Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.
ABSTRACT:The purpose of the present study was to give an overview of imported and traded gadoid fish species (Gadiformes) in the Czech Republic and to describe available methods for their authentication. Due to the increasing interest of customers in the purchase of buy fish meat and other seafood animals, it is necessary to have available analytical methods with discriminating power of respective fish species. With regard to different values and prices of various fish species, these may be adultered. Until recently, electrophoretic, chromatographic and immunological methods based on the analysis of proteins extracted from fish musculature seemed to be promising. Using these methods, various fish species can be identified in fresh, chilled and frozen products. However, they often fail in heat treated products. Molecular biology methods based on DNA analysis are more reliable and suitable for the analysis of fish products that have been heat treated during the production process.Keywords: cod fish; fish species identification; electrophoresis; PCR-RFLP; cytochrome b gene; food adulteration List of abbreviations AK = adenylate kinase; ARGK = arginine kinase; ATP = adenosine triphosphate; CE = capillary electrophoresis; CK = creatine kinase; CR = Czech Republic; DGGE = denaturating gradient gel electrophoresis; DHA = docosahexaen acid; DNA = deoxyribonucleotide acid; EPA = eicosapentaen acid; EU = European Union; FINS = forensically informative nucleotide sequencing; G 3-PD = glycerol 3-phosphate dehydrogenase; HPLC = high performance liquid chromatography; IEF = isoelectric focusing; LDH = lactate dehydrogenase; MDH = malate dehydrogenase; MS = mass spectrometry; mt cyt b = mitochondrial cytochrome b gene; Mw = molecular weight; NDKA = nucleoside diphosphate kinase A; NTSs = nontranscribed spacers; PCR = polymerase chain reaction; PCR-RAPD = polymerase chain reaction -random amplified polymorphic DNA; PCR-RFLP = polymerase chain reaction -restriction fragment length polymorphism; PCR-SSCP = polymerase chain reaction -single strand conformation polymorphism; pI = isoelectric point; RP-HPLC = reversed phased -high performance liquid chromatography; SDS-PAGE = sodium dodecyl sulphate -polyacrylamide gel electrophoresis; urea-IEF = urea isoelectric focusing; 2DE = two dimensional electrophoresis
The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food.
Fish species identification is important as the interest of consumers in sea-fish meat is increasing. The aim of this study was to determine hake species distribution on the Czech market by the PCR-RFLP and sequencing of mt cyt b, and to develop and optimise an alternative system for determination of hake species by sequencing and/or PCR-RFLP using Pan I sequence. Among 20 samples of hake obtained on the Czech market three species were identified: North Pacific hake (Merluccius productus), Argentine hake (Merluccius hubbsi) and South Pacific hake (Merluccius gayi). The approaches tested in our study represent a significant tool either for the differentiation of hake species from other gadoid species (PCR) or for intraspecies identification of different hake species (PCR-RFLP, sequencing). This knowledge can be applied in detection of fish species substitution within the consumers' rights protection.
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