Food fraud and mislabeling: development of a Real-Time PCR for rapid identification of Octopus vulgaris

The consumption of seafood products, in particular cephalopods, and their worldwide commercialization is increasing in last decade. The excessive exploitation of this type of resources is going to deplete seas and oceans, moreover the most appreciated species like Octopus vulgaris are widely substituted with others ( e.g. other Octopus and Amphioctopus species). The aim of this work was the comparison among barcoding (COI) and an in-house Real-Time PCR approaches for the identification of this fraud on products sold in north east Italy. The identification method was developed through an EvaGreen Real-Time PCR, as faster and cheaper technique in comparison of the classical barcoding approach (2 day vs. 4-5 day). One hundred of samples from different FAO areas (46 Octopus vulgaris and 56 non-O. vulgaris) were applied for the experimental set-up. This method can distinguish between Octopus vulgaris and other cephalopods species through the study of the amplification curves. Sensitivity and specificity tests on all samples showed two different clusters according to the ct (cycle threshold); the 20 ≥ Ct ≤ 30 levels identified O. vulgaris samples, while a <30 ct highlighted non-octopus samples. The non-octopus cluster (ct > 30) need a subsequent barcoding analysis for a detailed identification of genus and species using the same DNA extracts. After the method optimization, a market survey was conducted in order to collect data about commercial fraud. Seventy-seven prepared (e.g. cooked in oil) and unprepared products from different markets of 4 provinces (north-east Italy) were analyzed with both methods. Survey data shown that 51.2% of products labeled as Octopus vulgaris were substituted with non-Octopus vulgaris species (commercial fraud), 36.2% of products declared as other octopus species are mislabeled for a total of fraud/mislabeling estimated at 44.15%. The EvaGreen Real-Time PCR showed a specificity and sensitivity of 100% and 80% respectively. Furthermore there was a substantial agreement between Real-Time PCR and barcode methods (K Cohen value = 0.86). In conclusion, this technique allowed a simple and economic method to confirm Octopus vulgaris (< 48 h ) according to the variability of the samples tested and their different provenience areas. The EvaGreen Real-Time PCR could be a routinely system in diagnostic laboratories.