SIST EN 13784:2002

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



SIST EN 13784:2002



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



EN 13784:2001 (E)2ContentspageForeword.21Scope.32Normative references.33Principle.34Reagents.35Apparatus.56Procedure.67Evaluation.88Limitations.99Validation.1010Test report.11Annex A (informative)
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



EN 13784:2001 (E)31 ScopeThis European Standard specifies a screening method for foods which contain DNA. It is based on micro-gelelectrophoresis 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 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



EN 13784:2001 (E)44.4 Phosphate buffered saline (PBS), pH 7,4Dissolve 8,0 g of sodium chloride, 0,2 g of potassium chloride, 2,94 g of disodium hydrogen phosphatedodecahydrate (Na2HPO4 · 12 H2O) and 0,24 g of potassium dihydrogen phosphate (KH2PO4) in 900 ml water,adjust the pH to 7,4 with a few drops of hydrochloric acid (4.2) and adjust the volume with water to 1000 ml. Thesolution should be autoclaved or sterile-filtered.4.5 Coating agarose solution, 0,5% agarose in distilled waterDissolve 50 mg agarose in 10 ml water by boiling or microwaving (no flakes, clear solution). Keep the solution in awater bath at 45 °C for precoating the microscope slides.4.6 Casting gel solution, 0,8% agarose in PBSDissolve 80 mg of low melting temperature agarose, in 10 ml of PBS (4.4) by boiling or microwaving. Keep thesolution in a water bath at 45 °C, ready to be mixed with the cell suspension and to cast the gel on the slides.4.7 EDTA stock solution c(EDTA) = 0,5 mol/lAdd 93,05 g of ethylenediaminetetraacetic acid, disodium salt dihydrate to 300 ml of distilled water, mix well, andadjust the pH to 8,0 with 40 % sodium hydroxide solution. Dilute to 500 ml with distilled water, and autoclave.4.8 TBE stock solutionDissolve 54 g Tris(hydroxymethyl)aminomethane (Tris base) and 27,5 g of boric acid in 20 ml of EDTA stocksolution (4.7), dilute to 1000 ml with distilled water (TBE). This TBE stock solution can be stored in glass bottles atroom temperature. Discard any batches that develop a precipitate.4.9 Electrophoresis bufferDilute one volume part of the TBE stock solution (4.8) with nine volume parts of water. If necessary, adjust the pHto 8,4.4.10 Lysis bufferDissolve 25 g of sodium dodecylsulfate (SDS) in electrophoresis buffer (4.9) and adjust the volume to 1000 ml.4.11 Staining solutions4.11.1 Acridine orange stock solution2)Dissolve 100 mg of acridine orange in 100 ml of water. Keep in the dark at approximately 4 °C to 6 °C.4.11.2 Acridine orange staining solution2)Dilute 0,5 ml of acridine orange stock solution (4.11.1) to 100 ml with PBS (4.4). This solution may be stored at4 °C to 6 °C for up to one week.4.11.3 Propidium iodide stock solution2)Dissolve 100 mg of propidium iodide in 100 ml of water. Keep in the dark at approximately 4 °C to 6 °C.4.11.4 Propidium iodide staining solution2)Dilute 1 ml to 5 ml of propidium iodide stock solution (4.11.3) to 100 ml with PBS (4.4).
2) Acridine orange, ethidium bromide and propidium iodide are harmful substances and appropriate safety precautionsshould be taken.SIST EN 13784:2002



EN 13784:2001 (E)54.11.5 Ethidium bromide stock solution2)Dissolve 100 mg of ethidium bromide in 100 ml of water. Keep in the dark at approximately 4 °C to 6 °C.4.11.6 Ethidium bromide staining solution2)Dilute 2 ml of ethidium bromide stock solution (4.11.5) to 100 ml with water.4.11.7 Silver staining4.11.7.1 GeneralThe following silver staining solutions and procedure [33] has been employed in the interlaboratory trials [6]. Otherprocedures [34] as well as commercial silver staining kits for nucleic acids may be used, provided they have beenfound satisfactory.4.11.7.2 Fixing solution ATo 150 g of trichloroacetic acid add 50 g of zinc sulfate and 50 g of glycerol and dilute to 1000 ml with water.4.11.7.3 Staining solution BDissolve 12,5 g of sodium carbonate in water and adjust
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



EN 13784:2001 (E)65.8 Sieve cloth, 100 µm, 200 µm and 500 µm pore size, e.g. of nylon5.9 Microscope slides(76 mm x 26 mm) with one frosted end.5.10 Cover slips(24 mm x 60 mm).5.11 Staining jars5.12 MicroscopeIn the case of DNA silver staining a standard transmission microscope can be used, but using fluorescent staining,a microscope with epifluorescence illumination is needed, with a filter set of e.g. 460 nm to 485 nm (blue excitation)for acridine orange or a filter set of 515 nm to 560 nm (green excitation) combined with a barrier filter at 590 nm forpropidium iodide or ethidium bromide.The microscope should allow a magnification of 100x to 400x.6 Procedure6.1 Preparation of single cell suspensions6.1.1 GeneralFor a suitable evaluation of electrophoresed slides, the distribution of cells in the agarose gel should be even andnot overlapping each other. If too few cells are present, the amount of tissue can be increased, and vice versa. Thecell suspensions may be stored on ice but no longer than 10 min. By addition of DMSO to a final level of 5 % to10 % as a freeze protectant, the cell suspensions can be stored for extended periods at - 18 °C.6.1.2 Animal tissues6.1.2.1 Bone marrowSplit the bone (e.g. chicken leg) and transfer about 50 mg of bone marrow to a test tube with 3 ml of ice-cold PBS.Suspend the cells using a glass rod. Filter the cell suspension through sieve cloth with a pore size of 100 µm. Keepthe filtrate on ice. Use the supernatant for further analysis.6.1.2.2 Muscle tissueScrape off the tissue or cut it (without visible fat) in very thin slices with a scalpel and transfer about 1 g to a smallbeaker with 5 ml of ice-cold PBS. Cool the beaker in a larger one with crushed ice and stir for 5 min at about500 min-1. Filter the suspension sequentially through 500 µm and 200 µm sieve cloth. Leave to settle on ice forabout 5 min. Use the supernatant as cell extract.SIST EN 13784:2002



