CEN TS 16707-2014 Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Polymerase chain reaction (PCR) based screening strate.pdf

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1、BSI Standards PublicationFoodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Polymerase chain reaction (PCR) based screening strategiesPD CEN/TS 16707:2014National forewordThis Published Document is the UK implementation of CEN/TS 16707:2014.The UK

2、 participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions ofa contract. Us

3、ers are responsible for its correct application. The British Standards Institution 2014.Published by BSI Standards Limited 2014ISBN 978 0 580 83732 6ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of

4、theStandards Policy and Strategy Committee on 31 October 2014.Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 16707:2014TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16707 October 2014 ICS 67.050 English Version Foodstuffs

5、- Methods of analysis for the detection of genetically modified organisms and derived products - Polymerase chain reaction (PCR) based screening strategies Produits alimentaires - Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs - Stratgies de criblage bases

6、 sur lutilisation de la raction de polymrisation en chane (PCR) Lebensmittel - Verfahren zum Nachweis von gentechnisch vernderten Organismen und ihren Produkten - Strategien fr das Screening mit Polymerase-Kettenreaktion (PCR) This Technical Specification (CEN/TS) was approved by CEN on 28 June 2014

7、 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required

8、to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversi

9、on of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxemb

10、ourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussel

11、s 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16707:2014 EPD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 2 Contents Page Foreword 3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 Principle 6

12、 5 Reagents .7 5.1 General 7 5.2 PCR reagents .7 6 Apparatus and equipment 7 7 PCR analysis 7 7.1 General 7 7.2 Screening 7 7.2.1 General 7 7.2.2 Combination of targets .8 7.2.3 Analysis of the output of the first screening 9 7.2.4 Additional screening tests 9 7.3 GM event identification .9 7.3.1 Ev

13、ent specific tests .9 7.3.2 Additional tests . 10 7.4 Interpretation of PCR results . 10 7.4.1 General . 10 7.4.2 Interpretation of results at the limit of detection (LOD) 10 8 PCR method performance criteria and validation . 11 8.1 General . 11 8.2 Absolute limit of detection (LODabs) . 12 8.3 Spec

14、ificity and reference materials 12 8.4 Robustness . 13 8.5 False-positive rate and false-negative rate 13 8.6 Probability of Detection (POD) 13 Bibliography . 14 PD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 3 Foreword This document (CEN/TS 16707:2014) has been prepared by Technical Committee CEN/TC 275

15、 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. 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. Accord

16、ing to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germ

17、any, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 4 Introduction Largely, detection of materials

18、 derived from genetically modified organisms (GMOs) in a given sample employs polymerase chain reaction (PCR) analysis, specifically real-time PCR. A general strategy for GMO detection and identification by means of PCR analysis and a stepwise approach is described. In initial screening analysis, DN

19、A sequences of genetic elements common to many GMOs are targeted. According to its purpose, screening is a test to rapidly and reliably sort samples into groups. Once the samples are grouped, screening facilitates and potentially reduces subsequent analytical work and results interpretation. The scr

20、eening strategy should be adjusted to the scope (food, feed or seed, crop-specific etc.) of the test(s). This document takes the general principle of GMO detection strategies as a basis and describes the underlying analytical steps for complex screening (known as the matrix-approach 2). The document

21、 is written primarily for screening strategies applying real-time PCR methods. Other PCR methodologies may be applicable in the same way. The terms “screening method” and “screening strategies” are not interchangeable and have different meanings in this document. PD CEN/TS 16707:2014CEN/TS 16707:201

22、4 (E) 5 1 Scope This Technical Specification describes screening strategies for the detection of genetically modified (GM) DNA in food products by means of PCR methods. The strategies have been established for food matrices, but it can also be applied to other matrices (e.g. feed, seed and samples f

23、rom field grown plants). Detection of GM DNA is based on PCR methods targeting segments of transgenic DNA sequences (genetic elements, genetic constructs or insertion sites of transgenes). Various combinations of these PCR methods are involved in screening strategies. The methods are applied simulta

24、neously or hierarchically. The general strategy is based on the matrix approach. Examples for the implementation and application of this approach are described. In order to ensure reliable analytical results, the document also provides guidelines for the validation of the performance of qualitative

25、PCR methods applied in screening approaches. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of

26、 the referenced document (including any amendments) applies. EN ISO 21569, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods (ISO 21569) EN ISO 21570, Foodstuffs - Methods of analysis for the detection o

27、f genetically modified organisms and derived products - Quantitative nucleic acid based methods (ISO 21570) EN ISO 21571, Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction (ISO 21571) EN ISO 24276, Foodstuffs - Methods

28、 of analysis for the detection of genetically modified organisms and derived products - General requirements and definitions (ISO 24276) 3 Terms and definitions For the purposes of this document, the terms and definitions given in EN ISO 24276 and the following apply: 3.1 GMO method matrix relationa

29、l presentation (e.g. a table) of symbols or numbers Note 1 to entry: One dimension (e.g. columns) corresponds to genetic elements and genetic constructs detected by a defined PCR method and the other dimension (e.g. rows) corresponds to GM events. The entered symbols or numbers indicate the detectab

