A COIBar-RFLP strategy for the rapid detection of Engraulis encrasicolus in processed anchovy products

The species identity on anchovy products was tested through COI mitochondrial DNA sequences analysis in 50 samples belonging to 20 different commercial lots, such as anchovy fillets in vegetal oil, canned anchovies and anchovy paste. Seven samples (14%) were found to be not from Engraulis encrasicolus meats, confirming concerns of species substitution. Conventional COI-DNA barcoding revealed the presence of four different species, E. encrasicolus, Engraulis japonicus, Sardinella aurita and Sardina pilchardus, in the processed products labeled as European anchovy. The DNA barcoding was then used in combination with PCR-RFLP method to investigate labeling accuracy in processed anchovy products and to unveil putative fish fraud involving the replacement of the European anchovy, E. encrasicolus, with less valuable Engraulidae and Clupeidae species. We applied a COIBar-RFLP (Cytochrome Oxidase I Barcode-Restriction Fragment Length Polymorphism) analysis that yielded differential patterns allowing the unambiguous discrimination of European anchovy from the other species tested. The proposed molecular strategy relies on the efficiency of COI as a DNA barcode and proved very efficient and less expensive than DNA sequencing strategies. This approach may be useful in routine controls, especially in cases of large-scale sample screening.