EN 13784:2001
(Main)Foodstuffs - DNA Comet Assay for the detection of irradiated foodstuffs - Screening method
Foodstuffs - DNA Comet Assay for the detection of irradiated foodstuffs - Screening method
This European Standard specifies a screening method for foods which contain DNA. It is based on micro-gel electrophoresis of single cells or nuclei to detect DNA fragmentation presumptive to irradiation treatment [1] to [8]. The DNA Comet Assay is not radiation specific, therefore, it is recommended to confirm positive results using a standardized method to specifically prove an irradiation treatment of the respective food, e.g. EN 1784, EN 1785, EN 1786, EN 1787, EN 1788, EN 13708, and prEN 13751.
Interlaboratory studies have been successfully carried out with a number of food products, both of animal and plant origin such as various meats [9] to [11], seeds, dried fruits and spices [6], [12]. Other studies [13] to [32] demonstrate that the method is applicable to a large variety of foodstuffs, but also that limitations exist (see clause 8).
Lebensmittel - DNA-Kometentest zum Nachweis von bestrahlten Lebensmitteln - Screeningverfahren
Diese Europäische Norm legt ein Screeningverfahren für Lebensmittel, die DNA enthalten, fest. Das Verfahren basiert auf der Mikro-Gelelektrophorese einzelner Zellen oder Zellkerne und dient dem Nachweis einer DNA-Fragmentierung nach einer Strahlenbehandlung [1] bis [8]. Dieser DNA-Kometentest ist kein Verfahren für den spezifischen Nachweis einer Bestrahlung. Es wird daher empfohlen, positive Ergebnisse durch Anwendung eines genormten Verfahrens, z.B. EN 1784, EN 1785, EN 1786, EN 1787, EN 1788, EN 13708 und prEN 13751, zu bestätigen.
An einigen Lebensmitteln, sowohl tierischer als auch pflanzlicher Herkunft, wurden Ringversuche erfolgreich durchgeführt, z. B. an verschiedenen Fleischarten [9] bis [11] sowie an Samen, getrockneten Früchten und Gewürzen [6], [12]. Weitere Untersuchungen [13] bis [32] haben die Anwendbarkeit des Verfahrens auf eine große Anzahl von Lebensmitteln erwiesen, aber auch die Grenzen des Verfahrens aufgezeigt (siehe Abschnitt 8).
Produits alimentaires - Détection d'aliments ionisés en utilisant le test de comète d'ADN - Méthode par criblage
La présente Norme européenne décrit une méthode de criblage des produits alimentaires contenant de l'ADN. Cette méthode se base sur l'électrophorèse par micro-gel de cellules simples ou de noyaux, pour détecter la fragmentation éventuelle de l'ADN résultant d'une ionisation [1] à [8]. L'essai de comète d'ADN n'est pas spécifique au rayonnement. Par conséquent, il est recommandé de confirmer les résultats positifs à l'aide d'une méthode normalisée permettant de prouver spécifiquement que ces produits alimentaires ont bien été ionisés (par exemple EN 1784, EN 1785, EN 1786, EN 1787, EN 1788, EN 13708 et prEN 13751).
Des études interlaboratoires ont été menées avec succès sur plusieurs produits alimentaires, d'origines animale et végétale, qu'il s'agisse de viandes [9] à [11], de graines, de fruits séchés et d'épices [6], [12]. D'autres études [13] à [32] montrent que la méthode peut s'appliquer à une large gamme de produits alimentaires mais que certaines limites existent également (voir article 8).
