Differentiation of closely related species by DNA hybridization

The specificity of genomic DNA probes for species differentiation by slot blot hybridization has been investigated. Experiments have been performed investigating species differentiation between monkey and human and between cattle, goat and sheep.

It is demonstrated that cross hybridization between probe and DNA sequences from closely related species is reduced by addition of unlabelled DNA from the cross hybridizing species. Quantitative species differentiation is shown possible for all species although with different detection limits. For differentiation between cattle and sheep or goat the detection limits are determined to less than 0·01% whereas the detection limits for differentiation between the closely related species sheep and goat are about 10%.     

Differentiation of Sparidae species by DNA sequence analysis,PCR-SSCP and IEF of sarcoplasmic proteins

The increasing global trade of fishery and aquaculture products makes it necessary to develop methods for species identification in case of fish fillets or other highly processed seafood with external morphological characteristics (e.g. gills, fins) of the original fish being removed.     

Differentiation of cattle species in beef by PCR-RFLP of mitochondrial and satellite DNA

Methods currently used for the identification of the species origin of meat or tissue samples have not been validated for other bovine species than taurine cattle or water buffalo. These methods also do not discriminate between the different bovine species that are used as source of beef. Here, we describe two complementary methods for detection and differentiation of bovine species, which are based on mutations in mitochondrial DNA and centromeric satellite DNA, respectively. The analysis of satellite DNA is especially relevant for the identification of animals that are of hybrid origin.     

Applicability of DNA barcode for identification of fish species

DNA barcoding is a species identification technique, which uses a very short DNA sequence from a region of approximately 650 base-pairs in the 5'-end of the mitochondrial cytochrome c oxidase subunit I gene as a marker to identify species of mammals and fishes. The applicability of DNA barcoding for identification of fish species consumed in Japan was studied. Among thirty-one fresh or processed fishes were obtained from the market, two samples could not be identified due to lack of data in the Barcode of Life Data (BOLD) database. However, BLAST-search of 16S rRNA genes in the National Center for Biotechnology Information (NCBI) database and the PCR-RFLP method published by the Food and Agricultural Materials Inspection Center (FAMIC) were found to be applicable to identify these 2 fishes. The results show that the DNA barcoding technique is potentially useful as a tool for confirming the proper labeling of fish species in the Japanese market.     

Differentiation of sturgeon species by PCR-RFLP

A method for identification of sturgeon species in caviar has been developed based on the amplification of a region of the mitochondrial genome (tRNA^Glu/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several types of sturgeon caviar the obtained 462bp long PCR-products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphisms (RFLP). The method is suitable to differentiate between 10 species of Acipenser and Huso originating from Europe and Asia.     

Identification of Atlantic hake species by a simple PCR-based methodology employing microsatellite loci

Three Atlantic hake species (Merluccius merluccius, M. bilinearis, and M. hubbsi) were PCR typed for two microsatellite loci. A blind survey of the markers in samples of the three species determined the suitability of microsatellite loci for identification of hake product. All the analyzed samples were correctly assigned to the corresponding species. Typing of processed products (fish fingers, preprocessed frozen pieces) employing an automated sequencer was successful. This simple (PCR + fragment size) automated determination method is faster than any other method yet described for identification of hake commercial products.     

Tetrodotoxin detection and species identification of pufferfish in retail roasted fish fillet by DNA barcoding in China

This study identifies the pufferfish species and detects tetrodotoxin (TTX) in roasted fish fillet samples collected in Beijing, Qingdao and Xiamen, China. The cytochrome c oxidase I (COI) gene was used as the target gene for identification of the pufferfish species in the samples. Enzyme-linked immunosorbent assay (ELISA) screened the TTX levels in samples that had been detected as containing pufferfish by DNA barcode. A total of 125 samples were identified by DNA barcodes; 32 (26%) samples contained pufferfish composition and, among them, 26 (81%) were the highly toxic species Lagocephalus lunaris. All 32 samples containing the pufferfish composition were positive for TTX with levels ranging from 100 to 63 800 ng g^–1. Most of the 32 samples contained the highly toxic L. lunaris. Based on the results, we suggest that the monitoring of roasted fish fillet should be strengthened and the processing procedures should be standardised to minimise TTX poisoning caused by pufferfish.     

Identification of fish species in smoked fish products by electrophoresis and isoelectric focusing

Summary Different species of fresh fish and smoked fish products were analysed by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis and isoelectric focusing. It was possible to differentiate and identify species of fish in smoked samples and draw some conclusions about the type of smoking process employed.

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Artbestimmung in geräucherten Fischprodukten durch Elektrophorese und isoelektrischer Focussierung

Zusammenfassung Proben von verschiedenen Arten von Frischfisch und von geräucherten Fischprodukten wurden mit Hilfe von SDS-Polyacrylamid-Gelektrophorese und isoelektrischer Focussierung analysiert. Es war möglich in den geräucherten Proben zwischen verschiedenen Fischarten zu unterscheiden und die Fischart zu bestimmen. Ebenso waren Rückschlüsse auf den angewendeten Räucherungsprozeß möglich.

     

Identification of fish species by testing the minced-meat tissue

The detection is based on the fact that the sarcoplasmatic protein fractions of fish meat behave differently when subjected to starch-gel electrophoresis. This makes it possible to demonstrate the presence of another fish species in minced fish meat stated to contain but one fish species. In case of fish stuffings consisting of two fish species, fish identification is simple. Under favourable circumstances, it is also possible to detect and identify foreign additions in a mixture of more than two fish species.     

