A fish market survey using a novel PCR-sequencing-based protocols for the identification of commercial significant fish species

Abstract

This study developed a simple, specific, and affordable PCR-sequencing-COI gene-based protocol for the simultaneous identification of some important commercial fish species: Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasianodon hypophthalmus, Epinephelus spp. For this study, a local market survey on fish was carried out to evaluate the application of labelling laws and to detect fraudulent actions using the developed PCR protocols. Ten specimens of each fish species of interest were obtained from wholesale fishery plants and were utilized for the protocol development. DNA was extracted from the individual samples and quantified. DNA isolates were subjected to end-point PCR and the PCR products were sequenced. For the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software “Primer-BLAST” to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within the COI gene for M. merluccius, L. niloticus, G. morhua, R. pretiosus, P. hypophthalmus, Epinephelus spp., respectively. Single PCR was performed using DNA isolates and developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected sequences of the COI gene and showed a similarity ranging from 99 to 100%. Among 43 samples obtained for the survey, 19 (44.2%) were mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and fish marketplaces and 1 (2.32%) from hypermarket stores. Among fish samples purchased at local fisheries and fish marketplaces, fraudulent actions were observed more frequently in fish slices (100%) than fish fillets (65%). Regarding fish fillets, out of four samples labelled as grouper, three were L. niloticus and one P. hypophthalmus. Two fillets marketed as cod were substituted with L. niloticus. Five samples labelled as “fillet” and two samples labelled as “perch” were identified as P. hypophthalmus. Regarding fish slices, all samples marketed as grouper (E. marginatus) were slices of R. pretiosus. The single case of mislabelling detected from fishery products purchased at hypermarket stores was a sample of “Spinycheek grouper” (Epinephelus diacanthus) that was indicated on label as “Grouper” (Epinephelus marginatus). In summary, our work highlights the need for continuous surveillance of the commercialization of fishery products, to reduce the number of fraud cases that happen in the market. Furthermore, our protocols based on PCR techniques could be useful for quality control of fresh finfish and to strengthen controls on the most frequent fraudulent actions of marketed fishery products.

Keywords

• fishery products
• fish frauds
• Multiplex PCR
• COI gene