1、BSI Standards Publication PD ISO/TS 21569-3:2015 Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 3: Construct-specific real-time PCR method for detection of P35S-pat- sequence for screening genetica
2、lly modified organismsPD ISO/TS 21569-3:2015 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of ISO/TS 21569-3:2015. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations
3、represented 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 2015. Published by BSI Standards Limited 2015 ISBN
4、978 0 580 85097 4 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 31 January 2015. Amendments issued since publication Date Text affectedPD ISO/TS
5、21569-3:2015 ISO 2015 Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 3: Construct-specific real-time PCR method for detection of P35S-pat- sequence for screening genetically modified organisms Mtho
6、des horizontales danalyse molculaire de biomarqueurs Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs Partie 3: Mthode PCR en temps rel spcifique de la construction pour la dtection de la squence P35S-pat pour criblage des organismes gntiquement modifis TECH
7、NICAL SPECIFICATION ISO/TS 21569-3 First edition 2015-02-01 Reference number ISO/TS 21569-3:2015(E)PD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E)ii ISO 2015 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2015 All rights reserved. Unless otherwise specified, no part of this publication may be rep
8、roduced or utilized otherwise 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 request
9、er. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in SwitzerlandPD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E) ISO 2015 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Normative refe
10、rences 1 3 Terms and definitions . 1 4 Principle 1 5 Reagents and materials . 2 5.1 General . 2 5.2 PCR reagents . 2 6 Apparatus . 3 7 Procedure. 3 7.1 Test sample preparation . 3 7.2 Preparation of the DNA extracts 3 7.3 PCR setup . 3 7.4 Temperature-time programme . 4 8 Accept/reject criteria 4 8.
11、1 General . 4 8.2 Identification 4 8.3 Calculation of P35S-pat copy numbers . 4 9 Validation status and performance criteria . 5 9.1 Robustness of the method . 5 9.2 Collaborative trial for determination of LOD 5 9.3 Collaborative trial for quantification of the P35S-pat construct in rapeseed 6 9.4
12、Sensitivity 7 9.5 Specificity 7 10 Test report . 8 Bibliography 9PD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(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
13、 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-governmental, in liaison with ISO, also take part
14、 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 described in the ISO/IEC Directives, Part 1. In particul
15、ar 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 the possibility that some of the elements of this
16、 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 list of patent declarations received (see www.i
17、so.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 well as information about ISOs adherence to the
18、 WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary information The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 16, Horizontal methods for molecular biomarker analysis. ISO 21569 consists of the following p
19、arts, under the general title Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products: Part 2: Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products Technical Speci
20、fication Part 3: Construct-specific real-time PCR method for the detection of the P35S-patsequence for screening for compounds of genetically modified organisms Technical Specification ISO 21569:2005 is to be revised to become the future Part 1.iv ISO 2015 All rights reservedPD ISO/TS 21569-3:2015TE
21、CHNICAL SPECIFICATION ISO/TS 21569-3:2015(E) Horizontal methods for molecular biomarker analysis Methods of analysis for the detection of genetically modified organisms and derived products Part 3: Construct-specific real-time PCR method for detection of P35S-pat-sequence for screening genetically m
22、odified organisms 1 Scope This Technical Specification describes a procedure for the detection of the DNA transition sequence between the 35S promotor (P35S) from Cauliflower mosaic virus and a modified phoshinothricin- acetyltransferase gene (pat) from Streptomyces viridochromogenes. The P35S-pat c
23、onstruct is frequently found in genetically modified plants with tolerance for phosphinothricin-containing herbicides. The P35S-pat construct specific method is based on a real-time PCR and can be used for qualitative and quantitative screening purposes. For identification and quantification of a sp
24、ecific event, a follow-up analysis has to be carried out. This Technical Specification is applicable for the analysis of DNA extracted from foodstuffs. It may also be suitable for the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of this method requires
25、the extraction of an adequate quantity and quality of amplifiable DNA from the relevant matrix. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited app
26、lies. 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 based methods ISO 21571, Foodstuffs Methods of a
27、nalysis 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 the purposes of
