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.Purpose: To evaluate the influence of haemorheopheresis on anatomical and functional findings in patients with soft-drusen maculopathy. Methods: We investigated 29 eyes (16 patients) and randomized 25 eyes (16 controls) with soft-drusen maculopathy [soft, confluent and reticular drusen, drusenoid retinal pigment epithelium detachment (RPED)]. Each patient received a series of eight haemorheophereses (cascade filtration of 1.5 plasma volume) within 10 weeks. The patients were followed up using Early Treatment Diabetic Retinopathy Study (ETDRS) charts, optical coherence tomography, fluorescein angiography, electroretinography and measurements of pulsed ocular blood flow. Results: After the procedures, there was a substantial reduction in rheologically active substances [lipoproteins, immunoglobulin M (IgM), fibrinogen], plasma and blood viscosity. At the 1.5-year follow-up, we noticed soft drusen absorption; reattachment of drusenoid RPED and stabilization or improvement of visual acuity occurred in 72% of patients in comparison to only 39% of patients in the control group. Full-field electroretinograms showed significantly higher scotopic activity of treated patients in comparison with the control group, and mainly insignificant differences in photopic activity between both groups. Despite the significant increase of activity in the paramacular retina in treated patients, the differences in amplitudes of multifocal electroretinography (mfERG) average responses were insignificant between groups. Conclusion: Haemorheopheresis seems to be capable of changing the activity of promoters of the natural course of soft-drusen maculopathy, its development and progression. Visual acuity and electrical activity of the retina can be stabilized or even improved. The therapy has been shown to be effective and safe.

Pospiech M., Tremlová B., Renčová E., Randulová Z. A functional immunohistochemical method for soya proteins detection was developed. The procedure is based on the avidin-biotin complex (ABC) method that attains sufficient sensitivity. The method was verified by the analysis of the model samples of different forms of soya additives containing various concentrations of soya isolate. The detection limit of soya present in the model samples was 0.5%. Different possibilities of the background staining were tested. The best results were obtained with the background staining according to Calleja. The results were confirmed by the accredited indirect ELISA method. The method allows the identification of various forms of soya proteins such as isolates, texturates, concentrates, and flour.

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The conventional polymerase chain reaction (PCR) method to detect the major allergenic protein parvalbumin beta 2 of Atlantic herring (Clupea harengus) and Pacific herring (Clupea pallasii) was developed. The specific set of primers for the amplification of the partial genomic sequence of the pvalb 2 gene encoding the main fish allergen of both herrings was designed and applied to the investigation of 24 commercial fish products. The targeted amplicon size was 189 bp of pvalb 2 gene of Atlantic herring and Pacific herring. As the internal amplification control, the DNA of 18S rRNA gene for eukaryotes (141 bp) was successfully used. The specificity of designed primer pair using 26 various fish species was assessed. The intrinsic detection limit was 10 pg µl(-1) of the present specific DNA. Atlantic herring or Pacific herring allergenic parvalbumins were detected in 22 investigated fish products in conformity with the package declaration. Two fish products were negative in spite of the declaration. The proposed PCR method is specific enough and can be used for the detection of Atlantic and Pacific herrings' major allergen parvalbumin beta 2 in fish food products.

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

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.

Due to the menace of transmission of spongiform encephalopathies, feed components intended for ruminant nutrition must be checked for the presence of ruminant-derived materials. A sensitive method for the identification of bovine- and ovine- and also swine- and chicken-specific mitochondrial DNA sequences based on Polymerase Chain Reaction (PCR) has been developed. The specificity of the primers for PCR has been tested using samples of DNA of other vertebrate species, which may also be present in rendering plant products intended for feed manufacture. The method allows the detection in concentrate mixtures of 0.01% of the target species derived material. The identity of a sample containing 0.1% of bovine, ovine, swine, and chicken meat-and-bone meal has further been confirmed by sequencing.