1、Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 5: Real-time PCR based screening method for the detection of the FMV promoter (P-FMV) DNA sequence PD ISO/TS 21569-5:2016 BSI Standards Publication WB
2、11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National foreword This Published Document is the UK implementation of ISO/TS 21569-5:2016. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations represente
3、d 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 0 580
4、93943 3 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 PUBLISH
5、ED DOCUMENT PD ISO/TS 21569-5:2016 ISO 2016 Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 5: Real-time PCR based screening method for the detection of the FMV promoter (P-FMV) DNA sequence Mthodes
6、 horizontales danalyse molculaire de biomarqueurs Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs Partie 5: Mthode de dpistage PCR en temps rel pour la dtection de la squence ADN du promoteur FMV (P-FMV) TECHNICAL SPECIFICATION ISO/TS 21569-5 Reference numb
7、er ISO/TS 21569-5:2016(E) First edition 2016-11-01 PD ISO/TS 21569-5:2016 ISO/TS 21569-5:2016(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 o
8、therwise 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 off
9、ice 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 21569-5:2016 ISO/TS 21569-5:2016(E)Foreword iv 1 Scope . 1 2 Normative references 1 3 Terms and definitions . 1 4 Principle 2 5 Reagents and materials
10、 . 2 5.1 General . 2 5.2 PCR reagents . 2 6 Apparatus . 2 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 . 3 8 Accept/reject criteria 4 8.1 General . 4 8.2 Identification 4 9 Validation status and performance criteri
11、a . 4 9.1 General . 4 9.2 Robustness . 5 9.3 Collaborative trial . 5 9.4 Sensitivity 6 9.5 Specificity 7 10 Test report . 8 Annex A (informative) Detection of the Figwort mosaic virus (FMV) open reading frame VII .9 Bibliography .10 ISO 2016 All rights reserved iii Contents Page PD ISO/TS 21569-5:20
12、16 ISO/TS 21569-5:2016(E) 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 interested
13、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 Electrotechnical Commi
14、ssion (IEC) on all matters of electrotechnical standardization. The procedures 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 s
15、hould be noted. This document was drafted in accordance with the editorial rules 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
16、 identifying any or all such patent rights. Details of any patent rights identified 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 c
17、onvenience of users and does not constitute an endorsement. For an explanation 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)
18、 see the following URL: www.iso.org/iso/foreword.html. The committee responsible 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
19、rights reserved PD ISO/TS 21569-5:2016 TECHNICAL SPECIFICATION ISO/TS 21569-5:2016(E) Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 5: Real-time PCR based screening method for the detection of the
20、 FMV promoter (P-FMV) DNA sequence 1 Scope This document specifies a procedure for the detection of a DNA sequence used in genetically modified (GM) plants by means of a real-time PCR (polymerase chain reaction). The method detects a 78 base pairs long segment of the Figwort mosaic virus 34S promote
21、r DNA sequence. This segment in some GM plants is indicated as FMV promoter (P-FMV) and in other GM plants as FMV enhancer (E-FMV). The method was developed and validated for the analysis of DNA extracted from foodstuffs. It may be suitable also for analysis of other products such as feedstuffs and
22、seeds. The procedure requires the extraction of an adequate quantity and quality of amplifiable DNA from the test sample. The DNA sequence amplified by the P-FMV element-specific method can be detected in samples which contain DNA of the naturally occurring Figwort mosaic virus. For this reason, it
23、is necessary to confirm a positive 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.
24、 For dated references, only the 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
25、 based methods ISO 21570, Foodstuffs 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 ac
26、id extraction ISO 24276, Foodstuffs 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 and IEC maintain te
27、rminological 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 ISO 2016 All rights reserved 1 PD ISO/TS 21569-5:2016 ISO/TS 21569-5:2016(E) 4 Principle DNA i
28、s extracted from the test portion applying a suitable method (see ISO 21571). The DNA analysis consists of two parts: a) verification of the amount, quality and amplifiability of the extracted DNA, e.g. by a taxon-specific PCR assay (according to ISO 21569 and ISO 21570), see alsoReference 1; b) det
29、ection of the P-FMV DNA sequence in a real-time PCR, see Reference 2. 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
30、 by dissolving the corresponding reagents in water and be autoclaved. For all operations for which gloves are used it should be ensured that these are powder-free. The use of aerosol protected pipette tips (protection against cross contamination) is recommended. 5.2 PCR reagents 5.2.1 Thermostable D
31、NA polymerase (for hot-start PCR). 5.2.2 PCR buffer solution (containing magnesium chloride and deoxyribonucleoside triphosphates dNTPs. Ready-to-use reagent mixtures or mixtures of individual components can be used. Reagents and polymerases which lead to equal or better results may also be used. 5.
