1、August 2013 Translation by DIN-Sprachendienst.English price group 35No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS
2、 67.050!%(/“2052912www.din.deDDIN EN ISO 21569Foodstuffs Methods of analysis for the detection of genetically modified organismsand derived products Qualitative nucleic acid based methods (ISO 21569:2005 + Amd 1:2013);English version EN ISO 21569:2005 + A1:2013,English translation of DIN EN ISO 2156
3、9:2013-08Lebensmittel Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihrenProdukten Qualitative auf Nukleinsuren basierende Verfahren (ISO 21569:2005 + Amd 1:2013);Englische Fassung EN ISO 21569:2005 + A1:2013,Englische bersetzung von DIN EN ISO 21569:2013-08Produits alimentair
4、es Mthodes danalyse pour la dtection des organismes gntiquement modifis et desproduits drivs Mthodes qualitatives bases sur lutilisation des acides nucliques (ISO 21569:2005 +Amd 1:2013);Version anglaise EN ISO 21569:2005 + A1:2013,Traduction anglaise de DIN EN ISO 21569:2013-08SupersedesDIN EN ISO
5、21569:2005-09www.beuth.deDocument comprises 160 pagesIn case of doubt, the German-language original shall be considered authoritative.09.13 DIN EN ISO 21569:2013-08 2 A comma is used as the decimal marker. National foreword This document (EN ISO 21569:2005 + A1:2013) has been prepared by Technical C
6、ommittee ISO/TC 34 “Food products”, Subcommittee SC 16 “Horizontal methods for molecular biomarker analysis” in collaboration with Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), Working Group WG 11 “Genetically modified foodstuffs”, in accordance with
7、the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-04 AA Gentech
8、nisch modifizierte Lebensmittel. This standard includes Amendment A1 approved by CEN on 2013-03-14. The start and finish of text introduced or altered by amendment is indicated in the text by tags !“. The DIN Standards corresponding to the International Standards referred to in this document are as
9、follows: ISO 21571 DIN EN ISO 21571 ISO 24276 DIN EN ISO 24276 Amendments This standard differs from DIN EN ISO 21569:2005-09 as follows: a) the reference to EN ISO 21568 has been deleted; b) the reference to ISO 24276:2006 has been updated; c) specifications relating to the qualitative result have
10、been rendered more precise; d) Subclause 9.4 “Quality assurance requirements” has been rendered more precise; e) the informative Annexes A to D include ten new procedures in Subclauses A.5, A.6, B.6, B.7, B.8, B.9, C.6, C.7, C.8 and D.2; f) the document has been editorially revised (footnotes); g) t
11、he Bibliography has been supplemented. Previous editions DIN EN ISO 21569: 2005-09 DIN EN ISO 21569:2013-08 3 National Annex NA (informative) Bibliography DIN EN ISO 21571, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction
12、 DIN EN ISO 24276, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products General requirements and definitions DIN EN ISO 21569:2013-08 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 21569 June 2005 + A1 Ap
13、ril 2013 ICS 67.050 English Version Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods (ISO 21569:2005 + Amd 1:2013) Produits alimentaires - Mthodes danalyse pour la dtection des organismes gntiquement mo
14、difis et des produits drivs - Mthodes qualitatives bases sur lutilisation des acides nucliques (ISO 21569:2005 + Amd 1:2013) Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Qualitative auf Nukleinsuren basierende Verfahren (ISO 21569:2005 + Amd 1
15、:2013) EN ISO 21569:2005 was approved by CEN on 2005-06-06 and Amendment A1:2013 on 2013-03-14. 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-d
16、ate lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC 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 translatio
17、n under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Fo
18、rmer Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMI
19、T EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 21569:2005 + A1:2013 EContents Page Foreword to EN ISO 21569:20
20、05 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle of the method .6 5 Reagents .6 6 Apparatus and equipment 7 7 Procedure .7 8 Interpretation 10 9 Expression of results and quality assurance .10 10 Test report 11 Annex A (informative) Target-taxon-specific
21、 methods 12 Annex B (informative) Screening methods . 2 Annex C (informative) Construct-specific methods 92 Annex D (informative) Event-specific methods Bibliography DIN EN ISO 21569:2013-08 EN ISO 21569:2005 + A1:2013 (E) 2 4137151!Foreword to EN ISO 21569:2005/A1:2013 .3 Foreword to EN ISO 21569:2
22、005 This document (EN ISO 21569:2005) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 “Agricultural food products”. This European Standard shall be given the status of
23、 a national standard, either by publication of an identical text or by endorsement, at the latest by December 2005, and conflicting national standards shall be withdrawn at the latest by December 2005. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the fol
24、lowing countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Swed
25、en, Switzerland and United Kingdom. !Foreword to EN ISO 21569:2005/A1:2013 This document (EN ISO 21569:2005/Amd 1:2013) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration with Technical Committee CEN/TC 275 “Food analysis Horizontal method ces eht ”s tairater of whic
26、h is held by DIN. This Amendment to the European Standard EN ISO 21569:2005 shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by October 2013, and conflicting national standards shall be withdrawn at the latest by October 20
