Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh, marinated and cooked products

A conventional and a realtime multiplex PCR were developed to detect fraudulent substitutions of Bianchetto (juvenile form of Sardina philcardus) and Rossetto (Aphia minuta) with Icefish (Neosalanx spp.), which show similar morphological characteristics. Since it is the major by-catch species, Engraulis encrasicolus was also included in the analytical procedure. A common reverse primer and forward species-specific primer were designed on the mitochondrial cytochrome b gene to amplify sequences of different lengths by conventional PCR. Specific peaks were therefore also provided after melting temperature analysis in real-time PCR, thus enabling each species to be clearly differentiated. The two PCR methods were validated on fresh and processed products after preparing four typical dishes: two marinades (from raw or lightly boiled fish), a pasta sauce, and batter-fried fish cakes. All samples were correctly identified, although there was some DNA degradation after processing.