Živila - DNA komet analiza za detekcijo obsevane hrane - Informativna metoda
General Information
Standards Content (Sample)
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Živila - DNA komet analiza za detekcijo obsevane hrane - Informativna metodaLebensmittel - DNA-Kometentest zum Nachweis von bestrahlten Lebensmitteln - ScreeningverfahrenProduits alimentaires - Détection d'aliments ionisés en utilisant le test de comete d'ADN - Méthode par criblageFoodstuffs - DNA Comet Assay for the detection of irradiated foodstuffs - Screening method67.050Splošne preskusne in analizne metode za živilske proizvodeGeneral methods of tests and analysis for food productsICS:Ta slovenski standard je istoveten z:EN 13784:2001SIST EN 13784:2002en01-junij-2002SIST EN 13784:2002SLOVENSKI
STANDARD
EUROPEAN STANDARDNORME EUROPÉENNEEUROPÄISCHE NORMEN 13784November 2001ICS 67.050English versionFoodstuffs - DNA Comet Assay for the detection of irradiatedfoodstuffs - Screening methodProduits alimentaires - Détection d'aliments ionisés enutilisant le test de comète d'ADN - Méthode par criblageLebensmittel - DNA-Kometentest zum Nachweis vonbestrahlten Lebensmitteln - ScreeningverfahrenThis European Standard was approved by CEN on 29 September 2001.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the Management Centre or to any CEN member.This European Standard exists in three official versions (English, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the Management Centre has the same status as the officialversions.CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMITÉ EUROPÉEN DE NORMALISATIONEUROPÄISCHES KOMITEE FÜR NORMUNGManagement Centre: rue de Stassart, 36
B-1050 Brussels© 2001 CENAll rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 13784:2001 ESIST EN 13784:2002
Figures.12Bibliography.13ForewordThis European Standard has been prepared by Technical Committee CEN /TC 275 "Food analysis - Horizontalmethods", the secretariat of which is held by DIN.This European Standard shall be given the status of a national standard, either by publication of an identical text orby endorsement, at the latest by May 2002, and conflicting national standards shall be withdrawn at the latest byMay 2002.This European Standard was elaborated on the basis of a protocol developed following a concerted actionsupported by the Commission of European Union (XII C.). Experts and laboratories from E.U. and EFTA countries,contributed jointly to the development of this protocol.WARNING: The use of this standard may involve hazardous materials, operations and equipment. This standarddoes not purport to address all the safety problems associated with its use. It is the responsibility of the user of thisstandard to establish appropriate safety and health practices and determine the applicability of regulatorylimitations prior to use.Annex A is informative.This standard includes a Bibliography.According to the CEN/CENELEC Internal Regulations, the national standards organizations of the followingcountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland,France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden,Switzerland and the United Kingdom.SIST EN 13784:2002
[8].The DNA Comet Assay is not radiation specific, therefore, it is recommended to confirm positive results using astandardized method to specifically prove an irradiation treatment of the respective food, e.g. EN 1784, EN 1785,EN 1786, EN 1787, EN 1788, EN 13708, and prEN 13751.Interlaboratory studies have been successfully carried out with a number of food products, both of animal and plantorigin such as various meats [9] to [11], seeds, dried fruits and spices [6], [12]. Other studies [13] to [32]demonstrate that the method is applicable to a large variety of foodstuffs, but also that limitations exist (see clause8).2 Normative referencesThis European Standard incorporates by dated or undated reference, provisions from other publications. Thesenormative references are cited at the appropriate places in the text, and the publications are listed hereafter. Fordated references, subsequent amendments to or revisions of any of these publications apply to this EuropeanStandard only when incorporated in it by amendment or revision. For undated references the latest edition of thepublication referred to applies (including amendments).EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987).3 PrincipleDNA fragmentation can be caused by various chemical or physical treatments including ionizing radiation. Whenfood containing DNA is treated by ionizing radiation, modification of these large molecules occurs includingfragmentation either by single- or double-strand breaks. This fragmentation can be studied by microgel electro-phoresis of single cells or nuclei. These are embedded in agarose on microscope slides, lysed for disruption ofmembranes using a detergent and electrophoresed at a set voltage. DNA fragments will stretch or migrate out ofthe cells forming a tail in the direction of the anode giving the damaged cells the appearance of a comet. Thiscomet assay to measure DNA damage can be carried out under various conditions. Both alkaline and neutralprotocols exist. In general, under alkaline conditions both DNA single- and double-strand breaks and alkali-labilesites are measured, whereas under neutral conditions only DNA double-strand breaks are observed. However,using neutral conditions [1] single-strand breaks also exert an influence on the comet appearance, due torelaxation of supercoiled DNA in the nucleus [7], [8]. Irradiated cells will show an increased extension of the DNAfrom the nucleus towards the anode thus considerably longer comets (more fragmentation) than unirradiated cells.Unirradiated cells will appear nearly circular or with only slight tails (see Figure A.1).This European Standard describes the use of a simple agarose single-layer set-up employing neutral pH combinedwith a low voltage and short electrophoresis time.4 Reagents4.1 GeneralDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at leastgrade 1 according to EN ISO 3696.4.2 Hydrochloric acid, substance concentration c(HCl) = 1 mol/l4.3 Dimethylsulfoxide, DMSO1) (optional)
1) DMSO is a harmful substance and appropriate safety precautions should be taken.SIST EN 13784:2002
2) Acridine orange, ethidium bromide and propidium iodide are harmful substances and appropriate safety precautionsshould be taken.SIST EN 13784:2002
the volume to 250 ml.4.11.7.4 Staining solution CDissolve 100 mg of ammonium nitrat, 100 mg of silver nitrate and 500 mg of tungstosilic acid in water, add 250 µlof formaldehyde (min. 37%) and dilute to 500 ml with water.4.11.7.5 Staining solution DImmediately before use, add 68 ml of staining solution C (4.11.7.4) to 32 ml of a vigorously stirred staining solutionB (4.11.7.3).4.11.7.6 Stopping solution EDilute 10 ml glacial acetic acid to 1000 ml with water.5 ApparatusUsual laboratory apparatus and, in particular, the following:5.1 DNA horizontal submarine electrophoresis chamber,5.2 Power supply, e.g. 0 V to 100 V, 0 mA to 400 mA5.3 Stopwatch5.4 Balance5.5 Water bath5.6 Hot plate magnetic stirrer5.7 Microwave ovenSIST EN 13784:2002
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.