27、13. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organiza
28、tions of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Mal
29、ta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 21569:2005/Amd 1:2013 has been approved by CEN as EN ISO 21569:2005/A1:2013 without any modification.“ DIN EN ISO 21569:2013-08 EN ISO
30、 21569:2005 + A1:2013 (E) 3 ,Introduction The search for a genetically modified origin of ingredients is performed by means of the following successive (or simultaneous) steps. After sample collection, nucleic acids are extracted from the test portion. Extracted nucleic acids can be further purified
31、, simultaneously or after the extraction process. Afterwards, they are quantified (if necessary), diluted (if necessary) and subjected to analytical procedures (such as PCR). These steps are detailed in this International Standard and in the following series of International Standards with the gener
32、al title Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products: Quantitative nucleic acid based methods (ISO 21570); Nucleic acid extraction (ISO 21571). Further information about general requirements and definitions involving the steps cited above a
33、re collected in ISO 24276. The qualitative detection of DNA target sequences is performed in order to obtain a yes or no answer to the question whether a certain target sequence is detected or not relative to appropriate controls and within the detection limits of the analytical method used and test
34、 portion analysed. The specificity of the methods, as described in Annexes A to D, ranges from screening methods to detect common DNA sequences characteristic of GMOs, to specific identification of a genetic construct or a specific transformation event. The International Organization for Standardiza
35、tion (ISO) draws attention to the fact that it is claimed that compliance with this document may involve the use of a patent concerning the PCR technology. ISO takes no position concerning the evidence, validity and scope of this patent right. ISO has been informed that Applied Biosystems, Roche Mol
36、ecular Systems, Inc. and F. Hoffman La Roche Ltd. hold patent rights concerning the PCR technology. The companies have assured the ISO that they are willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the s
37、tatements of the holders of these patent rights are registered with ISO. Information may be obtained from: Licensing Department Applied Biosystems 850 Lincoln Centre Drive Foster City, CA 94404 USA and Roche Molecular Systems, Inc. Licensing Department 1145 Atlantic Avenue Alameda, CA 94501 USA Atte
38、ntion is drawn to the possibility that some of the elements of this document may be the subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights. !Deleted text“ DIN EN ISO 21569:2013-08 EN ISO 21569:2005 + A1:2013 (E)
39、 4 1 Scope This International Standard describes the procedure to qualitatively detect genetically modified organisms (GMOs) and derived products by analysing the nucleic acids extracted from the sample under study. The main focus is on polymerase chain reaction (PCR) based amplification methods. It
40、 gives general requirements for the specific detection and identification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified DNA sequence. Guidelines, minimum requirements and performance criteria laid down in this International Standard are intended to
41、ensure that comparable, accurate and reproducible results are obtained in different laboratories. This International Standard has been established for food matrices, but could also be applied to other matrices (e.g. feed and plant samples from the environment). Specific examples of methods are provi
42、ded in Annexes A to D. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO
43、 21571:2005, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 24276 apply. !ISO 24276:2006, Foodstuffs Methods of analysis
44、for the detection of genetically modified organisms and derived products General requirements and definitions“ DIN EN ISO 21569:2013-08 EN ISO 21569:2005 + A1:2013 (E) 5 4 Principle of the method 4.1 General Qualitative analysis consists of specific detection of target nucleic acid sequences in the
45、test samples. Each method shall specify the target sequence. 4.2 PCR amplification Amplification of the target sequence occurs in vitro through a reaction catalysed by a DNA polymerase in the presence of oligonucleotide primers and deoxyribonucleoside triphosphates in a defined reaction buffer1, 2.A
46、n important prerequisite for the amplification of the target sequence is that the reaction mixture contains no polymerase inhibitors. Amplification of the DNA is a cyclical process consisting of denaturation of the double-stranded DNA into single-stranded nucleic acid by means of heating, annealing
47、of the primers to the target sequence at a suitable temperature, and extension of the primers, which are bound to both single strands, by a DNA polymerase suitable for PCR, at an appropriate temperature. 4.3 Detection and confirmation of PCR products PCR products are detected by gel electrophoresis
48、or an appropriate alternative, if necessary, after isolation by means of a suitable separation procedure. The identity of any detected target sequence can be verified by an appropriate technique (e.g. by restriction enzyme analysis, by hybridization or by DNA sequence analysis). In the case of real-time PCR analysis, amplification and detection occur simultaneously. 5 Reagents It is generally advisable to store the reaction solutions required for the analytical method at approximatel
