1、BSI Standards PublicationPD ISO/TS 21569-3:2015Horizontal methods formolecular biomarker analysis Methods of analysis forthe detection of geneticallymodified organisms andderived productsPart 3: Construct-specific real-time PCRmethod for detection of P35S-pat-sequence for screening genetically modif
2、iedorganismsPD ISO/TS 21569-3:2015 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of ISO/TS21569-3:2015.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 beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 85097 4ICS 6
4、7.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31 January 2015.Amendments issued since publicationDate Text affectedPD ISO/TS 21569-3:2015 ISO 2015Horizont
5、al 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 organismsMthodes horizontales danalyse molcul
6、aire 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 modifisTECHNICAL SPECIFICATIONISO/TS21569-3F
7、irst edition2015-02-01Reference numberISO/TS 21569-3:2015(E)PD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
8、 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 officeCase postale 56 CH
9、-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandPD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E) ISO 2015 All rights reserved iiiContents PageForeword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Re
10、agents and materials . 25.1 General . 25.2 PCR reagents . 26 Apparatus .37 Procedure.37.1 Test sample preparation . 37.2 Preparation of the DNA extracts 37.3 PCR setup . 37.4 Temperature-time programme . 48 Accept/reject criteria 48.1 General . 48.2 Identification 48.3 Calculation of P35S-pat copy n
11、umbers . 49 Validation status and performance criteria .59.1 Robustness of the method . 59.2 Collaborative trial for determination of LOD 59.3 Collaborative trial for quantification of the P35S-pat construct in rapeseed 69.4 Sensitivity 79.5 Specificity 710 Test report . 8Bibliography 9PD ISO/TS 215
12、69-3:2015ISO/TS 21569-3:2015(E)ForewordISO (the International Organization for 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 interes
13、ted 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 in the work. ISO collaborates closely with the International Electrotechnical C
14、ommission (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 particular the different approval criteria needed for the different types of ISO document
15、s 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 document may be the subject of patent rights. ISO shall not be held responsible f
16、or 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.iso.org/patents).Any trade name used in this document is information given for the
17、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 WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Fore
18、word - Supplementary informationThe 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 parts, under the general title Horizontal methods for molecular biomarker analysis Meth
19、ods 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 Specification Part 3: Construct-specific real-time PCR method for the detection of the P35S
20、-patsequence for screening for compounds of genetically modified organisms Technical SpecificationISO 21569:2005 is to be revised to become the future Part 1.iv ISO 2015 All rights reservedPD ISO/TS 21569-3:2015TECHNICAL SPECIFICATION ISO/TS 21569-3:2015(E)Horizontal methods for molecular biomarker
21、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 organisms1 ScopeThis Technical Specification describes a procedure for the detec
22、tion 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 construct is frequently found in genetically modified plants with tolerance for phosphinothr
23、icin-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 specific event, a follow-up analysis has to be carried out.This Technical Specification is ap
24、plicable 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 the extraction of an adequate quantity and quality of amplifiable DNA from the relevant matr
25、ix.2 Normative referencesThe 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 applies. For undated references, the latest edition of the referenced document (including any ame
26、ndments) applies.ISO 21569, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methodsISO 21571, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid ex
27、tractionISO 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 24276 apply.4 PrincipleDNA is extracted from
28、 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 specific real-time PCR (see ISO 2157010), and ISO 2015 All rights reserved 1PD ISO/TS 21569-3:2015ISO/TS
29、 21569-3:2015(E)2) detection of the P35S-pat construct in a real-time PCR.1,25 Reagents and materials5.1 GeneralChemicals of recognized analytical grade, appropriate for molecular biology shall be used, as a rule. The water used shall be double distilled or PCR grade water (i.e. nuclease and nucleic
