Species Identification and Food Fraud Analysis in Commercial Fish Products Through DNA- based methods

Globalization of food supply chains has led to an increased uncertainty of the origin and safety of fish-based products. Barcoding can be used to validate the labelling of products and to trace their origin. “Fish fraud” has been discovered across the globe. Barcoding can also trace fish species as there can be human health hazards related to consumption of fish. The study evaluated the applicability of the mitochondrial genes cytochrome b (cytB), and cytochrome oxidase subunit I (COI) for the identification of fish and processed fish product by DNA barcoding. In the study, universal primers for mitochondrial cytB were used to discriminate fish species in raw and processed forms. The barcode primers were cross tested against collected fish product. In this study DNA barcoding was employed to identify fishery product collected from market and supermarket located in Apulia region (Southern Italy). We collected and analyses 90 samples for our study. For this project different varieties of fish samples were collected from different supermarkets and of different companies. DNA was isolated from all samples and amplified by PCR; the most intense amplified product was chosen for Sanger Sequencing. After sequencing, the sequences were matched with NCBI BLAST and FISH BOL. After obtaining the results species were identified and matched with the labelling of the products. Non-compliance between the species detected and the species declared in the label was detected in 10 out of 90 (11.1) % samples. The study provides further evidence of the need for increased traceability and assessment of food products authentication. Additionally, correct species denomination and traceability may increase the standard of management of hazards related to fish and food safety as well as ensuring product authenticity, providing reliable information to consumers. Another objective of the thesis was the development of loop-mediated isothermal amplification (LAMP) assay for rapid and direct screening of yellowfin tuna (Thunnus albacares) in commercial fish products. In this study, a loop mediated isothermal amplification (LAMP) assay was developed and validated targeting the mitochondrial cytochrome b gene for fast and direct detection of Thunnus albacares, which is a valuable species of tuna. The analytical specificity was confirmed using 18 target samples (Thunnus albacares) and 18 samples of non-target fish species. The analytical sensitivity of the LAMP assay was 540 fg DNA per reaction. In addition, a simple and direct swab method without time-consuming nucleic acid extraction procedures and the necessity for cost-intensive laboratory equipment was performed that allowed LAMP detection of Thunnus albacares samples within 13 minutes. Due to its high specificity and sensitivity, the LAMP assay can be used as a rapid and on-site screening method for the identification of Thunnus albacares, potentially providing a valuable monitoring tool for food authenticity control by the authorities