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1、 Reference number ISO 21571:2005(E) ISO 2005 INTERNATIONAL STANDARD ISO 21571 First edition 2005-02-15 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction Produits alimentaires Mthodes danalyse pour la dtection des organisme
2、s gntiquement modifis et des produits drivs Extraction des acides nucliques ISO 21571:2005(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 ar
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5、ating to it 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, wit
6、hout permission 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 ri
7、ghts reserved ISO 21571:2005(E) ISO 2005 All rights reserved iii Contents Page Forewordiv Introduction v 1 Scope1 2 Normative references .1 3 Principle.1 3.1 General.1 3.2 DNA extraction 2 3.3 DNA quantitation.2 4 General laboratory requirements 2 5 Procedure.2 5.1 Preparation of the test portion.2
8、5.2 DNA extraction/purification4 5.3 Quantitation of the extracted DNA5 5.4 Stability of extracted DNA6 6 Interpretation.6 7 Test report6 Annex A (informative) Methods for DNA extraction7 Annex B (informative) Methods for the quantitation of the extracted DNA34 Bibliography .41 -,-,- ISO 21571:2005(
9、E) iv ISO 2005 All rights reserved 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 in
10、terested 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 Electrotechni
11、cal 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 the
12、 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. ISO 21571 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food analy
13、sis Horizontal methods, in collaboration with Technical Committee ISO/TC 34, Food products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). -,-,- ISO 21571:2005(E) ISO 2005 All rights reserved v Introduction The search for genetically modified origin
14、 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, simultaneously or after the extraction process. Afterwards, they are quantified (if
15、necessary), diluted (if necessary) and subjected to analytical procedures (such as PCR). These steps are detailed in this and the following International Standards: ISO 21568, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Sampling. ISO 21569,
16、 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 acid based methods.
17、 Further information about definitions and general items involving the steps cited above are collected in: ISO 24276, Foodstuffs Nucleic acid based methods of analysis for the detection of genetically modified organisms and derived products General requirements and definitions. The International Org
18、anization 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 silica-based extraction method (No. EP 0389063/USP 5,234,809) given in Clause A.4. ISO takes no position concerning the evidence, valid
19、ity and scope of this patent right. The holder of this patent right has assured the ISO that he/she is willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the statement of the holder of this patent right is
20、 registered with ISO. Information may be obtained from: Jean Deleforge, BioMrieux Chemin de lOrme, 69280 Marcy-ltoile, France. Attention 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
21、 responsible for identifying any or all such patent rights. -,-,- INTERNATIONAL STANDARD ISO 21571:2005(E) ISO 2005 All rights reserved 1 Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction 1 Scope This International Standar
22、d provides general requirements and specific methods for DNA extraction/purification and quantitation. These methods are described in Annexes A and B. This International Standard has been established for food matrices, but could also be applicable to other matrices, such as grains and feed. It has b
23、een designed as an integral part of nucleic-acid-based analytical methods, in particular ISO 21569 on qualitative analytical methods, and ISO 21570 on quantitative analytical methods. 2 Normative references The following referenced documents are indispensable for the application of this document. Fo
24、r dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 24276:1), Foodstuffs Nucleic acid based methods of analysis for the detection of genetically modified organisms and derived products Gener
25、al requirements and definitions ISO 21568, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Sampling 3 Principle 3.1 General The objective of nucleic acid extraction methods is to provide nucleic acids suitable for subsequent analysis (see ISO 2
26、4276). NOTE The “quality” of DNA depends on the average length of the extracted DNA molecules, the chemical purity and the structural integrity of the DNA sequence and of the double helix (e.g. intra-, inter-strand linking between DNA bases, single-strand gaps, cross-linking with polyols, haemin, et
27、c). Moreover, such alterations are often sequence-specific and consequently not randomly distributed all over the genome (see References 1, 2, 3 and 4). Users of this International Standard should note that some methods (e.g. all silica-based methods), might be covered by patent rights. 1) To be pub
28、lished. ISO 21571:2005(E) 2 ISO 2005 All rights reserved 3.2 DNA extraction The basic principle of DNA extraction consists of releasing the DNA present in the matrix and further, concurrently or subsequently, purifying the DNA from polymerase chain reaction (PCR) inhibitors. DNA extraction/purificat
29、ion methods are described in Annex A. Method-selection is an experience-based choice of the user, taking into account the scope and examples of matrices as given in each method. Alternative protocols are suitable provided that the method has been validated on the respective matrix under investigatio
30、n. 3.3 DNA quantitation Quantitation of extracted DNA could be useful for subsequent PCR analysis. It may be performed by either physical (e.g. measure of absorbance at a specific wavelength), chemical- physical (e.g. use of intercalating or binding agents able to emit fluorescence), enzymatic (e.g.
