1、Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 4: Real-time PCR based screening methods for the detection of the P-nos and P-nos-nptII DNA sequences PD ISO/TS 21569-4:2016 BSI Standards Publication
2、 WB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National foreword This Published Document is the UK implementation of ISO/TS 21569-4:2016. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations represe
3、nted on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016. Published by BSI Standards Limited 2016 ISBN 978 0 5
4、80 93942 6 ICS 67.050 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the Standards Policy and Strategy Committee on 30 November 2016. Amendments/corrigenda issued since publication Date Text affected PUBL
5、ISHED DOCUMENT PD ISO/TS 21569-4:2016 ISO 2016 Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 4: Real-time PCR based screening methods for the detection of the P-nos and P-nos-nptII DNA sequences M
6、thodes horizontales danalyse molculaire de biomarqueurs Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs Partie 4: Mthodes de dpistage PCR en temps rel pour la dtection des squences ADN P-nos et P-nos-nptII TECHNICAL SPECIFICATION ISO/TS 21569-4 Reference nu
7、mber ISO/TS 21569-4:2016(E) First edition 2016-11-01 PD ISO/TS 21569-4:2016 ISO/TS 21569-4:2016(E)ii ISO 2016 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in Switzerland All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized
8、 otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright o
9、ffice Ch. de Blandonnet 8 CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 09 47 copyrightiso.org www.iso.org PD ISO/TS 21569-4:2016 ISO/TS 21569-4:2016(E)Foreword iv 1 Scope . 1 2 Normative references 1 3 Terms and definitions . 1 4 Principle 2 5 Reagents and materia
10、ls . 2 5.1 General . 2 5.2 PCR reagents . 2 6 Apparatus . 3 7 Procedure. 3 7.1 Preparation of test sample 3 7.2 Preparation of DNA extracts 3 7.3 PCR setup . 3 7.4 Temperature-time programme . 4 8 Accept/reject criteria 4 8.1 General . 4 8.2 Identification 4 9 Validation status and performance crite
11、ria . 5 9.1 General 5 9.2 Robustness of the method . 5 9.3 Collaborative trial . 5 9.4 Sensitivity 7 9.5 Specificity 7 10 Test report . 9 Bibliography .10 ISO 2016 All rights reserved iii Contents Page PD ISO/TS 21569-4:2016 ISO/TS 21569-4:2016(E) Foreword ISO (the International Organization for Sta
12、ndardization) 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 interested in a subject for which a technical committee has been established has the righ
13、t 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 Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures
14、 used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rul
15、es of the ISO/IEC Directives, Part 2 (see www.iso.org/directives). Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights ident
16、ified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso.org/patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation
17、 on the meaning of ISO specific terms and expressions related to conformit y assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso.org/iso/foreword.html. The committee responsib
18、le for this document is ISO/TC 34, Food products, Subcommittee SC 16, Horizontal methods for molecular biomarker analysis. A list of all the parts in the ISO/TS 21569 series can be found on the ISO website.iv ISO 2016 All rights reserved PD ISO/TS 21569-4:2016 TECHNICAL SPECIFICATION ISO/TS 21569-4:
19、2016(E) Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 4: Real-time PCR based screening methods for the detection of the P-nos and P-nos-nptII DNA sequences 1 Scope This document specifies a proced
20、ure for the detection of a DNA sequence of the promoter region of the nopaline synthase gene (P-nos) from Agrobacterium tumefaciens and a procedure for the detection of the DNA transition sequence between P-nos and the neomycin-phosphotransferase gene (nptII) from the Tn5 transposon of Escherichia c
21、oli K12. The nos-promoter and the P-nos-nptII-construct are frequently found in genetically modified plants. The P-nos and P-nos-nptII specific methods are based on real- time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific genetically modi
22、fied plant (event) a follow-up analysis has to be carried out. The methods described are applicable for the analysis of DNA extracted from foodstuffs. They may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of these methods requir
23、es the extraction of an adequate amount of amplifiable DNA from the relevant matrix. The DNA sequence amplified by the P-nos element-specific method can be detected in samples which contain DNA of the naturally occurring Ti-plasmid of A. tumefaciens. For this reason, it is necessary to confirm a pos
24、itive screening result. Further analyses are required using construct-specific or event specific methods. 2 Normative references The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of this document. For dated references, only t
25、he edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 21569, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methods ISO 21570, Foo
26、dstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methods ISO 21571:2005, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extraction ISO 24276, Food
27、stuffs Methods of analysis for the detection of genetically modified organisms and derived products General requirements and definitions 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 16577 apply. ISO 2016 All rights reserved 1 PD ISO/TS 21569-4:201
28、6 ISO/TS 21569-4:2016(E) ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at http:/ /www.electropedia.org/ ISO Online browsing platform: available at http:/ /www.iso.org/obp 4 Principle DNA is extracted from the test sam
29、ple applying a suitable method (see ISO 21571). The DNA analysis consists of two parts: a) verification of the amount and amplifiability of the extracted DNA, e.g. by means of a target taxon specific real-time PCR (according to ISO 21570), see also Reference 1; b) detection of the P-nos and/or P-nos
