus
Placopecten magellanicus
Placopecten magellanicus
Placopecten magellanicus
Pecten spp.
Placopecten magellanicus
Patinopecten yessoensis
Mizuhopecten yessoensis
1value of two replicates;
2samples were analysed with the MiSeq instrument;
3samples were analysed with the iSeq instrument
In example 2, the identification of Crustacean in food samples according to the invention is shown. The identification was done similar to example 1 using the primer set specific for the identification of seafood species of the respective family in a singleplex setup (two forward primer SEQ ID NO: 1 and SEQ ID NO: 2; and one reverse primer SEQ ID NO: 15). The final concentration of two forward Crustacean primers (SEQ ID NO: 1 and SEQ ID NO: 2) was 0.2 μM each and 0.4 μM for reverse Crustacean primer (SEQ ID NO: 15). Then, the primers of Crustacean und Cephalopods (two Crustacean forward primers (SEQ ID NO: 1 and SEQ ID NO: 2), one Crustacean reverse primer (SEQ ID NO: 15), one Cephalopod forward primer (SEQ ID NO: 3 respectively SEQ ID NO: 4 and SEQ ID NO: 5) and one Cephalopod reverse primer (SEQ ID NO: 16)) were combined in a duplex assay. Furthermore, duplex setups are shown for the combined identification of species of the family of Crustacean and Gastropoda (two forward Crustacean primer SEQ ID NO: 1 and SEQ ID NO: 2, one reverse Crustacean primer SEQ ID NO: 15, one Gastropoda primer SEQ ID NO: 6 and two reverse Gastropoda primer SEQ ID NO: 17 and SEQ ID NO: 18) and also for the combined identification of species of the family of Crustacean and Cephalopods (two Crustacean forward primers (SEQ ID NO: 1 and SEQ ID NO: 2), one Crustacean reverse primer (SEQ ID NO: 15), one Cephalopod forward primer (SEQ ID NO: 3 respectively SEQ ID NO: 4 and SEQ ID NO: 5) and one Cephalopod reverse primer (SEQ ID NO: 16)). Sanger Sequencing was performed as a control experiment for the identification of seafood species.
The results are shown in the following Tables 10 and 11.
Nephrops
Nephros
Nephros
Nephros
Nephros
norvegicus
norvegicus
norvegicus
norvegicus
norvegicus
Procambarus
Procambarus
Procambarus
Procambarus
Procambarus
clarkii
clarkii
clarkii
clarkii
clarkii
Panulirus argus
Panulirus argus
Panuliurs spp
Panuliurs argus
Panuliurs argus
Homarus
Homarus
Homarus
Homarus
Homarus
Homarus
americanus
americanus
americanus
americanus
americanus
americanus
Monomia
Monomia
Monomia
gladiator
gladiator
gladiator
Paralithodes
Paratithodes
Paratithodes
Paratithodes
Paratithodes
camtschaticus
camtschaticus
camtschaticus
camtschaticus
camtschaticus
Chionoecetes
Chionnoecetes
Chionnoecetes
Chionnoecetes
Chionnoecetes
opilio
opilio
Chionocetes
bairdi
Procambarus
Pontastacus
Pontastacus
Pontastacus
clarkii
leptodactylus
leptodactylus
leptodactylus
leptodactylus
Homarus
Homarus
Homarus
Homarus
Homarus
gammarus
gammarus
gammarus
gammarus
gammarus
Pleoticus
Pleoticus
Pleoticus
muelleri
muellerie
muellerie
Penaeus
Penaeus
Penaeus
monodon
monodon
monodon
Macrobrachium
Macrobrachium
Macrobrachium
rosenbergii
rosenbergii
rossenbergeri
Pandalus
Panadalus
Panadalus
borealis
borealis
borealis =
Panadaluseous
Crangon
Crangon
crangon
crangon
Litopenaeus
Penaeus
Litopenaeus
vannamei
vannamei
vanamei
Macrobrachium
Peanaeus
Peanaeus
rossenbergerii
monodon
monodon
Litopenaeus
Penaeus
Penaeus
vannamei
vannamei
vannamei
Metapenaeus
Mierspenaeopsis
Parapenaeopsis
monoceros
hardwickii
sculptilis
Xiphopenaeus
Parapenaeopsis
kroyeri
hardwickii
Heterocarpus
Heterocarpus
reedi
Penaeus
Penaeus
Peanaeus
monodon
vannamei
monodon
Macrobrachium
Peanaeus
Peanaeus
rossenbergerii
monodon
monodon
Penaeus
Panaeus
Panaeus
monodon
monodon
monodon
Crangon
Crangon
crangon
crangon
Crangon
Crangon
crangon
crangon
Aristaeopsis
Aristaeopsis
Aristaeopsis
edwardsiana
edwardsiana
edwardsiana
Aristaeomorpha
foliacea
Pleoticus
Pleoticus
muelleri
muelleri
Penaeus
Pleoticus
occidentalis
muelleri
Penaeus
Penaeus
Penaeus
notialis
duorarum
duorarum
Penaeus nr.
