Differenzierung von Pangasius (Pangasius hypophthalmus) und Asiatischem Rotflossenwels (Hemibagrus wyckioides) durch Protein- und DNA-Analyse

In the last years farmed Pangasius (Tra-Pangasius, Pangasius hypophthalmus) from Vietnam has reached a considerable market share, whereas aquaculture of Asian Redtail Catfish (Hemibagrus wyckioides) is in its infancy. Recently it has been detected by food control authorities in Hamburg, that Pangasius fillets have been mislabelled and sold as fillets produced from Asian Redtail catfish. The necessity to improve the analytical methods for differentiation of Pangasius and Redtail Catfish prompted us to evaluate the suitability of isoelectric focusing (IEF) and DNA-analysis for identification of the two species. IEF of water soluble proteins was found to be a fast, reliable and economical method for differentiation of raw fillets of Pangasius and Redtail Catfish, as long as reference material is available. PCR-based DNA analysis was performed as follows: (i) amplification of a 464 bp segment of the cytochrome b gene; (ii) sequencing of the PCR product; (iii) comparison of the sequence with entries in GenBank using BLAST. The sequences of both species differed considerably, allowing the unequivocal differentiation between P. hypophthalmus and H. wyckioides. Kurzfassung Pangasius (Schlankwels, Tra-Pangasius, Pangasius hypophthalmus) hat sich innerhalb weniger Jahre zu einem bedeutenden Zuchtfisch entwickelt, während die Aquakultur des Asiatischen Rotflossenwelses (Hemibagrus wyckioides) in Vietnam noch in einem relativ kleinen Maßstab stattfindet. Kürzlich wurde von der Lebensmittelüberwachung in Hamburg nachgewiesen, dass im Handel erhältliche Filets mit der Deklaration „Rotflossenwels“ aus Pangasius hergestellt worden waren. Vor diesem Hintergrund wurden zwei Methoden auf ihre Eignung zur Differenzierung von Pangasius und Rotflossenwels geprüft. Es zeigte sich, dass sowohl die isoelektrische Fokussierung (IEF) wasserlöslicher Proteine als auch die PCR-basierte DNA-Analyse zur Unterscheidung beider Arten gut geeignet ist. Die IEF stellt eine schnelle und kostengünstige Untersuchungsmethode dar, die allerdings Referenzmaterial benötigt. Mit Hilfe der PCR (Polymerase-Kettenreaktion) wurde ein Abschnitt des Cytochrom b-Gens vervielfältigt und sequenziert. Die Sequenzen von P. hypophthalmus und H. wyckioides wiesen beträchtliche Unterschiede auf. Es wird diskutiert, wie sich durch Vergleich dieser Sequenzen mit Einträgen in Gendatenbanken unbekannte Proben beider Arten sicher zuordnen lassen.

Differenzierung von Kammmuscheln durch DNA-Analyse

Abstract In the last years scallops have reached a considerable popularity and the import of scallops into the EU has increased about 20 % over the last fi ve years from some 50.000 t to nearly 63.000 t in the year 2010. Scallops are fi shed or farmed, and traded as fresh or deep frozen product. Recently investigation of scallop products of various origins by determining the species using molecular biological techniques showed that the species had been mislabelled in a considerable proportion of samples. Determination of the species was performed by PCR-based DNA-analysis of mitochondrial DNA followed by (i) sequencing the PCR product and (ii) comparison of the DNA sequence with entries in GenBank using BLAST. The deduced sequences of the analysed samples were considerably different from each other allowing the unambiguous assignment of samples to a certain species. Kurzfassung Die Nachfrage von Kammmuscheln in der EU hat in den letzten fünf Jahren erheblich zugenommen. Der Import stieg von knapp 53.000 t im Jahr 2005 um 20% auf annähernd 63.000 t im Jahr 2010. Gehandelt werden Kammmuscheln sowohl als frische als auch als Tiefkühlware aus Wildfängen und Aquakultur. Untersuchungen von Kammmuschel-Proben aus verschiedenen Ursprungsländern und Bestimmung der Spezies auf molekularbiologischer Basis zeigten, dass ein erheblicher Anteil der Proben falsch deklariert war. Die Bestimmung der Spezies erfolgte durch Vervielfältigung eines Abschnitts des 16S rRNA Gens durch Polymerase- Kettenreaktion (PCR), anschließender Sequenzanalyse der PCR-Produkte und Vergleich der DNA Sequenzen untereinander und mit Dateneintragungen in GenBank. Die DNA-Sequenzen der ermittelten Abschnitte der 16S rRNA der Proben unterschieden sich erheblich voneinander und erlaubten eine eindeutige Zuordnung zu jeweils einer Spezies.

