1、BRITISH STANDARD BS EN 13784:2002 Foodstuffs DNA Comet Assay for the detection of irradiated foodstuffs Screening method The European Standard EN 13784:2001 has the status of a British Standard ICS 67.050 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBS EN 13784:2002 This Bri
2、tish Standard, having been prepared under the direction of the Consumer Products and Services Sector Policy and Strategy Committee, was published under the authority of the Standards Policy and Strategy Committee on 31 January 2002 BSI 31 January 2002 ISBN 0 580 38993 6 National foreword This Britis
3、h Standard is the official English language version of EN 13784:2001. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Food analysis Horizontal method, which has the responsibility to: A list of organizations represented on this committee can be obtained on reques
4、t to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the
5、 BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid
6、enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comp
7、rises a front cover, an inside front cover, the EN title page, pages 2 to 17 and a back cover. The BSI copyright date displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN
8、 13784 November 2001 ICS 67.050 English version Foodstuffs DNA Comet Assay for the detection of irradiated foodstuffs Screening method Produits alimentaires Dtection daliments ioniss en utilisant le test de comte dADN Mthode par criblage Lebensmittel DNA-Kometentest zum Nachweis von bestrahlten Lebe
9、nsmitteln Screeningverfahren This 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 European Standard the status of a national standard without any alteration. Up-to-dat
10、e lists and bibliographical references concerning such national standards 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 translation under the re
11、sponsibility of a CEN member into its own language and notified to the Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Ne
12、therlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CEN All rights of exploitation in any form and by any means res
13、erved worldwide for CEN national Members. Ref. No. EN 13784:2001 EEN 13784:2001 (E) 2 Contents Page Foreword. 3 1 Scope . 4 2 Normative references .4 3 Principle . 4 4 Reagents 4 5A p p a r a t u s 7 6 Procedure 7 7 Evaluation 10 8 Limitations.10 9 Validation. 11 10 Test report .13 Annex A (informat
14、ive) Figures .14 Bibliography . 15EN 13784:2001 (E) 3 Foreword This European Standard has been prepared by Technical Committee CEN /TC 275, Food analysis Horizontal methods, the Secretariat of which is held by DIN. This European Standard shall be given the status of a national standard, either by pu
15、blication of an identical text or by endorsement, at the latest by May 2002, and conflicting national standards shall be withdrawn at the latest by May 2002. This European Standard was elaborated on the basis of a protocol developed following a concerted action supported by the Commission of Europea
16、n 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 standard does not purport to address all the safety problems associated
17、with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Annex A is informative. This standard includes a Bibliography. According to the CEN/CENELEC Internal Regula
18、tions, the national standards organizations of the following countries 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 Un
19、ited Kingdom.EN 13784:2001 (E) 4 1 Scope This European Standard specifies a screening method for foods that 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 sp
20、ecific, 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 w
21、ith 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). 2 Normative reference
22、s This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text, and the publications are listed hereafter. For dated references, subsequent amendments to or revisions of any of these
23、publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies (including amendments). EN ISO 3696, Water for analytical laboratory use Specification and test methods (ISO 3696:1987)
24、. 3 Principle DNA fragmentation can be caused by various chemical or physical treatments including ionizing radiation. When food containing DNA is treated by ionizing radiation, modification of these large molecules occurs including fragmentation either by single- or double-strand breaks. This fragm
25、entation can be studied by microgel electrophoresis of single cells or nuclei. These are embedded in agarose on microscope slides, lysed for disruption of membranes using a detergent and electrophoresed at a set voltage. DNA fragments will stretch or migrate out of the cells forming a tail in the di
26、rection of the anode giving the damaged cells the appearance of a comet. This comet assay to measure DNA damage can be carried out under various conditions. Both alkaline and neutral protocols exist. In general, under alkaline conditions both DNA single- and double-strand breaks and alkali-labile si
27、tes 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 to relaxation of supercoiled DNA in the nucleus 7, 8. Irradiated cells will show an increased e
28、xtension of the DNA from 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 se
29、t-up employing neutral pH combined with a low voltage and short electrophoresis time. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and water of at least grade 1 according to EN ISO 3696. 4.2 Hydrochloric acid, substance concent