30、-nptII sequence in a real-time PCR, see References 2, 3 and 4. 5 Reagents and materials 5.1 General For the purpose of this document, only chemicals and water of recognized analytical grade, appropriate for molecular biology shall be used. Unless stated otherwise, solutions should be prepared by dis
31、solving the corresponding reagents in water and be autoclaved. For all operations in which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected pipette tips as protection against cross contamination is recommended. 5.2 PCR reagents 5.2.1 Thermostable DNA pol
32、ymerase (for hot-start PCR). 5.2.2 PCR buffer solution (containing magnesium chloride and deoxyribonucleoside triphosphates, dNTPs). Ready-to-use reagent mixtures or mixes of individual components can be used. Reagents and polymerases which lead to equal or better results may also be used. 5.2.3 Oli
33、gonucleotides (see Tables 1 and 2). Table 1 Oligonucleotides for detection of P-nos element Name DNA sequence of the oligonucleotide Final concentration in the PCR P-nos as target sequence 4 : Primer p-nos-F1 5-AAg CAC ATA CgT CAg AAA CCA TTA TT-3 400 nmol/l Primer p-nos-R 5-TCA gTg gAg CAT TTT TgA
34、CAA gAA-3 400 nmol/l Probe p-nos-Tm 5-(FAM)-CgC gTT CAA AAg TCg CCT AAg gTC AC-(BBQ)-3 a 100 nmol/l aFAM: 6-Carboxyfluorescein, BBQ: BlackBerry Quencher. This information is given for convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent
35、 products from other manufacturers may be used if they can be shown to give equivalent or better results.2 ISO 2016 All rights reserved PD ISO/TS 21569-4:2016 ISO/TS 21569-4:2016(E) Table 2 Oligonucleotides for detection of P-nos-nptII construct Name DNA sequence of the oligonucleotide Final concent
36、ration in the PCR P-nos-nptII as target sequence 4 : Primer p-nos-F2 5-TTC CCC TCg gTA TCC AAT TAg Ag-3 400 nmol/l Primer NPTII-R 5-gAT TgT CTg TTg TgC CCA gTC A-3 400 nmol/l Probe NPTII-Tm2 5-(FAM)-AgC CgA ATA gCC TCT CCA CCC AAg C-(BBQ)-3 a 100 nmol/l aFAM: 6-Carboxyfluorescein, BBQ: BlackBerry Qu
37、encher. This information is given for convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or better results. 6 Apparatus Requirements concerning appa
38、ratus and materials shall be according to ISO 21569. In addition to the usual laboratory equipment, the following equipment is required. 6.1 Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence signals generated during PCR. 7 Procedure 7.1 Prep
39、aration of test sample It should be ensured that the test sample used for DNA extraction is representative of the laboratory sample, e.g. by grinding or homogenizing of the samples. Measures and operational steps to be taken into consideration are described in ISO 21571 and ISO 24276. 7.2 Preparatio
40、n of DNA extracts Concerning the preparation of DNA from the test sample, the general instructions and measures described in ISO 21571 shall be followed. It is recommended to choose one of the DNA extraction methods described in ISO 21571:2005, Annex A. 7.3 PCR setup The method is described for a to
41、tal volume of 25 l per PCR. The reaction setup is given in Table 3. Reagents are completely thawed at room temperature. Each reagent should be carefully mixed and briefly centrifuged immediately before pipetting. A PCR reagent mixture is prepared which contains all components except for the sample D
42、NA. The required amount of the PCR reagent mixture depends on the number of reactions to be performed, including at least one additional reaction as a pipetting reserve. Add 5 l of sample DNA to each reaction. Mix the PCR reagent mixture, centrifuge briefly and pipette 20 l into each reaction vial.
43、For the amplification reagent control, add 5 l of water into the respective reaction set-up. Pipette either 5 l of sample DNA or 5 l of the respective control solution (extraction blank control, positive DNA target control). If necessary, prepare a PCR inhibition control as described in ISO 24276. T
44、ransfer the reaction set-ups into the thermal cycler and start the temperature-time programme. ISO 2016 All rights reserved 3 PD ISO/TS 21569-4:2016 ISO/TS 21569-4:2016(E) Table 3 Components of the PCR reaction Total reaction volume 25 l Sample DNA (up to 200 ng) or controls 5 l PCR buffer solution
45、a(including MgCl 2 , dNTPs and hot-start DNA polymerase) 12,5 l Primers see Table 1 or 2 Probe see Table 1 or 2 Water add to obtain 25 l aIn the collaborative trial TaqMan Universal PCR Mastermix (Life Technologies) was used as PCR buffer solution. This information is given for convenience of users
46、of this document and does not constitute an endorsement by ISO of the product named. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or better results. If necessary, adapt the amounts of the reagents and the temperature-time programme. 7.4 Temperature
47、-time programme The temperature-time programme as outlined in Table 4 has been used in the validation study. The use of different reaction conditions and real-time PCR cyclers may require specific optimization. The time for initial denaturation depends on the master mix used. Table 4 Temperature-tim
48、e programme Step Parameter Temperature Time Fluorescence measurement Cycles 1 Initial denaturation 95 C 10 min no 1 2 Amplification Denaturation 94 C 15 s no 45 Annealing and elongation 60 C 60 s yes 8 Accept/reject criteria 8.1 General A corresponding real-time PCR device-specific data analysis pro
49、gramme is used for the identification of PCR products. The amplification results may be expressed in a different manner, depending on the device used. In the absence of detectable PCR products (e.g. negative controls) the result can be expressed as “undetermined”, “no amp”, or the maximum number of reaction cycles performed. If the amplification of the DNA target sequence in a sample (e.g. positive controls) occurred, a sigmoid shaped amplification curve shoul