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.     

Identification of Aloe species by random amplified polymorphic DNA (RAPD) analysis

Juice and integument of leaves of 3 Aloe species, Aloe vera, A. ferox and A. africana, are not allowed to be used as food according to the Pharmaceutical Affairs Law in Japan. On the other hand, whole leaves of A. arborescens can be used as food. The present study was designed to distinguish Aloe species by random amplified polymorphic DNA (RAPD) analysis. DNA was isolated from fresh and dried leaves of the 4 Aloe species. Five out of 32 different 10-mer primers examined were useful for analysis. By comparison of the characteristic bands of PCR products on agarose gel, it was possible to distinguish the 4 species. Thus, the botanical species of Aloe in commercial food products can be identified by RAPD analysis.     

Differentiation of animal species in food by oligonucleotide microarray hybridization

Oligonucleotide microarray hybridization analysis of polymerase chain reaction (PCR) products from the mitochondrial cytochrome b gene DNA was applied to identify different animal species in meat and cheese food samples. A pair of universal primers binding to conserved regions of the vertebrate mitochondrial cytochrome b gene was used to amplify a 377 bp fragment with internal regions of high inter-species variability. PCR products of cattle, pig, chicken, turkey, sheep and goat were unequivocally identified by hybridization with species-specific probe sequences immobilized on an oligonucleotide microarray. In meat samples, 0.1% admixtures of beef or chicken meat were still detectable. By using this new PCR-based DNA chip hybridization for the analysis of 24 commercial food samples from routine control, the simultaneous species composition of mixtures with up to four different species could be determined in a single experiment. The results agreed well with those from the reference methods performed at the local food control authority, which are a combination of enzyme-linked immunosorbent assay (ELISA), species-specific PCR and PCR–RFLP (restriction fragment length polymorphism). Thus, the DNA chip hybridization analysis of cytochrome b PCR products offers a new way for rapid and sensitive species differentiation in food.     

Identification of fish species by 5S rRNA gene amplification

The 5S ribosomal RNA is a very suitable target for easy, rapid and inexpensive fish species identification due to its structure, consisting of a conserved region followed by a species-specific noncoding region called ‘nontranscribed spacer’. We have exploited this species-specificity in length and sequence to discriminate among fish species which can be subjected to substitution in the fish markets. After sequencing and alignment of the corresponding portions of the 5S rRNAs of different fish species, we have designed the primer pairs necessary for PCR amplification on the DNA traits which most diverged and a primer pair on conserved regions. Our results have shown the feasibility, simplicity and reliability of the proposed approach for the detection of mislabelling or fraudulent substitution of fish species.     

Identification of some Aegean fish species by native isoelectric focussing

In this study, native protein patterns and band analysis of 20 Aegean and 2 North Atlantic fish species were obtained by isoelectric focussing (IEF) by determining their protein (in extract), TMA and TVB-N contents. Band specifications of the samples have been determined for comparing the different species of fish used as raw material. Aegean Sea has around 40 commercial fish species and 20 of them were taken into investigation. Building up a database for characterization of these species may help to find a way for further studies.     

Identification of fish species in dried fish products by immunostaining using anti-myosin light chain antiserum

In order to immunologically detect fish species used in boiled and dried fish products (dried fish sticks), the availability of antiserum against myosin light chains was examined. After dissolving the samples in the presence of 8 M urea and 1% sodium dodecyl sulfate (SDS), the solubilized matters were applied to SDS–polyacrylamide gel electrophoresis and electroblotted onto PVDF membranes. Subsequently, protein bands concerned were stained using anti-myosin light chain (alkali light chain 1) rabbit antiserum and horseradish peroxidase-conjugated anti-rabbit immunoglobulin. Most of the fish species could be identified by fingerprinting of immunostained patterns even after drastic processing, even though the protein staining patterns were not clear enough for species identification.     

Production and quality of fish fingers from different fish species

Abstract Production of fish fingers was achieved by using fish species such as sardine (Sardina pilchardus, Walbaum, 1792), whiting (Merlangius merlangus, Linnaeus, 1758) and pike perch (Sander lucioperca, Linnaeus, 1758). Quality changes of battered fish patties during a period of 8 months at –18 °C were investigated. According to the results of microbiological and chemical analysis, fish fingers were found to be within acceptable limits during frozen storage for 8 months. However, sensory analysis showed that, at the end of the frozen storage, fish fingers made from sardine could not be consumed because of rancidity.     

DNA-based identification of fish species implicated in Puffer fish poisoning

A method for identification of fish species using three different mitochondrial DNA regions, 16S rRNA, cytochrome b and cytochrome c gene fragments, was investigated. The combined use of all three regions enabled reliable species identification in not only raw fish, but also dried, seasoned and boiled fish, products. Furthermore, the method was applicable even to vomitus from a patient involved in a puffer fish poisoning incident. However, further improvement is necessary to discriminate between closely related species such as Takifugu rubripes and T. chinensis, because they showed close similarity in the nucleotide sequences in the three gene fragments analyzed in this study.     

Improvement of chromosomal DNA extraction from different yeast species by analysis of single preparation steps

A modified procedure is proposed for chromosomal DNA extraction based on a cell-wall lytic enzyme never applied before in pulsed field gel electrophoresis. Protoplasting efficiency is retained under very challenging conditions for enzyme activity, such as those required for non-Saccharomyces yeasts often characterized by cell walls highly resistant to lysis.