28、 this document, the terms and definitions given in ISO 24276 apply. 4 Principle DNA is extracted from the test portion applying a suitable method. The DNA analysis consists of two parts, namely, 1) verification of the amount and amplifiability of the extracted DNA, e.g. by means of a target taxon sp
29、ecific real-time PCR (see ISO 21570 10 ), and ISO 2015 All rights reserved 1PD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E) 2) detection of the P35S-pat construct in a real-time PCR. 1,2 5 Reagents and materials 5.1 General Chemicals of recognized analytical grade, appropriate for molecular biology sha
30、ll be used, as a rule. The water used shall be double distilled or PCR grade water (i.e. nuclease and nucleic acid free). 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
31、 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 (dATP , dCTP , dGTP and dUTP). Ready-to-use reagent mixtures or mixes of individual components can be used. Reagents
32、and polymerases which lead to equal or better results may also be used. 5.2.3 Oligonucleotides (see Table 1). Table 1 Oligonucleotides Name DNA sequence of the oligonucleotide Final concentration in the PCR P35S-pat construct as the target sequence: 1,2 Primer 35SP03.f 5-AAg TTC ATT TCA TTT ggA gAg
33、gAC A-3 200 nmol/l Primer pat-7.r 5-Cgg CCA TAT CAg CTg CTg TAg-3 200 nmol/l Probe GSS01.s 5-(FAM)-CCg gAg Agg AgA CCA gTT gAg ATT Agg C-(TAMRA)-3 a 100 nmol/l aFAM: 6-Carboxyfluorescein, TAMRA: 6-Carboxytetramethylrhodamine NOTE Equivalent reporter dyes and/or quencher dyes can be used for the prob
34、e if they can be shown to yield similar or better results. 5.2.4 Standard DNA for calibration A standard DNA solution of a known concentration (ng/l) can be used to calculate the copy number of the P35S-pat target sequence. When using genomic plant DNA as the standard DNA, the number of haploid geno
35、me equivalents should be calculated on the basis of the molecular mass of the plant haploid genome by applying the Formula (1): Number of genome equivalents per l = (1) On the basis of the genome equivalents, the respective copy number for the P35S-pat sequence can be calculated. In doing so, the nu
36、mber of integrations into the plant genome as well as the degree of zygosity of the plant material used shall be taken into consideration.2 ISO 2015 All rights reservedPD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E) 6 Apparatus Regarding the apparatus and materials, see ISO 21569. In addition to the us
37、ual 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. 7 Procedure 7.1 Test sample preparation It should be ensured that the test sample used for DNA
38、 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 shall be as described in ISO 21571 and ISO 24276. 7.2 Preparation of the DNA extracts Concerning the preparation of DNA from the test
39、 portion, the general instructions and measures described in ISO 21571 should be followed. It is recommended to choose one of the DNA extraction methods described in ISO 21571, Annex A. 7.3 PCR setup The method is described for a total volume of 25 l per PCR. The reaction set-up is given in Table 2.
40、 Completely thaw reagents at room temperature. Each reagent should be carefully mixed and briefly centrifuged immediately before pipetting. Prepare a PCR reagent mixture which contains all components except for the sample DNA. The required amount of the PCR reagent mixture depends on the number of r
41、eactions 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 set-up for the amplificiation Overall reaction volume 25 l Sample DNA (up to 200 ng) or controls 5 l PCR buffer solution a(including MgCl 2 , dNTPs an
42、d hot-start DNA polymerase) 12,5 l Primer 35SP03.f and pat-7.r see Table 1 Probe GSS01.s see Table 1 Water To 25 l aIn the collaborative study, depending on the real-time PCR devices, different PCR buffer solutions were used TaqMan Universal PCR Mastermix (Life Technologies, Darmstadt), QuantiTect M
43、ultiplex PCR NoROX or QuantiTect Probe PCR Mastermix (Qiagen GmbH, Hilden). This information is given for the convenience of users of this document and does not constitute an endorsement by ISO. Equivalent products from other manufacturers may be used if they yield similar or better results. If nece
44、ssary, 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 water into the respective reaction set-up. Pipette either 5 l of sample DNA or 5 l of
45、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 set-ups into the thermal cycler and start the temperature-time programme. ISO 2015 All rights reserved 3PD ISO/TS 2
46、1569-3:2015ISO/TS 21569-3:2015(E) 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 cyclers may require specific optimization. The time for initial denaturation depend
47、s on the master mix used. Table 3 Temperature-time programme Step Parameter Temperature Time Fluorescence measurement Cycles 1 UNG activation (optional) 50 C 2 min no 1 2 Initial denaturation 95 C 10 min no 1 3 Amplification Denaturation 95 C 15 s no 45 Annealing and elongation 60 C 60 s yes 8 Accep
48、t/reject criteria 8.1 General A 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
49、 control), the result can be expressed as undetermined, no amp, or the maximum number of possible cycles. If the amplification of the DNA target sequence occurs in a sample (e.g. positive control), a sigmoid-shaped amplification curve can be observed and the cycle number is calculated at which a predetermined fluorescence threshold value is exceeded (C tvalue or C pvalue). If, due to atypical fluorescence measureme
