1、Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived productsPart 5: Real-time PCR based screening method for the detection of the FMV promoter (P-FMV) DNA sequencePD ISO/TS 21569-5:2016BSI Standards PublicationWB1188
2、5_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National forewordThis Published Document is the UK implementation of ISO/TS21569-5:2016.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this
3、 committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions ofa contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 93943 3ICS 67.
4、050Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 30 November 2016.Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD ISO/TS 2
5、1569-5:2016 ISO 2016Horizontal 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 sequenceMthodes horizontales danalyse mo
6、lculaire 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 SPECIFICATIONISO/TS21569-5Reference numberISO/TS 21569-5:2016(E)First
7、 edition2016-11-01PD ISO/TS 21569-5:2016ISO/TS 21569-5:2016(E)ii ISO 2016 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2016, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means
8、, 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 officeCh. de Blandonnet 8 CP 401CH-1214
9、Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgPD ISO/TS 21569-5:2016ISO/TS 21569-5:2016(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 25 Reagents and materials . 25.1 General . 25.2 PCR reagents . 26 Apparatus
10、 . 27 Procedure. 37.1 Preparation of test sample 37.2 Preparation of DNA extracts 37.3 PCR setup . 37.4 Temperature-time programme . 38 Accept/reject criteria 48.1 General . 48.2 Identification 49 Validation status and performance criteria . 49.1 General . 49.2 Robustness . 59.3 Collaborative trial
11、. 59.4 Sensitivity 69.5 Specificity 710 Test report . 8Annex A (informative) Detection of the Figwort mosaic virus (FMV) open reading frame VII . 9Bibliography .10 ISO 2016 All rights reserved iiiContents PagePD ISO/TS 21569-5:2016ISO/TS 21569-5:2016(E)ForewordISO (the International Organization for
12、 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 in a subject for which a technical committee has been established has the
13、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 Commission (IEC) on all matters of electrotechnical standardization.The procedu
14、res 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
15、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 identifying any or all such patent rights. Details of any patent rights ide
16、ntified 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 conformity 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 responsible
18、 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 reservedPD ISO/TS 21569-5:2016TECHNICAL SPECIFICATION ISO/TS 21569-5:2016(
19、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 FMV promoter (P-FMV) DNA sequence1 ScopeThis document specifies a procedure for the
20、 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 promoter DNA sequence. This segment in some GM plants is indicated as FMV promoter (P-FMV) an
21、d 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 seeds. The procedure requires the extraction of an adequate quantity and quality of amp
22、lifiable 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 is necessary to confirm a positive screening result. Further analyses are required using
23、 construct-specific or event specific methods.2 Normative referencesThe 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 the edition cited applies. For undated references, the latest
24、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 methodsISO 21570, Foodstuffs Methods of analysis for the detection of genetically mo
25、dified organisms and derived products Quantitative nucleic acid based methodsISO 21571:2005, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid extractionISO 24276, Foodstuffs Methods of analysis for the detection of genetically modif
26、ied organisms and derived products General requirements and definitions3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO 16577 apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: a
27、vailable at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp ISO 2016 All rights reserved 1PD ISO/TS 21569-5:2016ISO/TS 21569-5:2016(E)4 PrincipleDNA is extracted from the test portion applying a suitable method (see ISO 21571). The DNA analysis consists o
28、f 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) detection of the P-FMV DNA sequence in a real-time PCR, see Reference 2.5 Reagents and materials5.1 GeneralFor
29、 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 dissolving the corresponding reagents in water and be autoclaved. For all operations for which gloves are
30、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 reagents5.2.1 Thermostable DNA polymerase (for hot-start PCR).5.2.2 PCR buffer solution (containing magnesium chloride and deoxyribonucleosi
31、de 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.5.2.3 Oligonucleotides (see Table 1)1).Table 1 OligonucleotidesName DNA sequence of the oligonucleotideFinal concentr
32、ation in PCRP-FMV as the target sequence (GeneBank accession number X061662,3):pFMV-F 5-CAA AAT AAC GTG GAA AAG AGC T3 340 nmol/lpFMV-R 5-TCT TTT GTG GTC GTC ACT GC3 340 nmol/lProbe pFMV 5-(FAM)-CTG ACA GCC CAC TCA CTA ATG C-(BHQ1)-3a120 nmol/laFAM: 6-Carboxyfluorescein, BHQ-1: Black Hole Quencher 1
33、 (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. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or better results.6 ApparatusRequir
34、ements 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-time PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence signals generated during PCR.
35、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 until the start of the interlaboratory trial). Per aliquot 375 l PCR grade water was added and allowed to settle.2 ISO 2016 All rights r
36、eservedPD ISO/TS 21569-5:2016ISO/TS 21569-5:2016(E)7 Procedure7.1 Preparation of test sampleIt 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 laboratory sample. Measures and operational steps to be ta
37、ken into consideration should be according to ISO 21571 and ISO 24276.7.2 Preparation of DNA extractsConcerning the preparation of DNA from the test portion the general instructions and measures described in ISO 21571 shall be followed. It is recommended to choose one of the DNA extraction methods d
38、escribed in ISO 21571:2005, Annex A.7.3 PCR setupThe method described applies for a total volume of 25 l per PCR. The reaction setup is given in Table 2.Reagents are completely thawed at room temperature. Each reagent should be carefully mixed and briefly centrifuged immediately before pipetting. A
39、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 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
40、2 Reaction setup for the amplificationTotal reaction volume 25 lSample DNA (up to 200 ng) or controls 5 lPCR buffer solutiona(including MgCl2, dNTPs and hot-start DNA polymerase) 12,5 lPrimer pFMV-F and pFMV-R see Table 1Probe pFMV see Table 1Water to 25 laIn the collaborative trial the QuantiTect M
41、ultiplex PCR NoROX Kit (Qiagen GmbH, Hilden/Germany) was used. 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 yield similar or better results. If
42、necessary, adapt the amounts of the reagents and the temperature-time programme.Mix the PCR reagent mixture, centrifuge briefly and pipette 20 l 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
43、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.Transfer the reaction setups into the thermal cycler and start the temperature-time programme.7.4 Temperature-time programmeThe tempe
44、rature-time programme as outlined in Table 3 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. ISO 2016 All rights reserved 3PD ISO/TS 21569-5:
45、2016ISO/TS 21569-5:2016(E)Table 3 Temperature-time programmeStep Parameter Temperature Time Fluorescence measurementCycles1 UNG activation (optional) 50 C 2 min no 12 Initial denaturation 95 C 15 min no 13 AmplificationDenaturation 95 C 15 s no45Annealing and elongation 60 C 60 s yes8 Accept/reject
46、criteria8.1 GeneralA corresponding real-time PCR device-specific data analysis programme 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)
47、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 should be observed. The cycle number at the crossing point
48、of the amplification curve and the fluorescence threshold is calculated (Ctvalue or Cpvalue).If, due to atypical fluorescence 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 t
49、he 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 presence of a GM plant derived product additional tests using construct-specific or event specific methods are required for confirmation.8.2 IdentificationThe target sequence is c
