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Food Business Operators (FBOs) rely on laboratory analysis to ensure seafood traceability. DNA barcoding and Forensically Informative Nucleotide Sequencing may represent a support within self-checking programs finalized to suppliers’ qualification and products identity certification. The present study aimed at verifying the usefulness of a decisional procedure (decision tree) set up at the FishLab (Department of Veterinary Sciences, University of Pisa, Italy) for seafood species identification by DNA analysis, to cope with FBOs’ needs. The decision tree was applied to the analysis of 182 seafood (fish and molluscs) products, conferred to the FishLab by different FBOs between 2014 and 2015 as result of their self-checking activities. The analysis relied on a standard COI gene fragment eventually integrated by the analysis of alternative or supportive molecular targets (cytb and 16S rRNA). It also included a mini-DNA barcoding approach for processed products. Overall, 96.2% of the samples were unambiguously identified at species level using the elective target alone (92.4%) or a multitarget approach (3.8%). The lack of species identification (3.8%) was attributable to the absence of reference sequences or to the low resolution of the molecular targets. Nonetheless, all the molecular results were deemed adequate to evaluate the sample’s compliance to the label information. Noncompliances were highlighted in 18.1% of the products. The protocol was proven as an effective supportive tool for the seafood identity verification within the supply chain self-checking activities. In addition, a considerable fraud rate was confirmed and the species most frequently involved in substitution were pointed out.
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