《ISO-21570-2005.pdf》由会员分享，可在线阅读，更多相关《ISO-21570-2005.pdf（110页珍藏版）》请在三一文库上搜索。
1、 Reference number ISO 21570:2005(E) ISO 2005 INTERNATIONAL STANDARD ISO 21570 First edition 2005-11-01 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methods Produits alimentaires Mthodes danalyse pour la dtecti
2、on des organismes gntiquement modifis et des produits drivs Mthodes quantitatives bases sur lutilisation des acides nucliques Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo re
3、production or networking permitted without license from IHS -,-,- ISO 21570:2005(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed
4、 to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobes licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the s
5、oftware products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to i
6、t is found, please inform the Central Secretariat at the address given below. ISO 2005 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permi
7、ssion in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2005 All rights reser
8、ved Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO 21570:2005(E) ISO 2005 All rights reserved iii Conte
9、nts Page Foreword. v Introduction. vi 1 Scope . 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 2 4.1 General. 2 4.2 Amplification, detection and confirmation of PCR products. 2 4.3 Quantitation of PCR products . 2 5 Reagents 2 6 Apparatus and equipment . 2 7 Guidelines concernin
10、g the procedure 3 7.1 General. 3 7.2 Target sequence stability. 3 7.3 Calibration of the analysis. 3 7.4 Quantitation considerations 3 7.5 Quality assurance requirements. 3 8 Interpretation. 4 9 Expression of results . 4 10 Test report . 5 Annex A (informative) Target taxon-specific methods. 6 A.1 T
11、arget taxon-specific method for the absolute quantitation of the adh1 gene DNA of maize using real-time PCR. 6 Annex B (informative) Screening methods 12 B.1 Screening method for the relative quantitation of the 35S-promoter DNA of soya bean line GTS 40-3-2 using real-time PCR 12 Annex C (informativ
12、e) Construct-specific methods . 20 C.1 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using real-time PCR (Method 1). 20 C.2 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using real-time PCR (Method 2). 27 C.3 Construct-specific meth
13、od for the quantitation of Event176 maize DNA using real-time PCR. 34 C.4 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using real-time PCR 41 C.5 Construct-specific method for the quantitation of maize line MON 810 DNA using real-time PCR 49 C.6 Construct-specific
14、method for the quantitation of maize line Event176 DNA using real-time PCR 56 C.7 Construct-specific method for the quantitation of maize line Bt11 DNA using real-time PCR. 63 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/11111
15、11164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO 21570:2005(E) iv ISO 2005 All rights reserved C.8 Construct-specific method for the quantitation of maize line GA21 DNA using real-time PCR.71 C.9 Construct-specific method for the
16、 quantitation of maize line T25 DNA using real-time PCR 78 Annex D (informative) Event-specific methods.87 D.1 Event-specific method for the absolute and relative quantitation of maize line Bt11 DNA based on real-time PCR87 D.2 Event-specific method for the relative quantitation of maize line MON 81
17、0 DNA using real-time PCR.93 Bibliography .100 Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO 21570:200
18、5(E) ISO 2005 All rights reserved v Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body i
19、nterested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechn
20、ical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by th
21、e technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights
22、. ISO shall not be held responsible for identifying any or all such patent rights. ISO 21570 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food Analysis Horizontal methods, in collaboration with Technical Committee ISO/TC 34, Food products, in accor
23、dance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted w
24、ithout license from IHS -,-,- ISO 21570:2005(E) vi ISO 2005 All rights reserved Introduction The search for ingredients of genetically modified origin is performed by means of the following successive (or simultaneous) steps. After sample collection, nucleic acids are extracted from the test portion
25、. Extracted nucleic acids can be further purified, 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 the present and in the following International S
26、tandards: ISO 21569, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methods ISO 21570, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic
27、acid based methods Further information about definitions and general items involving the steps cited above are collected in: ISO 24276, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products General requirements and definitions. The International Orga
28、nization for Standardization (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 these patent rights. ISO has been informed that App
