Development of a DNA Metabarcoding Method for the Identification of Bivalve Species in Seafood Products

Abstract

The production of bivalve species has been increasing in the last decades. In spite of strict requirements for species declaration, incorrect labelling of bivalve products has repeatedly been detected. We present a DNA metabarcoding method allowing the identification of bivalve species belonging to the bivalve families Mytilidae (mussels), Pectinidae (scallops), and Ostreidae (oysters) in foodstuffs. The method, developed on Illumina instruments, targets a 150 bp fragment of mitochondrial 16S rDNA. We designed seven primers (three primers for mussel species, two primers for scallop species and a primer pair for oyster species) and combined them in a triplex PCR assay. In each of eleven reference samples, the bivalve species was identified correctly. In ten DNA extract mixtures, not only the main component (97.0–98.0%) but also the minor components (0.5–1.5%) were detected correctly, with only a few exceptions. The DNA metabarcoding method was found to be applicable to complex and processed foodstuffs, allowing the identification of bivalves in, e.g., marinated form, in sauces, in seafood mixes and even in instant noodle seafood. The method is highly suitable for food authentication in routine analysis, in particular in combination with a DNA metabarcoding method for mammalian and poultry species published recently.

A next-generation sequencing approach for the detection of mixed species in canned tuna

“…In the area of seafood identification, an increasing number of studies on metabarcoding approaches have been published in recent years, with focus mainly on qualitative identification of diverse species in processed surimi, fish products, and bivalve products in which thresholds for species detection ranged between 0.5 and 1 % (e.g. Baetscher et al, 2021 , Gense et al, 2021 , Giusti et al, 2019 ). Several NGS-based or metabarcoding methods also exist in the field of meat analysis ( Ballin et al, 2009 ).…”

Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin? A systematic review

“…In fact, most of the SPs (n = 17; 77.2%) applied the monotarget approach, in which a unique gene was used for the authentication. Only four of them (23.5%) relied on the use of more than one primer pair (two or three) to amplify the same target gene (Gense et al, 2021;Ho et al, 2020;Kappel et al, 2017;Preckel et al, 2021). The five SPs (22.7%) applying the multi-target approach coupled two genes (Ribani et al, 2018a(Ribani et al, , 2018bCarvalho et al, 2017), three genes (Paracchini et al, 2017), or eight genes (Paracchini et al, 2019).…”

“…The number of replicates should be established based on the type of investigated matrix. In this case, it was especially seafood, ranging from canned tuna (Kappel et al, 2017;Klapper et al, 2023), surimi (Noh et al, 2021), mussel-based products (Gense et al, 2021) and commercial sea cucumber (Xing et al, 2021), and only in one case, heavily processed meat products (Pan et al, 2020). Given the higher number of commercial seafood species with respect to meat, the use of replicates is especially useful.…”

Identification of Squid Species Using DNA Barcoding and Real-time PCR

“…However, when the squid is processed into sh llets, canned food, sh balls, and other products, its unique morphological characteristics hardly exist, so this method has certain limitations. Meanwhile, Squid species authentication can be achieved using several analytical methods (Gense et al 2021;Shi et al 2020). For animal species authentication, the ribosome 16S rRNA gene (16S rRNA), the mitochondrial cytochrome c oxidase subunit I gene (COI), and the cytochrome b gene (cyt-b) have also been widely discussed (Giusti et al 2017).…”