notialis
Penaeus
Pandalus
Pandalus eous =
borealis
borealis
Pandalus borealis
Dendrobranchiata
Pleoticus
Pleoticus
muellerie
muellerie
Penaeus
Penaeus
monodon
monodon
Macrobrachium
Macrobrachium
rosenbergii
rosenbergii
Pandalus spp.
Pandalus spp.
Crangon crangon
Crangon crangon
Crangon
crangon
Penaeus
Litopenaeus
Litopenaeus
vannamei
vannamei
vannamei
Penaeus spp.
Genus Penaeus
Penaeus spp.
Rosenbergerii
Rosenbergerii
Rosenbergerii
Penaeus
Penaeus
Penaeus
vannamei
vannamei
vannamei
Ganjampenaeopsis
Ganjampenaeopsis
Ganjampenaeopsis
uncta
uncta
uncta
Heterocarpus
Heterocarpus
Heterocarpus
Penaeus
Penaeus
Penaeus
vannamei
vannamei
vannamei
Genus Penaeus
Genus Penaeus
Genus Penaeus
Rosenbergerii
Rosenbergerii
Rosenbergerii
Penaeus spp.
Penaeus spp.
Crangon crangon
Crangon crangon
Crangon crangon
Crangon crangon
Crangon crangon
Crangon crangon
Aristaeopsis
Aristaeopsis
Aristaeopsis
Pleoticus
muellerie
Pleoticus muelleri
Pleoticus
Pleoticus
muellerie
muellerie
Penaeus spp.
Penaeus spp.
Pandalus spp.
Pandalus spp.
Penaeus
Penaeus
Penaeus
vannamei
vannamei
vannamei
In example 3, the identification of Cephalopods in food samples according to the invention is shown. The identification was done similar to example 1 using the primer set specific for the identification of Cephalopods seafood species of the respective family in a singleplex setup (one forward primer SEQ ID NO: 3 respectively SEQ ID NO: 4 and SEQ ID NO: 5; and one reverse primer SEQ ID NO: 16). The final concentration of forward (SEQ ID NO: 3 respectively SEQ ID NO: 4 and SEQ ID NO: 5) and reverse primer (SEQ ID NO: 16) was 0.2 μM. Furthermore, a duplex setup is shown for the combined identification of species of the family of Crustacean and Cephalopods. The primers of Crustacean und Cephalopods (two Crustacean forward primers (SEQ ID NO: 1 and SEQ ID NO: 2), one Crustacea reverse primer (SEQ ID NO: 15), one Cephalopod forward primer (SEQ ID NO: 3 respectively SEQ ID NO: 4 and SEQ ID NO: 5) and one Cephalopod reverse primer (SEQ ID NO: 16)) were combined in a duplex assay. Sanger Sequencing was performed as a control experiment for the identification of seafood species.
The results are shown in the following Table 12.
Loligo edulis
Illex illecebrosus
Uroteuthis
Uroteuthis
chinensis
chinensis
Octopus vulgaris
Octopus vulgaris
Loligo chinensis
Doryteuthis
Loligo duvauceli
Loligo duvauceli
Loligo gahi
Sepiella japonica
Sepiella inermis
Sepiella inermis
Octopus vulgaris
Octopus vulgaris
Octopus vulgaris
Sepia officinalis
Sepia pharaonis
Sepia pharaonis
Loligo vulgaris
Doryteuthis
Loligo gahi
Octopus aegina
Amphioctopus
Octopus aegina
aegina
Loligo opalescens
Doryteuthis
opalescens
Sepiella inermis
Sepiella inermis
Sepiella inermis
Octopus maya
Octopus maya
Octopus maya
Sepiella inermis
Sepiella inermis
Sepiella inermis
Loligo edulis
Illex illecebrosus
Loligo
Uroteuthis
chinensis
chinensis
Eledone
Amphioctopus
Amphioctopus
moschata
aegina
aegina
Loligo chinensis
Illex illecebrosus
Loligo chinensis
Uroteuthis
chinensis
Loligo galhi
Doryteuthis
Loligo gali
Uroteuthis
duvaucelii
Dosidicus gigas
Dosidicus gigas
Dosidicus gigas
Octopus
membranaceus
Illex illecebrosus
Uroteuthis
chinensis
Octopus spp
Octopus spp.