29、lied Biosystems, Roche Molecular Systems, Inc. and Hoffman-La Roche hold patent rights concerning 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
30、respect, the statements 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
31、 94501, USA Attention is drawn to the possibility that some of the elements of this document may be subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights. Copyright International Organization for Standardization Pr
32、ovided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted without license from IHS -,-,- INTERNATIONAL STANDARD ISO 21570:2005(E) ISO 2005 All rights reserved 1 Foodstuffs Methods of analysis for the detectio
33、n of genetically modified organisms and derived products Quantitative nucleic acid based methods 1 Scope This International Standard provides the overall framework of quantitative methods for the detection of genetically modified organisms (GMOs) in foodstuffs, using the polymerase chain reaction (P
34、CR). It defines general requirements for the specific amplification of DNA target sequences, in order to quantify the relative GMO-derived DNA content and to confirm the identity of the amplified DNA sequence. Guidelines, minimum requirements and performance criteria laid down in this International
35、Standard are intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories. This International Standard has been established for food matrices, but is also applicable to other matrices, e.g. feed and plant samples from the environment. Specific examples
36、 of methods are provided 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 ame
37、ndments) applies. ISO 21569:2005, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methods ISO 21571, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic
38、acid extraction ISO 24276:1), Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products General requirements and definitions ISO Guide 32, Calibration in analytical chemistry and use of certified reference materials 3 Terms and definitions For the purpos
39、es of this document, the terms and definitions given in ISO 24276 apply. 1) To be published. Copyright International Organization for Standardization Provided by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitte
40、d without license from IHS -,-,- ISO 21570:2005(E) 2 ISO 2005 All rights reserved 4 Principle 4.1 General Quantitative analysis consists of the quantitation of target DNA sequences in the test samples. Each method specifies the target sequences(s). Quantitation may be performed using competitive 1,2
41、 or real-time PCR 3,4. A quantitative analysis should clearly express the quantity of the target genetic element, relative to the quantity of a specific reference, appropriate calibrants and controls, and be within the dynamic range of the analytical method used and the test portion analysed. The an
42、alysis generally consists of amplification of one or more specific target sequences, detection and confirmation of the specificity of the PCR product(s), and quantitation of the amplified fragments relative to calibrants. NOTE In the case of real-time PCR analysis, amplification, detection and confi
43、rmation occur simultaneously. 4.2 Amplification, detection and confirmation of PCR products See ISO 21569 for the principles of amplification, detection and confirmation of the DNA sequences. 4.3 Quantitation of PCR products The principle of quantitation is usually to determine the ratio (expressed
44、as a percent) of two DNA target sequences; i.e. a sequence representing the genetically modified organism of interest and an (endogenous) target taxon-specific sequence. However, in some cases, quantitation can also be carried out relative to a specified amount of food matrix (e.g. when detecting GM
45、 microorganisms in foods). Calibrants (calibration materials) used for quantitation should be traceable to certified reference materials (CRMs), if available. If not available, other suitable reference material should be used. Example guidance is provided in Reference 5. Information on validation st
46、udies and measurement uncertainty has been gathered from international studies 6,7,8,9. 5 Reagents All reagents and materials used in the analysis should be identical, or equivalent, to those specified in the method. Otherwise, all reagents and materials should be of molecular biology grade. These r
47、eagents shall be stored and used as recommended by the supplier or according to the laboratory quality assurance specifications. For a list of reagents, see the specific annex. 6 Apparatus and equipment See Annexes A to D and ISO 24276. Copyright International Organization for Standardization Provid
48、ed by IHS under license with ISO Licensee=HP Monitoring/1111111164 Not for Resale, 12/04/2007 20:53:27 MSTNo reproduction or networking permitted without license from IHS -,-,- ISO 21570:2005(E) ISO 2005 All rights reserved 3 7 Guidelines concerning the procedure 7.1 General General considerations relevant to PCR amplification for the detection of GMOs are described in ISO 21569. Annexes A to D specify PCR detection me