1、Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modifiedorganisms and derived productsPart 4: Real-time PCR based screening methods for the detection of the P-nos and P-nos-nptII DNA sequencesPD ISO/TS 21569-4:2016BSI Standards PublicationWB11
2、885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National forewordThis Published Document is the UK implementation of ISO/TS21569-4:2016. The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on t
3、his 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 93942 6ICS
4、67.050Compliance 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/T
5、S 21569-4:2016 ISO 2016Horizontal 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 sequencesMthodes horizontales danal
6、yse 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-nptIITECHNICAL SPECIFICATIONISO/TS21569-4Reference numberISO/TS 21569-4:2016(E)Fir
7、st edition2016-11-01PD ISO/TS 21569-4:2016ISO/TS 21569-4:2016(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 mea
8、ns, 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-121
9、4 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgPD ISO/TS 21569-4:2016ISO/TS 21569-4:2016(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 Apparat
10、us . 37 Procedure. 37.1 Preparation of test sample 37.2 Preparation of DNA extracts 37.3 PCR setup . 37.4 Temperature-time programme . 48 Accept/reject criteria 48.1 General . 48.2 Identification 49 Validation status and performance criteria . 59.1 General 59.2 Robustness of the method . 59.3 Collab
11、orative trial . 59.4 Sensitivity 79.5 Specificity 710 Test report . 9Bibliography .10 ISO 2016 All rights reserved iiiContents PagePD ISO/TS 21569-4:2016ISO/TS 21569-4:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
12、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 right to be represented on that committee. International organizations, govern
13、mental 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 used to develop this document and those intended for its further maintenan
14、ce 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 rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention
15、 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 identified during the development of the document will be in the Introduction and
16、/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 on the meaning of ISO specific terms and expressions related to conformity as
17、sessment, 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 for this document is ISO/TC 34, Food products, Subcommittee SC 16, Horizontal
18、 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 21569-4:2016TECHNICAL SPECIFICATION ISO/TS 21569-4:2016(E)Horizontal methods for molecular biomarker analysis Methods of analysis for
19、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 sequences1 ScopeThis document specifies a procedure for the detection of a DNA sequence of the promoter region of the nopaline synthas
20、e 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 coli K12. The nos-promoter and the P-nos-nptII-construct are frequently found in geneti
21、cally 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 modified plant (event) a follow-up analysis has to be carried out.The methods described are
22、 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 requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.The
23、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 positive screening result. Further analyses are required using construct-specific or event s
24、pecific 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 edition of the referenced docu
25、ment (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 modified organisms and derived p
26、roducts Quantitative nucleic acid based methodsISO 21571:2005, Foodstuffs M ethods 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 modified organisms and derived pro
27、ducts General requirements and definitions3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO 16577 apply. ISO 2016 All rights reserved 1PD ISO/TS 21569-4:2016ISO/TS 21569-4:2016(E)ISO and IEC maintain terminological databases for use in standardization a
28、t the following addresses: IEC Electropedia: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso.org/obp4 PrincipleDNA is extracted from the test sample applying a suitable method (see ISO 21571). The DNA analysis consists of two parts:a) verification of
29、 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-nptII sequence in a real-time PCR, see References 2, 3 and 4.5 Reagents and materials5.1 GeneralFor the pu
30、rpose 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 in which gloves are used, it
31、 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 reagents5.2.1 Thermostable DNA polymerase (for hot-start PCR).5.2.2 PCR buffer solution (containing magnesium chloride and deoxyribonucleoside tri
32、phosphates, 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 Oligonucleotides (see Tables 1 and 2).Table 1 Oligonucleotides for detection of P-nos elementName DNA sequence of the
33、oligonucleotideFinal concentration in the PCRP-nos as target sequence4:Primer p-nos-F1 5-AAg CAC ATA CgT CAg AAA CCA TTA TT-3 400 nmol/lPrimer p-nos-R 5-TCA gTg gAg CAT TTT TgA CAA gAA-3 400 nmol/lProbe p-nos-Tm 5-(FAM)-CgC gTT CAA AAg TCg CCT AAg gTC AC-(BBQ)-3a100 nmol/laFAM: 6-Carboxyfluorescein,
34、 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 products from other manufacturers may be used if they can be shown to give equivalent or better results.2 ISO 2016 All rights
35、reservedPD ISO/TS 21569-4:2016ISO/TS 21569-4:2016(E)Table 2 Oligonucleotides for detection of P-nos-nptII constructName DNA sequence of the oligonucleotideFinal concentration in the PCRP-nos-nptII as target sequence4:Primer p-nos-F2 5-TTC CCC TCg gTA TCC AAT TAg Ag-3 400 nmol/lPrimer NPTII-R 5-gAT T
36、gT CTg TTg TgC CCA gTC A-3 400 nmol/lProbe NPTII-Tm2 5-(FAM)-AgC CgA ATA gCC TCT CCA CCC AAg C-(BBQ)-3a100 nmol/laFAM: 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 name
37、d. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or better results.6 ApparatusRequirements concerning apparatus and materials shall be according to ISO 21569. In addition to the usual laboratory equipment, the following equipment is required.6.1 Rea
38、l-time PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence signals generated during PCR.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 grindi
39、ng or homogenizing of the samples. Measures and operational steps to be taken into consideration are described in ISO 21571 and ISO 24276.7.2 Preparation of DNA extractsConcerning the preparation of DNA from the test sample, the general instructions and measures described in ISO 21571 shall be follo
40、wed. It is recommended to choose one of the DNA extraction methods described in ISO 21571:2005, Annex A.7.3 PCR setupThe method is described for a total 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
41、 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 of reactions to be performed, including at least one additional reaction as a p
42、ipetting 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. 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 co
43、ntrol solution (extraction blank control, positive DNA target control). If necessary, prepare a PCR inhibition control as described in ISO 24276.Transfer the reaction set-ups into the thermal cycler and start the temperature-time programme. ISO 2016 All rights reserved 3PD ISO/TS 21569-4:2016ISO/TS
44、21569-4:2016(E)Table 3 Components of the PCR reactionTotal 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 lPrimers see Table 1 or 2Probe see Table 1 or 2Water add to obtain 25 laIn the collaborative trial Ta
45、qMan Universal PCR Mastermix (Life Technologies) was used as PCR buffer solution. 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 g
46、ive equivalent or better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.7.4 Temperature-time programmeThe 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 c
47、yclers may require specific optimization. The time for initial denaturation depends on the master mix used.Table 4 Temperature-time programmeStep Parameter Temperature Time Fluorescence measurementCycles1 Initial denaturation 95 C 10 min no 12 AmplificationDenaturation 94 C 15 s no45Annealing and el
48、ongation 60 C 60 s yes8 Accept/reject 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
49、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 should be observed. The cycle number at the crossing point of the amplification curve and the fluorescence threshold is calculated (Ctvalue or Cpvalue).If, due to atypical fluorescence