EN 13784:2001 (E)76.1.3 Plant tissues6.1.3.1 Seeds, nuts and spicesCrush about 0,25 g of the samples with a mortar and pestle (if present remove outer shell before grinding,sometimes immersion in water facilitates the removal) and transfer to a small beaker with 3 ml of ice-cold PBS.Cool the beaker in a larger one with crushed ice and stir for 5 min at about 500 min-1. Filter the suspensionsequentially through 200 µm and 100 µm sieve cloth. Leave to settle on ice for 15 min to 60 min. A longer timegives a suspension with less contaminants, but also with less cells/cell nuclei. Use the supernatant for furtheranalysis.6.1.3.2 StrawberriesIsolate the achenes of strawberries just by picking or by blending the strawberries in a large amount of water,allowing the heavier achenes to settle. Weigh about 0,25 g of achenes and proceed as for seeds, nuts and spices(6.1.3.1).6.1.3.3 PotatoesCut the meristem of potato in very thin slices with a scalpel and transfer about 4 g to a small beaker with 5 ml ofice-cold PBS, and proceed as for seeds, nuts and spices (6.1.3.1).6.1.3.4 OnionsCut the meristem of onions in very thin slices with a scalpel and transfer about 2 g to a small beaker with 4 ml ofice-cold PBS, and proceed as for seeds, nuts and spices (6.1.3.1).6.2 Pre-coating slidesTo improve adhesion of the agarose gel to the slide, the latter is pre-coated with a thin agarose layer. Beforecoating, the slides are freed from fat by immersion overnight in methanol, and are allowed to air dry. Precoat thecleaned dustfree slide by spreading one drop (approximately 50 µl) of the warm coating agarose solution (4.5) witha second slide across the first slide and allow air dry for about 30 min. Precoating can also be done by dipping andcleaning one side with paper tissue. Coated slides can be stored dustfree for several weeks.6.3 Casting the gelsMix 100 µl of cell suspension with about 1 ml of warm casting gel solution (4.6). Transfer 100 µl of this mixture on aprecoated slide and spread it roughly by the pipette tip. Cover immediately with a cover slip in such a way that thegel is spread evenly and avoid air bubbles. Place the slide on ice for 5 min to solidify the agarose gel. Move thecover slip aside with the tip of a scalpel, and gently slide the slip off the agarose. The gel shall be even withoutbubbles. Several slides can be prepared in parallel using the same gel solution.6.4 Lysis of the cellsDNA fragments migrate out of the cells during electrophoresis under the condition that the cell membranes arepermeable. Therefore, the lysis of the cells is a necessary prerequisite for the application of the comet assay.Immerse the casted slides completely in lysis buffer in a staining jar for at least 5 min for animal cells and at least15 min for plant cells at room temperature. Do not touch the agarose layer. (To check for complete lysis, cells maybe stained and observed with a microscope: lysed cells will show diffusion of DNA out of the cells.)6.5 ConditioningImmerse the slides after lysis in electrophoresis buffer (4.9) for 5 min.6.6 ElectrophoresisPlace the slides in the horizontal electrophoresis chamber side by side, avoiding spaces and with the frosted endfacing the cathode. Fill the tank with fresh electrophoresis buffer (4.9) to a level approximately 2 mm to 4 mmabove the slides (do not displace the slides). Conduct electrophoresis at room temperature at a potential of 2 V/cmSIST EN 13784:2002



EN 13784:2001 (E)8(voltage applied/ distance between electrodes) for 2,0 min. After the current is switched off, remove the slidescarefully from the tank and immerse them in water for 5 min. Let them air dry for about 1 h or dry them in alaboratory oven at 40 °C to 50 °C.6.7 Staining6.7.1 Staining with fluorescent dyesThe slides shall be stained immediately prior to observation as the dye fades during storage.6.7.1.1 Acridine orangeImmerse the slides in acridine orange staining solution (4.11.2) for 3 min to 5 min. Wash the slides by submergingin water for 0,5 min to 1 min. Before viewing with the fluorescence microscope, mount a cover slip on the wet slideand blot off excess water. Observe immediately, since drying of the slides impair the view of the cells.Avoid prolonged exposure to light, since fading may occur. Overstaining may cause a high backgroundfluorescence which can be reduced by further washing.6.7.1.2 Propidium iodideSubmerge the slides in propidium iodide staining solution (4.11.4) for 5 min to 10 min. Wash and continue as foracridine orange (6.7.1.1).6.7.1.3 Ethidium bromideUse the same procedure as for propidium iodide (6.7.1.2).6.7.2 Silver stainingImmerse the slides in fixing solution A (4.11.7.2) for 10 min. Rinse for about 1 min with water. Dry the gel for 1 h inan oven at 40 °C to 50 °C or in air at room temperature (can be done overnight). Submerge the slides in stainingsolution D (4.11.7.5) for 10 min to 20 min. Repeat the staining step once or twice with fresh staining solution D for5 min to 10 min until a greyish-brown colour develops on the slides. Rinse for about 1 min with water. Stop stainingreaction with stopping solution E (4.11.7.6) for 5 min and rinse again for about 1 min with water. Dry the inclinedslides at room temperature. The stained slides do not fade and can be
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