30、ility or non-detectability of the target sequence for the GM event. Note 2 to entry: The term matrix is commonly used for a defined composition of food, but this definition is not relevant here. 3.2 GMO target matrix relational presentation (e.g. a table) of symbols or numbers Note 1 to entry: One d

31、imension corresponds to genetic elements or genetic constructs present in a GMO and the other dimension (e.g. rows) corresponds to GM events. The entered symbols or numbers indicate the presence or absence of the target for the GM event and copy number, if available. Note 2 to entry: In contrast to

32、GMO method matrix, the GMO target matrix is independent from a detection method. PD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 6 3.3 screening method method that rapidly and reliably eliminates (screens) a large number of negative (or positive) test samples and restricts the number of test samples requi

33、ring the application of a rigorous method Note 1 to entry: In this document, a screening method refers to PCR methods detecting the presence of several GMOs in one test. 3.4 element-specific method method that targets a single discrete DNA sequence of a specific genetic element Note 1 to entry: A ge

34、netic element is a part of a gene, for example a promoter, terminator, intron or a coding sequence. Elements are often derived from naturally occurring viruses, bacteria, plants, etc However, elements in GMOs are commonly modified at the sequence level, relative to the original (natural) source, e.g

35、. by altered codon-usage or specific single nucleotide polymorphisms (SNPs). This sequence modification may be based on adaptation of the nucleotide composition to the new host genome. 3.5 construct specific method method that targets a combination of inserted DNA sequences that are only found in GM

36、O-derived material composed of at least two elements that do not naturally co-exist in this conformation, and where the 5 and 3 end of the sequence are derived from separate genetic elements 3.6 event specific method method that detects a specific sequence that is only present in that event Note 1 t

37、o entry: The event may be the result of either a rearrangement or unique combination of insert and insertion locus during the transformation. Note 2 to entry: This is commonly targeted at the integration-border region. 3.7 probability of detection (POD) probability of a positive analytical outcome f

38、or a qualitative method for a given matrix at a given concentration Note 1 to entry: It is estimated by the expected proportion of positive results for the given matrix at the given analyte concentration. 4 Principle Total DNA is extracted from the sample by a suitable extraction method. The DNA qua

39、ntity and quality shall be checked as specified in EN ISO 21571 to ensure the presence of sufficient analyte and to assess the presence of PCR inhibitors that could inhibit PCR amplification. Subsequently, a set of validated PCR methods are selected and applied in a decision tree approach, for detec

40、tion and identification of genetic modifications linked to the sample. This approach enables the user to choose between different courses of action. Commonly, screening starts with element-specific PCR tests prior to construct-specific and/or event-specific tests. Before, in parallel or after screen

41、ing, taxon-specific tests are recommended. Based on the combination of results at each level of the decision tree the total number of tests required for the analysis is reduced. PD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 7 5 Reagents 5.1 General The requirements and conditions for nucleic acid extrac

42、tion and PCR analysis laid down in detail in EN ISO 24276, EN ISO 21569, EN ISO 21570 and EN ISO 21571 shall be followed. Only analytical grade reagents suitable for molecular biology shall be used. 5.2 PCR reagents 5.2.1 Concentrated PCR buffer solution1)(containing reaction buffer, dNTPs, MgCl2and

43、 Hotstart Taq polymerase) or equivalent. 5.2.2 Oligonucleotides at the concentrations specified in the method protocol. 6 Apparatus and equipment See EN ISO 24276, EN ISO 21569, EN ISO 21570 and EN ISO 21571. 7 PCR analysis 7.1 General A large number of validated PCR methods applicable for the detec

44、tion of GM DNA in food samples are available in EN ISO 21569, EN ISO 21570 or are compiled in 3 and 4. The complexity of these methods can be divided in four categories according to the targeted DNA sequence: taxon-specific (for the detection and identification of DNA of a species or a taxonomical g

45、roup); element-specific (for the detection and identification of genetic elements present in more than one GMO); construct-specific (for the detection and identification of specific combinations of genetic elements present in a GMO as a result of genetic engineering); event-specific (for the detecti

46、on and identification of the insertion site specific for a GM event). Testing laboratories develop a strategy for selecting the methods according to their scope (food, feed or seed testing), and requirements of their clients (e.g. government authority or company). In addition, the incidence of produ

47、cts on the market containing or consisting of new GMOs may require the ad hoc implementation of new methods into an existing screening strategy in order to incorporate the detection and identification of the respective GM target DNA. To confirm the identity of the PCR product generated by the PCR me

48、thod, the method shall include a step for verification of the amplicon by an appropriate technique (e.g. probe hybridization, subsequent DNA sequence analysis or restriction enzyme digestion of the PCR product, respectively). 7.2 Screening 7.2.1 General The first step of PCR based screening analysis

49、 is the selection of a defined set of methods targeting DNA-sequences present in several GM events. The set of methods selected depends on the availability of (validated) methods, the capability of the laboratory and the scope of the testing. 1)Ready-to-use reagent mixtures or single components may be used as PCR buffer solution. If other reagent mixtures are used than the ones stated in the method validation report, they should give comparable to or better results. PD CEN/TS 16707:2014CEN/TS 16707:2014 (E) 8 The selection of methods shou