30、 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 is recommended.5.2 PCR reagents5.2.1 Thermostable DNA polymerase, for hot-start PCR.5.2.2 PCR buffer solution
31、, 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 and polymerases which lead to equal or better results may also be used.5.2.3 Oligonucleotides (see Table 1).Table 1
32、OligonucleotidesName DNA sequence of the oligonucleotideFinal concentration in the PCRP35S-pat construct as the target sequence:1,2Primer 35SP03.f 5-AAg TTC ATT TCA TTT ggA gAg gAC A-3 200 nmol/lPrimer pat-7.r 5-Cgg CCA TAT CAg CTg CTg TAg-3 200 nmol/lProbe GSS01.s5-(FAM)-CCg gAg Agg AgA CCA gTT gAg
33、 ATT AggC-(TAMRA)-3a100 nmol/laFAM: 6-Carboxyfluorescein, TAMRA: 6-CarboxytetramethylrhodamineNOTE Equivalent reporter dyes and/or quencher dyes can be used for the probe if they can be shown to yield similar or better results.5.2.4 Standard DNA for calibrationA standard DNA solution of a known conc
34、entration (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 genome equivalents should be calculated on the basis of the molecular mass of the plant haploid genome by applying the Formula (1):Number
35、of genome equivalents per l = DNA concentrationng/lhaploidgenomemasspg1000(1)On the basis of the genome equivalents, the respective copy number for the P35S-pat sequence can be calculated. In doing so, the number of integrations into the plant genome as well as the degree of zygosity of the plant ma
36、terial used shall be taken into consideration.2 ISO 2015 All rights reservedPD ISO/TS 21569-3:2015ISO/TS 21569-3:2015(E)6 ApparatusRegarding the apparatus and materials, see ISO 21569. In addition to the usual laboratory equipment, the following equipment is required.6.1 Real-time PCR device, suitab
37、le for the excitation of fluorescent molecules and the detection of fluorescence signals generated during PCR.7 Procedure7.1 Test sample preparationIt 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 sa
38、mples. 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 extractsConcerning the preparation of DNA from the test portion, the general instructions and measures described in ISO 21571 should be followed. It is rec
39、ommended to choose one of the DNA extraction methods described in ISO 21571, Annex A.7.3 PCR setupThe method is described for a total volume of 25 l per PCR. The reaction set-up is given in Table 2.Completely thaw reagents at room temperature. Each reagent should be carefully mixed and briefly centr
40、ifuged 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 reactions to be performed, including at least one additional reaction as a pipetting reserve. Add 5 l of
41、 sample DNA to each reaction.Table 2 Reaction set-up for the amplificiationOverall 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 35SP03.f and pat-7.r see Table 1Probe GSS01.s see Table 1Water To 25
42、laIn the collaborative study, depending on the real-time PCR devices, different PCR buffer solutions were used TaqMan Universal PCR Mastermix (Life Technologies, Darmstadt), QuantiTect Multiplex PCR NoROX or QuantiTect Probe PCR Mastermix (Qiagen GmbH, Hilden). This information is given for the conv
43、enience 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 necessary, adapt the amounts of the reagents and the temperature-time programme.Mix the PCR reagent mixture, centrifug
44、e 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 the respective control solution (extraction blank control, positive DNA target control). If necessary, prepare a PC
45、R 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 21569-3:2015ISO/TS 21569-3:2015(E)7.4 Temperature-time programmeThe temperature-time programme as outlined in Table 3
46、 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 3 Temperature-time programmeStep Parameter Temperature Time Fluorescence measurementCyc
47、les1 UNG activation (optional) 50 C 2 min no 12 Initial denaturation 95 C 10 min no 13 AmplificationDenaturation 95 C 15 s no45Annealing and elongation60 C 60 s yes8 Accept/reject criteria8.1 GeneralA corresponding real-time PCR device-specific data analysis programme is used for the identification
48、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 control), the result can be expressed as undetermined, no amp, or the maximum number of possible cycles. If the amplification of t
49、he 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 (Ctvalue or Cpvalue).If, due to atypical fluorescence measurement data, the automatic interpretation does not provide a meaningful result, it might be necessary to set the baseline and the threshold manually prior to interpreting the data. In this case,
