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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 LAW Licensed Copy: sheffield
2、un sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS EN 13784:2002 This British 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 an
3、d Strategy Committee on 31 January 2002 BSI 31 January 2002 ISBN 0 580 38993 6 National foreword This British 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, whic
4、h has the responsibility to: A list of organizations represented on this committee can be obtained on request 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
5、 the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the 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 app
6、lication. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid 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 relate
7、d international and European developments and promulgate them in the UK. Summary of pages This document comprises 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. Am
8、endments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 13784 November 2001 ICS 67.050 English version Foodstuffs DNA Comet Assay for the de
9、tection 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 Lebensmitteln Screeningverfahren This European Standard was approved by CEN on 29 Septemb
10、er 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-date lists and bibliographical references concerning such national standards may be obta
11、ined 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 responsibility of a CEN member into its own language and notified to the Management Cen
12、tre 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, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN
13、COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2001 CENAll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 13784:2001 E Licensed Copy: shef
14、fieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13784:2001 (E) 2 Contents Page Foreword.3 1Scope .4 2Normative references .4 3Principle.4 4Reagents4 5Apparatus 7 6Procedure 7 7Evaluation10 8Limitations.10 9Validation.11 10Test report .13 Annex A (informativ
15、e) Figures .14 Bibliography.15 Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13784:2001 (E) 3 Foreword This European Standard has been prepared by Technical Committee CEN /TC 275, Food analysis Horizontal methods, the Secretariat of whi
16、ch is held by DIN. This European Standard shall be given the status of a national standard, either by publication 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
17、on the basis of a protocol developed following a concerted action supported 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,
18、 operations and equipment. This standard does not purport to address all the safety problems associated 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. Ann
19、ex A is informative. This standard includes a Bibliography. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, I
20、celand, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13784:2001 (E) 4 1 Scope This European Standard specifies a screening me
21、thod 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 specific, therefore, it is recommended to confirm positive results using a standardized me
22、thod 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 t
23、o 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 references This European Standard incorporates by dated or undated reference, provisions from oth
24、er 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 publications apply to this European Standard only when incorporated in it by amendment o
25、r 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). 3 Principle DNA fragmentation can be caused by various chemical or physical treatments
26、 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 fragmentation can be studied by microgel electrophoresis of single cells or nuclei. These are
27、 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 direction of the anode giving the damaged cells the appearance of a comet. This comet assa
28、y 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 sites are measured, whereas under neutral conditions only DNA double-strand breaks are obs
29、erved. 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 extension of the DNA from the nucleus towards the anode thus considerably longer comets (
30、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 combined with a low voltage and short electrophoresis time. 4
31、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 concentration c(HCl) = 1 mol/l Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10
32、GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13784:2001 (E) 5 4.3 Dimethylsulfoxide, DMSO1) (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 (Na2HPO4 12 H2O) and 0,24 g of potas
33、sium 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 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 agar
34、ose in 10 ml 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.
35、 Keep the solution 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
36、,0 with 40 % 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
37、 solution can 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 sodi
38、um dodecylsulfate (SDS) in electrophoresis buffer (4.9) and adjust the volume to 1 000 ml. 4.11 Staining solutions 4.11.1 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. 1) DMSO is a harmful substance and appropria
39、te safety precautions should be taken. 2) Acridine orange, ethidium bromide and propidium iodide are harmful substances and appropriate safety precautions should be taken. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncontrolled Copy, (c) BSI EN 13784:2001 (E) 6 4
40、.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 at 4 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
41、 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). 4.11.5 Ethidium bromide stock solution2) Dissolve 100 mg of ethidium bromide in 100 ml of water. Keep in the dark at approximately 4 C t
42、o 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 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
43、well as commercial silver staining kits for nucleic acids may be used, provided they have been found satisfactory. 4.11.7.2 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
44、 of sodium carbonate in water and adjust the volume to 250 ml. 4.11.7.4 Staining solution C Dissolve 100 mg of ammonium 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 Imme
45、diately before use, add 68 ml of staining solution C (4.11.7.4) to 32 ml of a vigorously stirred staining solution B (4.11.7.3). 4.11.7.6 Stopping solution E Dilute 10 ml glacial acetic acid to 1 000 ml with water. Licensed Copy: sheffieldun sheffieldun, na, Mon Oct 30 01:53:10 GMT+00:00 2006, Uncon
46、trolled Copy, (c) BSI 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 Stopwatch 5.4 Balance 5.5 Water bath 5.6 Hot plate magnetic stirrer 5.7 Mic
47、rowave 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 silver staining a standard transmission microscope can be used, but using f
48、luorescent 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 for propidium iodide or ethidium bromide. Th
49、e 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 gel 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 su