Identification of larval sea basses (Centropristis spp.) using ribosomal DNA-specific molecular assays

The identification of sea bass (Centropristis) larvae to species is difficult because of similar morphological characters, spawning times, and overlapping species ranges. Black sea bass (Centropristis striata) is an important fishery species and is currently considered to be overfished south of Cape Hatteras, North Carolina. We describe methods for identifying three species of sea bass larvae using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) assays based on species-specific amplification of rDNA internal transcribed spacer regions. The assays were tested against DNA of ten other co-occurring reef fish species to ensure the assay's specificity. Centropristis larvae were collected on three cruises during cross-shelf transects and were used to validate the assays. Seventy-six Centropristis larva were assayed and 69 (91%) were identified successfully. DNA was not amplified from 5% of the larvae and identification was inconclusive for 3% of the larvae. Those assays can be used to identify sea bass eggs and larvae and will help to assess spawning locations, spawning times, and larval dispersal.

Rapid identification of European (Anguilla anguilla) and North American eel (Anguilla rostrata) by polymerase chain reaction.

A rapid and cost effective DNA test is described to identify European eel (Anguilla anguilla) and North American eel (Anguilla rostrata). By means of polymerase chain reaction (PCR) technique parts of the mitochondrial cytochrome b gene are amplified with species specific primers which are designed to produce PCR fragments of different characteristic sizes for European and American eel. The size differences can easily be made visible by agarose gel electrophoresis

Fish species identification in Canned Tuna by DNA Analysis (PCR-SSCP)

DNA in canned tuna is degraded into short fragments of a rew hundred base pairs. The polymerase chain reaction (PCR) was used to amplify short sequences of mitochondrial DNA, which were denatured and analysed by polyacrylamide gel electrophoresis (native PAGE) for detection of single strand conformation polymorphisms. Species specific patterns of DNA bands were obtained for a number of tuna and bonito species. DE: In Thunfischkonserven liegt die DNA in Form kurzkettiger Fragmente von wenigen Hundert Basenpaaren Länge vor. Mit Hilfe der Polymerase-Kettenreaktion (PCR) wurden kurze Sequenzen der mitochondrialen DNA vervielfältigt. Anschließend wurde die gebildete DNA in Einzelsträngen überführt, die durch eine native Polyacrylamidgel-Elektrophorese (PAGE) aufgetrennt wurde. Für eine Reihe von Thunfischen und Boniten ergaben die Einzelstränge artspezifische Bandenmuster, die auf unterschiedliche Konformationen der DNA-Stränge der einzelnen Fischarten zurückzuführen sind.

Identifizierung der Welsart in Filetware durch Protein- und DNA-Analyse

Zusammenfassung Zur Identifizierung der folgenden vier Welsarten bzw. zwei Hybriden (Clarias gariepinus, Pangasius hypophthalmus, Pseudoplatystoma spp., Silurus glanis, Claresse® und Melander®) wurden die isolektrische Fokussierung (IEF) der wasserlöslichen Muskelproteine und die Polymerase-Kettenreaktion (PCR) zur Vervielfältigung und Sequenzierung eines Abschnittes aus dem Cytochrom b – Gen eingesetzt. Die IEF ergab artspezifische Proteinmuster mit hitzestabilen Proteinbanden im anodalen Gelbereich. Der afrikanische Wels (C. gariepinus) und das Hybriderzeugnis Melander® wiesen das gleiche Proteinmuster auf. Mittels DNA-Analyse ließen sich die Welsarten anhand ihrer Cytochrom b Gensequenzen eindeutig identifizieren. Auch hier zeigte der Welshybrid Melander® ein identisches Ergebnis wie der afrikanische Wels. Die Schwierigkeiten der Identifizierung von Tigerwelsen südamerikanischer Herkunft aus der Gattung Pseudoplatystoma werden diskutiert. Abstract Isoelectric focusing (IEF) of water soluble proteins and PCR-based DNA- analysis were used to differentiate between four catfish species (Clarias gariepinus, Pangasius hypophthalmus, Pseudoplatystoma spp., Silurus glanis) and two hybrids Claresse® and Melander®. Specific protein patterns have been obtained for all species and Claresse®, but in case of Melander® the identical pattern was observed as for the African catfish Clarias gariepinus. By sequencing the PCR products and application of BLAST, authenticity of the different catfish samples was confirmed. The cytochrome b gene sequences of Melander® and African catfish were identical. The difficulties of identifying catfishes of the genus Pseudoplatystoma are discussed.