Doryteuthis
Doryteuthis
Loligo spp.
Loligo spp.
Sepiella inermis
Sepiella inermis
Octopus spp.
Octopus spp.
Sepia spp.
Sepia spp
Doryteuthis
Doryteuthis
Amphioctopus
Amphioctopus
aegina
aegina
Doryteuthis
Doryteuthis
opalescens
opalescens
Sepiella inermis
Sepiella inermis
Octopus maya
Octopus maya
Sepiella inermis
Sepiella inermis
Uroteuthis
Uroteuthis
chinensis
chinensis
llex ilecebrosus
llex ilecebrosus
Amphioctopus
Amphioctopus
aegina
aegina
Uroteuthis
Uroteuthis
chinensis
chinensis
llex ilecebrosus
llex ilecebrosus
Doryteuthis
Doryteuthis
Amerigo
gahi
Doryteuthis
Octopus cyanea
Uroteuthis spp.
Dosidicus gigas
Dosidicus gigas
Todarodes
pacificus
Todarodes
pacificus
Amphioctopus
Dosidicus gigas
In example 4, the identification of Gastropoda in food samples according to the invention is shown. The identification was done similar to example 1 using the primer set specific for the identification of seafood species of the respective family in a singleplex setup (one forward primer SEQ ID NO: 6 and two reverse primer SEQ ID NO: 17 and SEQ ID NO: 18). The final concentration of forward Gastropoda primer (SEQ ID NO: 6) was 0.4 μM and 0.2 μM each for the two reverse Gastropoda primer (SEQ ID NO: 17 and SEQ ID NO: 18). Furthermore, a duplex setup is shown for the combined identification of species of the family of Crustacean and Gastropoda. Sanger Sequencing was performed as a control experiment for the identification of seafood species.
The results are shown in the following Table 13.
Helix lucorum
Helix lucorum
Helix spp.
Helix spp
Achatina reticulata
Achantina spp
Achantina spp.
Helix lucorum
Helix promatia
Helix promatia
Helix spp.
Helix spp.
Helix spp.
Helix thessalica
Helix spp.
Helix spp.
Achantina fulica
In example 5, the identification of Veneridae in food samples according to the invention is shown. The identification was done similar to example 1 using the primer set specific for the identification of seafood species of the respective family in a singleplex setup (five forward primer SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 and three reverse primer SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21). The final concentration of forward primer (SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11) was 0.2 μM each, 0.2 μM each for two reverse primer (SEQ ID NO: 20 and SEQ ID NO: 21) and 0.6 μM for the reverse primer (SEQ ID NO: 19). Sanger Sequencing was performed as a control experiment for the identification of seafood species.
The results are shown in the following Table 14.
Ensis ensis
Ensis directus
Ensis directus
Ensis spp
Ensis terranovensis
Ruditapes philippinarum
Ruditapes philippinarum
Ruditapes philipinarum
Cerastoderma edule
Cerastoderma edule
Meretrix lyrata
Meretrix lyrate
Meretrix lyrata
Meretrix lyrata
Callista Chione
Solen marinatus
Ensis directus
Ensis directus
Ensis spp
Ensis terranovensis
Ruditapes philippinarum
Ruditapes philippinarum
kein Ergbenis
Ruditapes philipinarum
Cerastoma edulis
Cerastoma edulis
Cerastoma edulis
Cerastoderma edule
Chamelea gallina
Chamelea gallina
Chamelea gallina
Chamelea gallina
Cerastoderma edule
Cerastoderma edule
Ruditapes philipinarum
Ruditapes philipinarum
Ensis direktus
Ensis spp
Venus spp
Ruditapes philipinarum
Metrix lyrate
Metrix lyrate
Chamelea gallina
Ruditapes philipinarum
Number | Date | Country | Kind |
---|---|---|---|
21204456.4 | Oct 2021 | EP | regional |
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2022/079764 | 10/25/2022 | WO |