Differential detection of small pelagic fish in Tunisian canned products by PCR-RFLP: An efficient tool to control the label information

Correct identification of fish species including their origin requires a compilation of recognized material to verify the conformity of the product with the labelling requirement. Sardine is among the internationally disputed example of species identification conflicts, with the lack of genetic reference material at the Southern part of the Mediterranean. Therefore a method was developed to discriminate between Tunisian small pelagic fish: Sardina pilchardus, Sardinella aurita and Engraulis encrasicolus. DNA extraction from fresh fish and from 12 canned sardine and 2 anchovy-type products, was followed by a PCR method that specifically amplifies a 252 bp fragment of the cytochrome b gene. The new sequences which were deposited in the NCBI GenBank, were searched against a nucleotide sequence database (GenBank) and phylogenetically analyzed with the MEGA software. Multiple alignments of 3 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned anchovy and sardine (<0.05), whereas mean interspecific variability was 0.23. A phylogenetic tree was constructed, and the calculated bootstrap values (BP, 71-100%) were used as indicators of the correct assignment of unknown canned samples to reference species. Postamplification digestion with HaeIII and ALuI restriction enzymes, yielded specific profiles that enabled anchovy to be distinguished from either of the sardine species. This PCR-RFLP was found to be reliable for species identification of canned fish products.

► We used PCR to amplify and sequence a short fragment of 252 bp of the mtDNA gene. ► We constructed a phylogenetic tree, the bootstrap values were calculated. ► Results showed that all sardine canned products did not match Sardina pilchardus. ► RFLP was used to differentiate S. pilchardus from Sardina aurita and Engraulis encrasicolus. ► Such procedure allowed the authentication of commercial canned sardine and anchovy.