Surface mucous as a source of genomic DNA from Atlantic billfishes (Istiophoridae) and swordfish (Xiphiidae)

Procedures for sampling genomic DNA from live billfishes involve manual restraint and tissue excision that can be difficult to carry out and may produce stresses that affect fish survival. We examined the collection of surface mucous as a less invasive alternative method for sourcing genomic DNA by comparing it to autologous muscle tissue samples from Atlantic blue marlin (Makaira nigricans), white marlin (Tetrapturus albidus), sailfish (Istiophorus platypterus), and swordfish (Xiphias gladius). Purified DNA from mucous was comparable to muscle and was suitable for conventional polymerase chain reaction, random amplified polymorphic DNA analysis, and mitochondrial and nuclear locus sequencing. The nondestructive and less invasive characteristics of surface mucous collection may promote increased survival of released specimens and may be advantageous for other marine fish genetic studies, particularly those involving large live specimens destined for release.

Microbial community analysis of Acropora palamata mucus swabs, water and sediment samples from Hawksnest Bay, St. John, U.S. Virgin Islands

Colonies of the scleractinian coral Acropora palmata, listed as threatened under the US Endangered Species Act in 2006, have been monitored in Hawksnest Bay, within Virgin Islands National Park, St. John, from 2004 through 2010 by scientists with the US Geological Survey, National Park Service, and the University of the Virgin Islands. The focus has been on documenting the prevalence of disease, including white band, white pox (also called patchy necrosis and white patches), and unidentified diseases (Rogers et al., 2008; Muller et al., 2008). In an effort to learn more about the pathologies that might be involved with the diseases that were observed, samples were collected from apparently healthy and diseased colonies in July 2009 for analysis. Two different microbial assays were performed on Epicentre Biotechnologies DNA swabs containing A. palmata coral mucus, and on water and sediment samples collected in Hawksnest Bay. Both assays are based on polymerase chain reaction (PCR) amplification of portions of the small rRNA gene (16S). The objectives were to determine 1) if known coral bacterial pathogens Serratia marcescens (Acroporid Serratiosis), Vibrio coralliilyticus (temperature-dependent bleaching, White Syndrome), Vibrio shiloi (bleaching, necrosis), and Aurantimonas coralicida (White Plague Type II) were present in any samples, and 2) if there were any differences in microbial community profiles of each healthy, unaffected or diseased coral mucus swab. In addition to coral mucus, water and sediment samples were included to show ambient microbial populations. In the first test, PCR was used to separately amplify the unique and diagnostic region of the 16S rRNA gene for each of the coral pathogens being screened. Each pathogen test was designed so that an amplified DNA fragment could be seen only if the specific pathogen was present in a sample. A positive result was indicated by bands of DNA of the appropriate size on an agarose gel, which separates DNA fragments based on the size of the molecule. DNA from pure cultures of each of the pathogens was used as a positive control for each assay.

Nuclear and mitochondrial DNA markers for specific identification of istiophorid and xiphiid billfishes

Independent molecular markers based on mitochondrial and nuclear DNA were developed to provide positive identification of istiophorid and xiphiid billfishes (marlins, spearfishes, sailfish, and swordfish). Both classes of markers were based on amplification of short segments (<1.7 kb) of DNA by the polymerase chain reaction and subsequent digestion with informative restriction endonucleases. Candidate markers were evaluated for their ability to discriminate among the different species and the level of intraspecific variation they exhibited. The selected markers require no more than two restriction digestions to allow unambiguous identification, although it was not possible to distinguish between white marlin and striped marlin with any of the genetic characters screened in our study. Individuals collected from throughout each species’ range were surveyed with the selected markers demonstrating low levels of intraspecific character variation within species. The resulting keys provide two independent means for the forensic identification of fillets and for specific identification of early life history stages.