1、Horizontal 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-sequences PD ISO/TS 21569-6:2016 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- 6:2016. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations represent
3、ed 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、 93944 0 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 PUBLIS
5、HED DOCUMENT PD ISO/TS 21569-6:2016 ISO 2016 Horizontal 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 sequences Mthode
6、s horizontales danalyse molculaire 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-cry TECHNICAL SPECIFICATION ISO/TS 21569-6 Reference number
7、ISO/TS 21569-6:2016(E) First edition 2016-11-01 PD ISO/TS 21569-6:2016 ISO/TS 21569-6:2016(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 othe
8、rwise 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 office
9、 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 21569-6:2016 ISO/TS 21569-6:2016(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 . 3 7 Procedure. 3 7.1 Preparation of test samples . 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 criteri
11、a . 4 9.1 General . 4 9.2 Robustness . 5 9.3 Collaborative trial . 5 9.4 Sensitivity 7 9.5 Specificity 8 10 Test report . 9 Bibliography .10 ISO 2016 All rights reserved iii Contents Page PD ISO/TS 21569-6:2016 ISO/TS 21569-6:2016(E) Foreword ISO (the International Organization for Standardization)
12、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 right to be repres
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14、op 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 rules of the ISO/
15、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 identified during t
16、he 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 on the meanin
17、g 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 responsible for this do
18、cument 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 21569-6:2016 TECHNICAL SPECIFICATION ISO/TS 21569-6:2016(E) Horizo
19、ntal 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 sequences 1 Scope This document specifies a procedure for the detec
20、tion of a DNA sequence of the modified cry1Ab/Ac gene and a procedure for the detection 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. Bo
21、th detection 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. This document is applicable for the analysis of DNA extracted from fo
22、odstuffs. It 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. 2 Normative references The following documents are referre
23、d 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 document (including any amendments) applies. ISO 21569, Foodstuffs Methods of
24、 analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid 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, Foodstu
25、ffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid 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
26、 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: available at http:/www.electropedia.org/ ISO Online browsing platform: available at http:/www.iso
27、.org/obp ISO 2016 All rights reserved 1 PD ISO/TS 21569-6:2016 ISO/TS 21569-6:2016(E) 4 Principle DNA 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
28、means of a target taxon specific real-time PCR (according to ISO 21570, see alsoReference 1; b) detection of the cry1Ab/Ac and/or the Pubi-cry DNA sequence in a real-time PCR, see References 2 and 3. 5 Reagents and materials 5.1 General For the purpose of this document, only chemicals and water of r
29、ecognized 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 should be ensured that these are powder-free. The us
30、e of aerosol-protected pipette tips as protection against cross contamination is recommended. 5.2 PCR reagents 5.2.1 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
31、 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 cry1Ab/Ac Name DNA sequence of the oligonucleotide Final concentration in the PCR cr
32、y1Ab/Ac as target sequence 2 : Bt-F1(mod) 5-gAg gAA ATg CgT ATT CAA TTC AAC-3 400 nmol/l Bt-R 5-TTC Tgg ACT gCg AAC AAT gg-3 400 nmol/l Bt-P 5-(FAM)-ACA TgA ACA gCg CCT TgA CCA CAg C-(NFQ)-3 a 100 nmol/l aFAM: 6-Carboxyfluorescein, NFQ: non-fluorescent quencher (dark quencher). Table 2 Oligonucleoti
33、des for detection of Pubi-cry Name DNA sequence of the oligonucleotide Final concentration in the PCR Pubi-cry as target sequence 2 : Pubi-F2 5-ATT TgC TTg gTA CTg TTT CTT TTg TC-3 300 nmol/l Cry-rr-R 5-TTg TTg TCC ATg gAT CCT CTA gAg T-3 600 nmol/l Pubi-T2 5- (FAM)- ACC CTg TTg TTT ggT gTT ACT TCT
34、gCA-(NFQ)-3 a,b 100 nmol/l aFAM: 6-Carboxyfluorescein, NFQ: non-fluorescent quencher (dark quencher). bThe Pubi-T2 probe is three bases shorter than the probe described in Reference 3 but identical specificity and sensitivity is achieved.2 ISO 2016 All rights reserved PD ISO/TS 21569-6:2016 ISO/TS 2
35、1569-6:2016(E) Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield similar or better results. 6 Apparatus Requirements concerning apparatus and materials shall be according to ISO 21569. In addition to the usual laboratory equipment, the following eq
36、uipment 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 Preparation of test samples It should be ensured that the test portion used for DNA extraction is representative of the
37、laboratory sample, e.g. by grinding or homogenizing of the samples. Measures and operational steps to be taken into consideration should be as described in ISO 21571 and ISO 24276. 7.2 Preparation of DNA extracts Concerning the preparation of DNA from the test portion, the general instructions and m
38、easures 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 total volume of 25 l per PCR. The reaction setup is given in Table 3. Completely thaw reagents at room tem
39、perature. 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 of reactions to be performed, includi
40、ng at least one additional reaction as a pipetting reserve. Add 5 l of sample DNA to each reaction. Table 3 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 and hot-start DNA polymerase) 12,5 l Pr
41、imers Bt-F1(mod) and Bt-R or primers Pubi-F2 and Cry-rr-R see Table 1 or 2 Probe Bt-P or probe Pubi-T2 see Table 1 or 2 Water add to obtain 25 l aIn the interlaboratory trial TaqMan Universal PCR Mastermix (Life Technologies) was used as PCR buffer solution. This information is given for convenience
42、 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 can be shown to give equivalent or better results. If necessary, adapt the amounts of the reagents and the temperature-time programme. Mix t
43、he 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 l of the respective control solution (extraction blank control, positive DNA target c
44、ontrol). 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. ISO 2016 All rights reserved 3 PD ISO/TS 21569-6:2016 ISO/TS 21569-6:2016(E) 7.4 Temperature-time programme The temperatur
45、e-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-time programme Step Parameter Tem
46、perature 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 programme is used for the identif
47、ication 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
48、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 (C tvalue or C pvalue). If, due to atypi
49、cal 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 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 Identification The cry1Ab/Ac or Pubi-cry target sequence is considered as detected, if by using the specific primers Bt-F1
