A validated DNA metabarcoding approach for routine authentication of processed bivalves

Species mislabeling in commercial bivalves represents a growing concern for food authenticity. Given the diversity of traded bivalves, broad taxonomic coverage is essential for untargeted screening. To address this challenge, we developed and validated a DNA metabarcoding approach employing two PCR assays, a singleplex and a duplex, to amplify mitochondrial 16S rDNA fragments (160-203 bp) across seven families: Ostreidae, Pectinidae, Mytilidae, Pharidae, Veneridae, Glycymerididae, and Cardiidae. Taxa were identified at the species or genus level in 38 reference samples and four model food samples, with individual species detected at concentrations as low as 0.008-0.014% (w/w). All main and most minor components were detected in 40 DNA extract mixtures, with few false negatives, presumably resulting from primer-template mismatches causing amplification bias. A total of 70 commercial food products were analyzed. Considering both species substitution and adulteration, 29% of samples were determined to be mislabeled, with scallops being the most frequently affected family. These results highlight the method's utility for detecting species substitution and adulteration. This study presents the first DNA metabarcoding method targeting a broad taxonomic range of bivalves. The validated approach is well-suited for qualitative screening in routine food authentication and can support laboratories and regulatory agencies in enforcing international monitoring strategies.