1、Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived productsPart 6: Real-time PCR based screening methods for the detection of cry1Ab/Acand Pubi-cry DNA-sequencesPD ISO/TS 21569-6:2016BSI Standards PublicationWB11885
2、_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06National forewordThis Published Document is the UK implementation of ISO/TS 21569-6:2016.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 93944 0ICS 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、1569-6:2016 ISO 2016Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 6: Real-time PCR based screening methods for the detection of cry1Ab/Ac and Pubi-cry DNA sequencesMthodes horizontales danalyse mo
6、lculaire de biomarqueurs Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs Partie 6: Mthodes de dpistage PCR en temps rel pour la dtection de squences ADN cry1Ab/Ac et Pubi-cryTECHNICAL SPECIFICATIONISO/TS21569-6Reference numberISO/TS 21569-6:2016(E)First edi
7、tion2016-11-01PD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(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, el
8、ectronic 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 Vern
9、ier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgPD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(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 . 3
10、7 Procedure. 37.1 Preparation of test samples . 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 . 49.1 General . 49.2 Robustness . 59.3 Collaborative trial .
11、 59.4 Sensitivity 79.5 Specificity 810 Test report . 9Bibliography .10 ISO 2016 All rights reserved iiiContents PagePD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies).
12、 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, governmental and non-
13、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 maintenance are describe
14、d 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 is drawn to th
15、e 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/or on the ISO
16、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 assessment, as we
17、ll 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 methods for mo
18、lecular 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 21569-6:2016TECHNICAL SPECIFICATION ISO/TS 21569-6:2016(E)Horizontal methods for molecular biomarker analysis Methods of analysis for the detection o
19、f genetically modified organisms and derived products Part 6: Real-time PCR based screening methods for the detection of cry1Ab/Ac and Pubi-cry DNA sequences1 ScopeThis document specifies a procedure for the detection of a DNA sequence of the modified cry1Ab/Ac gene and a procedure for the detection
20、 of the DNA transition sequence between the maize ubiquitin promoter (Pubi) and the cry1Ab/Ac gene. The modified cry1Ab/Ac gene and the Pubi-cry construct are frequently found in genetically modified Bt plants. Both detection methods are based on real-time PCR and can be used for qualitative screeni
21、ng purposes. For identification and quantification of a specific genetically modified plant (event) a follow-up analysis has to be carried out.This document is applicable for the analysis of DNA extracted from foodstuffs. It may also be suitable for the analysis of DNA extracted from other products
22、such as feedstuffs and seeds. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.2 Normative referencesThe following documents are referred to in the text in such a way that some or all of their content constitutes requirements
23、of this document. 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 Qualitat
24、ive nucleic acid based methodsISO 21570, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Quantitative nucleic acid based methodsISO 21571:2005, Foodstuffs M ethods of analysis for the detection of genetically modified organisms and derived prod
25、ucts Nucleic acid extractionISO 24276, Foodstuffs Methods of analysis for the detection of genetically modified 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
26、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 ISO 2016 All rights reserved 1PD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(E)4 PrincipleDN
27、A is extracted from the test portion 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 alsoReference 1;b) detect
28、ion of the cry1Ab/Ac and/or the Pubi-cry DNA sequence in a real-time PCR, see References 2 and 3.5 Reagents and materials5.1 GeneralFor the purpose of this document, only chemicals and water of recognized analytical grade, appropriate for molecular biology shall be used. Unless stated otherwise, sol
29、utions should be prepared by dissolving 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 reag
30、ents5.2.1 Thermostable DNA polymerase, (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 result
31、s may also be used.5.2.3 Oligonucleotides (see Tables 1 and 2).Table 1 Oligonucleotides for detection of cry1Ab/AcName DNA sequence of the oligonucleotideFinal concentration in the PCRcry1Ab/Ac as target sequence2:Bt-F1(mod) 5-gAg gAA ATg CgT ATT CAA TTC AAC-3 400 nmol/lBt-R 5-TTC Tgg ACT gCg AAC AA
32、T gg-3 400 nmol/lBt-P 5-(FAM)-ACA TgA ACA gCg CCT TgA CCA CAg C-(NFQ)-3a100 nmol/laFAM: 6-Carboxyfluorescein, NFQ: non-fluorescent quencher (dark quencher).Table 2 Oligonucleotides for detection of Pubi-cryName DNA sequence of the oligonucleotideFinal concentration in the PCRPubi-cry as target seque
33、nce2:Pubi-F2 5-ATT TgC TTg gTA CTg TTT CTT TTg TC-3 300 nmol/lCry-rr-R 5-TTg TTg TCC ATg gAT CCT CTA gAg T-3 600 nmol/lPubi-T2 5- (FAM)- ACC CTg TTg TTT ggT gTT ACT TCT gCA-(NFQ)-3a,b100 nmol/laFAM: 6-Carboxyfluorescein, NFQ: non-fluorescent quencher (dark quencher).bThe Pubi-T2 probe is three bases
34、 shorter than the probe described in Reference 3 but identical specificity and sensitivity is achieved.2 ISO 2016 All rights reservedPD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(E)Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield similar or better res
35、ults.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 Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence signals
36、 generated during PCR.7 Procedure7.1 Preparation of test samplesIt should be ensured that the test portion 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 should be as d
37、escribed in 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 described in ISO 21571:2005, Annex
38、 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.Completely thaw reagents at room temperature. Each reagent should be carefully mixed and briefly centrifuged immediately before pipetting. A PCR reagent mixture is prepared which contai
39、ns 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 3 Reaction setup for the amplificationTotal
40、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 Bt-F1(mod) and Bt-R or primers Pubi-F2 and Cry-rr-R see Table 1 or 2Probe Bt-P or probe Pubi-T2 see Table 1 or 2Water add to obtain 25 laIn the interla
41、boratory trial TaqMan 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 names. Equivalent products from other manufacturers may be used if they
42、can be shown to give equivalent 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 into each reaction vial. For the amplification reagent control, add 5 l of water into the respective
43、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 described in ISO 24276.Transfer the reaction setups into the thermal cycler and start the temperature-
44、time programme. ISO 2016 All rights reserved 3PD ISO/TS 21569-6:2016ISO/TS 21569-6:2016(E)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 cyclers may require specific
45、 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 elongation 60 C 60 s yes8 Acc
46、ept/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 PCR products (e.g. negative
47、 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 cros
48、sing point 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 int
49、erpreting the data. In this case, the device-specific instructions given in the manual regarding the use of the interpretation software should be applied.8.2 IdentificationThe cry1Ab/Ac or Pubi-cry target sequence is considered as detected, if by using the specific primers Bt-F1(mod) and Bt-R and the probe Bt-P or the specific primers Pubi-F2 and Cry-rr-R and the probe Pubi-T2, a sigmoid shaped amplification curve is observed and a Ctvalue or Cpvalue is calcul