30、ration c(HCl) = 1 mol/lEN 13784:2001 (E) 5 4.3 Dimethylsulfoxide, DMSO 1)(optional) 4.4 Phosphate buffered saline (PBS), pH 7,4 Dissolve 8,0 g of sodium chloride, 0,2 g of potassium chloride, 2,94 g of disodium hydrogen phosphate dodecahydrate (Na 2 HPO 4 12 H 2 O) and 0,24 g of potassium dihydrogen
31、 phosphate (KH 2 PO 4 ) 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 1 000 ml. The solution should be autoclaved or sterile-filtered. 4.5 Coating agarose solution, 0,5 % agarose in distilled water Dissolve 50 mg agarose in 10 m
32、l water by boiling or microwaving (no flakes, clear solution). Keep the solution in a water bath at 45 C for precoating the microscope slides. 4.6 Casting gel solution, 0,8 % agarose in PBS Dissolve 80 mg of low melting temperature agarose, in 10 ml of PBS (4.4) by boiling or microwaving. Keep the s
33、olution 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/l Add 93,05 g of ethylenediaminetetraacetic acid, disodium salt dihydrate to 300 ml of distilled water, mix well, and adjust the pH to 8,0 with 40
34、% sodium hydroxide solution. Dilute to 500 ml with distilled water, and autoclave. 4.8 TBE stock solution Dissolve 54 g Tris(hydroxymethyl)aminomethane (Tris base) and 27,5 g of boric acid in 20 ml of EDTA stock solution (4.7), dilute to 1 000 ml with distilled water (TBE). This TBE stock solution c
35、an be stored in glass bottles at room temperature. Discard any batches that develop a precipitate. 4.9 Electrophoresis buffer Dilute one volume part of the TBE stock solution (4.8) with nine volume parts of water. If necessary, adjust the pH to 8,4. 4.10 Lysis buffer Dissolve 25 g of sodium dodecyls
36、ulfate (SDS) in electrophoresis buffer (4.9) and adjust the volume to 1 000 ml. 4.11 Staining solutions 4.11.1 Acridine orange stock solution 2) Dissolve 100 mg of acridine orange in 100 ml of water. Keep in the dark at approximately 4 C to 6 C.1)DMSO is a harmful substance and appropriate safety pr
37、ecautions should be taken. 2)Acridine orange, ethidium bromide and propidium iodide are harmful substances and appropriate safety precautions should be taken.EN 13784:2001 (E) 6 4.11.2 Acridine orange staining solution 2) Dilute 0,5 ml of acridine orange stock solution (4.11.1) to 100 ml with PBS (4
38、.4). This solution may be stored at 4 C to 6 C for up to one week. 4.11.3 Propidium iodide stock solution 2) 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 solution 2) Dilute 1 ml to 5 ml of propidium iodide stoc
39、k solution (4.11.3) to 100 ml with PBS (4.4). 4.11.5 Ethidium bromide stock solution 2) 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 solution 2) Dilute 2 ml of ethidium bromide stock solution (4.11.5) to 100 ml
40、 with water. 4.11.7 Silver staining 4.11.7.1 General The following silver staining solutions and procedure 33 has been employed in the interlaboratory trials 6. Other procedures 34 as well as commercial silver staining kits for nucleic acids may be used, provided they have been found satisfactory. 4
41、.11.7.2 Fixing solution A To 150 g of trichloroacetic acid add 50 g of zinc sulfate and 50 g of glycerol and dilute to 1 000 ml with water. 4.11.7.3 Staining solution B Dissolve 12,5 g of sodium carbonate in water and adjust the volume to 250 ml. 4.11.7.4 Staining solution C Dissolve 100 mg of ammon
42、ium nitrate, 100 mg of silver nitrate and 500 mg of tungstosilic acid in water, add 250 l of formaldehyde (min. 37 %) and dilute to 500 ml with water. 4.11.7.5 Staining solution D Immediately before use, add 68 ml of staining solution C (4.11.7.4) to 32 ml of a vigorously stirred staining solution B
43、 (4.11.7.3). 4.11.7.6 Stopping solution E Dilute 10 ml glacial acetic acid to 1 000 ml with water.EN 13784:2001 (E) 7 5 Apparatus Usual 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 mA 5.3
44、 Stopwatch 5.4 Balance 5.5 Water bath 5.6 Hot plate magnetic stirrer 5.7 Microwave oven 5.8 Sieve cloth, 100 m, 200 m and 500 m pore size, e.g. of nylon 5.9 Microscope slides (76 mm x 26 mm) with one frosted end. 5.10 Cover slips (24 mm x 60 mm). 5.11 Staining jars 5.12 Microscope In the case of DNA
45、 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
46、with a barrier filter at 590 nm for propidium iodide or ethidium bromide. The microscope should allow a magnification of 100x to 400x. 6 Procedure 6.1 Preparation of single cell suspensions 6.1.1 General For a suitable evaluation of electrophoresed slides, the distribution of cells in the agarose ge
47、l should be even and not overlapping each other. If too few cells are present, the amount of tissue can be increased, and vice versa. The cell suspensions may be stored on ice but no longer than 10 min. By addition of DMSO to a final level of 5 % to 10 % as a freeze protectant, the cell suspensions
48、can be stored for extended periods at -18 C.EN 13784:2001 (E) 8 6.1.2 Animal tissues 6.1.2.1 Bone marrow Split 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 c
49、loth with a pore size of 100 m. Keep the filtrate on ice. Use the supernatant for further analysis. 6.1.2.2 Muscle tissue Scrape off the tissue or cut it (without visible fat) in very thin slices with a scalpel and transfer about 1 g to a small beaker with 5 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 suspension sequentially through 500 m and 200 m sieve cloth. Leave to settle on ice for about 5 min. Use the supernatant as cell