31、 bioluminescence detection) methods or by quantitative PCR. The latter method is especially suitable for composite matrices or for samples with a low DNA content or whose DNA is degraded. There are several methods available to quantify the DNA present in a solution, as described in Annex B. It is fo
32、r the user to choose the most appropriate one to be applied, depending on the amount and quality of DNA to be quantified and, consequently, on the matrix from which the DNA has been extracted. Alternative protocols are suitable, provided that the method has been validated on the respective matrix un
33、der investigation. 4 General laboratory requirements Accidental contamination of DNA can originate from dust and spreading aerosols. As a consequence, the organization of the work area in the laboratory is logically based on the systematic containment of the methodological steps involved in the prod
34、uction of the results, and a “forward flow” principle for sample handling. The latter ensures that the DNA to be analysed and the amplified DNA generated by PCR remain physically segregated. Further details can be found in ISO 24276. 5 Procedure 5.1 Preparation of the test portion 5.1.1 General Comm
35、odity-specific variables (e.g. humidity) and processing can impact the amount and quality of DNA extracted from the material under investigation. Therefore the performance characteristics of a given DNA extraction method depend on the matrix. Take appropriate measures to ensure that the test portion
36、 is representative of the laboratory sample. The test portion shall be of sufficient size and shall contain a sufficient number of particles to be representative of the laboratory sample (e.g. 3 000 particles at an LOD of 0,1 %) to allow a statistically valid conclusion to be made (see ISO 21568). -
37、,-,- ISO 21571:2005(E) ISO 2005 All rights reserved 3 For practical/technical reasons, it is not recommended to exceed a size of 2 g. Any restrictions that arise from the size of the test portion which prevent it from being representative shall be reported and taken into consideration in the interpr
38、etation of the analytical results. The methods for DNA extraction in Annex A describe test portions from 200 mg to 500 mg, which are usually adequate for DNA-rich raw materials (e.g. ground grains, flour). However, for certain matrices containing very low amounts or degraded DNA, insufficient DNA su
39、itable for analysis can be extracted. In these cases, the test portion may be increased. DNA extractions shall be carried out at least on two test portions. Storage of standards, samples and test portions shall comply with ISO 24276 and shall be organized in such a way as to preserve the biochemical
40、 parameters to be analysed (for details, see ISO/IEC 17025). 5.1.2 Samples All operations for the preparation of test samples (e.g. grinding, homogenization, division, drying) shall be carried out in accordance with the procedures described in ISO 24276, taking care to prevent all contamination of t
41、he sample or modification of its composition. Laboratory samples shall be sufficiently homogeneous before reducing the laboratory sample and taking the test portion. For liquid samples, shake the vessel containing the sample to improve the homogenization of the product. In the case of non-homogenous
42、 products like raw oils, check that the sediments have been completely removed from the walls of the vessel. For solid matrices that cannot easily be suspended, the matrix shall be ground to reduce the particle size and/or facilitate the extractability of DNA. In such a case, attention shall be paid
43、 to the particle size. The test portion subjected to extraction shall contain a minimum number of particles as specified in ISO 21568. Milling/grinding devices should be capable of being thoroughly cleaned and shall be selected in order to achieve the expected particle number and particle size distr
44、ibution within the test portion as defined in ISO 21568. If components of the laboratory sample have been removed prior to extraction, then such procedures shall be reported. Final food products that are solid or paste and have high lipid contents are often not easy to grind to the desired particle
45、size in a single step. Several procedures may therefore be added, such as lipid removal using hexane after intermediate grinding, freezing or freeze-drying before grinding. In order to facilitate the grinding of paste or viscous products, it is possible to apply one of the following treatments to ce
46、rtain matrices: heating to a maximum temperature of 40 C; dissolving in an appropriate liquid such as water; freezing at a temperature below or equal to 20 C. Homogenize the whole laboratory sample. Sample the two test portions, taking into account possible dilutions or concentrations. During millin
47、g/grinding, precautions should be taken to ensure that the heating of the sample is kept to a minimum since heating can have a negative impact on the quality of the extracted DNA. Milling/grinding techniques with a high risk of cross-contamination (such as the combined use of liquid nitrogen and mor
48、tar) shall be avoided as far as possible. As a rule of good practice, any dust-producing methodological step should be contained from all other analytical steps. -,-,- ISO 21571:2005(E) 4 ISO 2005 All rights reserved If salts, spices, powdered sugars and/or other substances that could potentially interfere with the extraction or analytical method are present, appropriate purification steps should be considered according to the selected method (see Annex A). For example, in samples from composite matrices, the target matrix (e.g. the breading layer of fish