32、2.3 Oligonucleotides (see Table 1) 1) . Table 1 Oligonucleotides Name DNA sequence of the oligonucleotide Final concentration in PCR P-FMV as the target sequence (GeneBank accession number X06166 2,3 ): pFMV-F 5-CAA AAT AAC GTG GAA AAG AGC T3 340 nmol/l pFMV-R 5-TCT TTT GTG GTC GTC ACT GC3 340 nmol/
33、l Probe pFMV 5-(FAM)-CTG ACA GCC CAC TCA CTA ATG C-(BHQ1)-3 a 120 nmol/l aFAM: 6-Carboxyfluorescein, BHQ-1: Black Hole Quencher 1 (non-fluorescent chromophore). This information is given for convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equ
34、ivalent products from other manufacturers may be used if they can be shown to give equivalent or better results. 6 Apparatus Requirements concerning apparatus and materials shall be according to ISO 21569. In addition to the usual laboratory equipment, the following equipment is required. 6.1 Real-t
35、ime PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence signals generated during PCR. 1) In the interlaboratory trial performed for P-FMV, participants were provided with dried aliquots (per 50 reactions) of primer/probe -mixes (to be stored in dark unt
36、il the start of the interlaboratory trial). Per aliquot 375 l PCR grade water was added and allowed to settle.2 ISO 2016 All rights reserved PD ISO/TS 21569-5:2016 ISO/TS 21569-5:2016(E) 7 Procedure 7.1 Preparation of test sample It should be ensured that the test sample used for DNA extraction is r
37、epresentative of the laboratory sample, e.g. by grinding or homogenizing of the laboratory sample. Measures and operational steps to be taken into consideration should be according to ISO 21571 and ISO 24276. 7.2 Preparation of DNA extracts Concerning the preparation of DNA from the test portion the
38、 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 described applies for a total volume of 25 l per PCR. The reaction setup is given in Table 2. Reag
39、ents 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 DNA. The required amount of the PCR reagent mixture depends on the number
40、 of reactions to be performed, including at least one additional reaction as a pipetting reserve. Add 5 l of sample DNA to each reaction. Table 2 Reaction setup for the amplification Total reaction volume 25 l Sample DNA (up to 200 ng) or controls 5 l PCR buffer solution a(including MgCl 2 , dNTPs a
41、nd hot-start DNA polymerase) 12,5 l Primer pFMV-F and pFMV-R see Table 1 Probe pFMV see Table 1 Water to 25 l aIn the collaborative trial the QuantiTect Multiplex PCR NoROX Kit (Qiagen GmbH, Hilden/Germany) was used. This information is given for convenience of users of this document and does not co
42、nstitute an endorsement by ISO of the product named. Equivalent products from other manufacturers may be used if they yield similar or better results. If necessary, adapt the amounts of the reagents and the temperature-time programme. Mix the PCR reagent mixture, centrifuge briefly and pipette 20 l
43、into each reaction vial. For the amplification reagent control, add 5 l of water into the respective reaction setup. 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 d
44、escribed in ISO 24276. Transfer the reaction setups into the thermal cycler and start the temperature-time programme. 7.4 Temperature-time programme The temperature-time programme as outlined in Table 3 has been used in the validation study. The use of different reaction conditions and real-time PCR
45、 cyclers may require specific optimization. The time for initial denaturation depends on the master mix used. ISO 2016 All rights reserved 3 PD ISO/TS 21569-5:2016 ISO/TS 21569-5:2016(E) Table 3 Temperature-time programme Step Parameter Temperature Time Fluorescence measurement Cycles 1 UNG activati
46、on (optional) 50 C 2 min no 1 2 Initial denaturation 95 C 15 min no 1 3 Amplification Denaturation 95 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 programme is used for the identification of PCR p
47、roducts. 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 amplificatio
48、n of the DNA target sequence in a sample (e.g. positive controls) occurred, a sigmoid shaped amplification curve should be observed. The cycle number at the crossing point of the amplification curve and the fluorescence threshold is calculated (C tvalue or C pvalue). If, due to atypical fluorescence
49、 measurement data, the automatic interpretation does not provide a meaningful result, it may be required to set the baseline and the threshold manually prior to interpreting the data. In this case, the device-specific instructions given in the manual regarding the use of the interpretation software should be applied. A positive result can be also obtained if the sample contains DNA derived from the Figwort mosaic virus, which can naturally infect plants. To proof the pr
