Esther Carrera

Polymerase chain reaction-restriction fragment length polymorphism analysis of a short fragment of the cytochrome b gene for identification of flatfish species

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

Identification of Atlantic Salmon (Salmo salar) and Rainbow Trout (Oncorhynchus mykiss) by Using Polymerase Chain Reaction Amplification and Restriction Analysis of the Mitochondrial Cytochrome b Gene

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the identification of fresh and smoked samples of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Digestion of the 359-bp PCR product with the endonucleases EcoRV and TaqI yielded specific banding patterns for salmon and trout. This genetic marker can be very useful for detecting fraudulent substitution of the cheaper smoked trout for the more expensive smoked salmon.

Immunostick colorimetric ELISA assay for the identification of smoked salmon, trout and bream

A colorimetric ELISA using immunostick tubes has been developed for the rapid identification of smoked salmon (Salmo salar), trout (Oncorhynchus mykiss) and bream (Brama raii). The assay uses polyclonal antibodies raised in rabbits against muscle-soluble proteins of smoked salmon (anti-SSP), trout (anti-TSP) and bream (anti-BSP) and rendered species-specific by blocking them with the heterologous species of smoked fish. The blocked antibodies were used for the immunorecognition of the smoked fish samples bound to the paddles of immunostick tubes and the immunocomplex detected with peroxidase-conjugated goat anti-rabbit immunoglobulins. The blue colour developed by conversion of the peroxidase substrate allows unequivocal identification of S. salar, O mykiss and B. raii.

Genetic differentiation between sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides) by PCR–RFLP analysis of a 12S rRNA gene fragment

PCR-RFLP analysis was applied to the identification of two closely related flatfish species: sole (Solea solea) and Greenland halibut (Reinhardtius hippoglossoides). Amplification of DNA isolated from fish muscle samples was carried out using a set of primers flanking a region of 321 base pairs (bp) from the mitochondrial 12S rRNA gene. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for AciI and MwoI endonucleases. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with AciI and MwoI enzymes allowed the unequivocal identification of sole and Greenland halibut species.

Development of an enzyme-linked immunosorbent assay for the identification of smoked salmon (Salmo salar), trout (Oncorhynchus mykiss) and bream (Brama raii)

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of smoked meat from salmon ( Salmo salar ), trout ( Oncorhynchus mykiss ), and bream ( Erama raii ). The assay uses polyclonal antibodies raised in rabbits against soluble proteins of muscle from salmon (anti-SSP), trout (anti-TSP), and bream (anti-BSP) which are rendered species-specific by blocking them with the heterologous soluble muscle proteins. The blocked antibodies were used to detect the samples from smoked fish bound to the wells of a microtiter plate. Immunorecognition of polyclonal antibodies adsorbed to fish samples was made with goat anti-rabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic coversion of the substrate allowed clear species identification of smoked meat of salmon, trout, and bream.

PCR-RFLP of the mitochondrial cytochrome oxidase gene: a simple method for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss)

A DNA-based method (PCR-RFLP) has been developed for discrimination between Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). The polymerase chain reaction (PCR) was used for amplification of a 464 bp fragment of the mitochondrial cytochrome oxidase subunit II (COII) gene. Digestion of the products with endonucleases Nci I and Sau 3AI, followed by agarose gel electrophoresis of the digested products, yielded specific restriction profiles that enabled direct visual identification of the species analysed. This PCR-RFLP methodology allowed clear discrimination of Atlantic salmon and rainbow trout samples both in raw and smoked products. © 1999 Society of Chemical Industry

Indirect enzyme-linked immunosorbent assay for the identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides).

Polyclonal antibodies produced against soluble muscle protein extracts from sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides) were used in an indirect enzyme-linked immunosorbent assay for the specific identification of fillets from these flatfish species. The assay was performed in two different formats: microtiter plates and immunostick tubes. Immunorecognition of antibodies adsorbed to their specific fish samples was made with goat antirabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymatic conversion of the substrate allowed unequivocal identification of all flatfish species studied.

ELISA-based detection of mislabeled albacore (Thunnus alalunga) fresh and frozen fish fillets

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of albacore (Thunnus alalunga) and its differentiation from other less-valued scombrid species such as yellowfin tuna (Thunnus albacares), bullet tuna (Auxis rochei), atlantic bonito (Sarda sarda), bigeye tuna (Thunnus obesus), little tunny (Euthynnus alleteratus) and skipjack tuna (Katsuwonus pelamis). The assay uses polyclonal antibodies raised in rabbits against soluble muscle protein extract from fresh albacore. These polyclonal antibodies were rendered species-specific by blocking them with the heterologous soluble muscle proteins, allowing discrimination between fresh albacore and the rest of the scombrid species, except for yellowfin tuna. A total of 40 commercial albacore fresh and frozen fillets were analysed, revealing an incorrect labelling in 32.5% of the albacore samples. However, positive samples (67.5%) could be albacore or yellowfin tuna and should require a DNA assay as discriminatory technique.