Identification and semi-quantification of Atlantic salmon in processed and mixed seafood products using Droplet Digital PCR (ddPCR).

Fishery products are often subject to substitution fraud, which is hard to trace due to a lack of morphologic traits when processed, gutted, or decapitated. Traditional molecular methods (DNA barcoding) fail to identify products containing multiple species and cannot estimate original weight percentages. As a proof of concept, an Atlantic salmon (Salmo salar) specific ddPCR assay was designed to authenticate mixed food products. The method proved to be specific and able to accurately quantify S. salar when using DNA extracts, even in the presence of DNA from closely related salmon species. The ddPCR estimates correlated well with the percentage of S. salar in artificially assembled tissue mixtures. The effect of common salmon processing techniques (freezing, smoking, poaching with a "Bellevue" recipe and marinating with a 'Gravad lax' recipe) on the ddPCR output was investigated and freezing and marinating appeared to lower the copies detected by the ddPCR. Finally, the assay was applied to 46 retail products containing Atlantic or Pacific salmon, and no indications of substitution fraud were detected. The method allows for a semi-quantitative evaluation of the S. salar content in processed food products and can rapidly screen Atlantic salmon products and flag potentially tampered samples for further investigation.

A fast HRMA tool to authenticate eight salmonid species in commercial food products.

Atlantic and Pacific salmon are frequently consumed species with very different economic values: farmed Atlantic salmon is cheaper than wild-caught Pacific salmons. Species replacements occur with the high valued Pacific species (Oncorhynchus keta, O. gorbuscha, O. kisutch, O. nerka and O. tshawytscha) substituted by cheaper farmed Atlantic salmon (Salmo salar) and Atlantic salmon by rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta). Here we use High-Resolution Melting Analysis (HRMA) to identify eight salmonid species. We designed primers to generate short amplicons of 72 and 116 bp from the fish barcode genes CO1 and CYTB. The time of analysis was under 70 min, after DNA extraction. Food processing of Atlantic salmon (fresh, "Bellevue", "gravadlax", frozen and smoked) did not impact the HRMA profiles allowing reliable identification. A blind test was conducted by three different institutes, showing correct species identifications irrespective of the laboratory conducting the analysis. Finally, a total of 82 retail samples from three European countries were analyzed and a low substitution rate of 1.2% was found. The developed tool provides a quick way to investigate salmon fraud and contributes to safeguard consumers.

A high-quality genetic reference database for European commercial fishes reveals substitution fraud of processed Atlantic cod (Gadus morhua) and common sole (Solea solea) at different steps in the Belgian supply chain.

Seafood is an important component of the human diet. With depleting fish stocks and increasing prices, seafood is prone to fraudulent substitution. DNA barcoding has illustrated fraudulent substitution of fishes in retail and restaurants. Whether substitution also occurs in other steps of the supply chain remains largely unknown. DNA barcoding relies on public reference databases for species identification, but these can contain incorrect identifications. The creation of a high quality genetic reference database for 42 European commercially important fishes was initiated containing 145 Cytochrome c oxidase subunit I (COI) and 152 Cytochrome b (cytB) sequences. This database was used to identify substitution rates of Atlantic cod (Gadus morhua) and common sole (Solea solea) along the fish supply chain in Belgium using DNA barcoding. Three out of 132 cod samples were substituted, in catering (6%), import (5%) and fishmongers (3%). Seven out of the 41 processed sole samples were substituted, in wholesale (100%), food services (50%), retailers (20%) and catering (8%). Results show that substitution of G. morhua and S. solea is not restricted to restaurants, but occurs in other parts of the supply chain, warranting for more stringent controls along the supply chain to increase transparency and trust among consumers.