1、 ISO 2012 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 Mthodes horizontales danalyse molcul
2、aire de biomarqueurs Mthodes danalyse pour la dtection des organismes gntiquement modifis et des produits drivs Partie 2: Mthode PCR en temps rel spcifique de la construction pour la dtection dun vnement FP967 dans les graines de lin et les produits base de graines de lin TECHNICAL SPECIFICATION ISO
3、/TS 21569-2 First edition 2012-09-01 Reference number ISO/TS 21569-2:2012(E) ISO/TS 21569-2:2012(E) ii ISO 2012 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO 2012 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any m
4、eans, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail cop
5、yrightiso.org Web www.iso.org Published in Switzerland ISO/TS 21569-2:2012(E) ISO 2012 All rights reserved iii Contents Page Foreword iv 1 Scope . 1 2 Normative references 1 3 Terms and definitions . 1 4 Principle 1 5 Reagents and materials . 2 5.1 PCR reagents . 2 6 Apparatus . 3 6.1 General . 3 6.
6、2 PCR device . 3 7 Sampling 3 8 Procedure. 3 8.1 Test sample preparation . 3 8.2 Preparation of the DNA extracts 3 8.3 DNA extraction 3 8.4 PCR setup . 3 8.5 Temperature time programme 4 9 Accept/reject criteria 4 9.1 General . 4 9.2 Identification 5 10 Validation status and performance criteria . 5
7、 10.1 Robustness of the method . 5 10.2 Intralaboratory trial . 5 10.3 Collaborative trial . 5 10.4 Sensitivity 7 10.5 Specificity 7 11 Test report . 8 Bibliography 9 ISO/TS 21569-2:2012(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standa
8、rds 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 represented on that committee. International organ
9、izations, governmental 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. International Standards are drafted in accordance with the rules given i
10、n the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 %
11、 of the member bodies casting a vote. In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical e
12、xperts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical committee and is accepted for publication if it
13、 is approved by 2/3 of the members of the committee casting a vote. An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed, it is re
14、viewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn. 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 identifyin
15、g any or all such patent rights. ISO/TS 21569-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16, Horizontal methods for molecular biomarker analysis. ISO/TS 21569 consists of the following parts, under the general title Horizontal methods for molecular biomarker anal
16、ysis M e t h o d s o f a n a l y s i s f o r t h e d e t e c t i o n o f g e n e t i c a l l y m o d i f i e d o r g a n i s m s a n d derived products: Part 2: Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products iv ISO 2012 All rights reserved TECHNI
17、CAL SPECIFICATION ISO/TS 21569-2:2012(E) 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 1 Sco
18、pe This method describes a procedure for the detection of a DNA sequence present in a genetically modified linseed (Linum usitatissimum) line (event FP967, also named as “CDC Triffid”). For this purpose, extracted DNA is used in a real-time PCR and the genetic modification (GM) is specifically detec
19、ted by amplification of a 105 bp DNA sequence representing the transition between the nopalin synthase gene terminator (Tnos) from Agrobacterium tumefaciens and the dihydrofolate reductase gene (dfrA1) from a Class 1 integron of Escherichia coli. The method described is applicable for the analysis o
20、f 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 amount of amplifiable DNA from the relevant matrix for the purpose of analysis. 2 Normati
21、ve references ISO 21569, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Qualitative nucleic acid based methods ISO 21571:2005, Foodstuffs Methods of analysis for the detection of genetically modified organisms and derived products Nucleic acid
22、 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 this document, the terms and definitions given in ISO 24276 apply. 4 Principle DNA is extrac
23、ted from the test sample applying a suitable method. The DNA analysis consists of two parts: a) Verification of the amount, quality and amplifiability of the extracted DNA, e.g. by means of a target taxon specific real-time PCR with primers amplifying a 68 bp long fragment from the linseed-specific
24、(Linum usitatissimum) stearoyl-acyl carrier protein desaturase 2 gene (SAD) (Reference 1). b) Detection of the Tnos-dfr construct in a real-time PCR (Reference 1). ISO 2012 All rights reserved 1 ISO/TS 21569-2:2012(E) 5 Reagents and materials Chemicals of recognized analytical grade, appropriate for
25、 molecular biology shall be used, as a rule. The water used shall be double distilled or of an adequate quality. Unless stated otherwise, solutions should be prepared by dissolving the corresponding reagents in water and autoclaved. For all operations in which gloves are used, it should be ensured t
26、hat these are powder-free. The use of aerosol-protected pipette tips serves as protection against cross-contamination. 5.1 PCR reagents 5.1.1 Thermostable DNA polymerase (for hot-start PCR). 5.1.2 PCR buffer solution (contains magnesium chloride and deoxyribonucleoside triphosphate dATP , dCTP , dGT
27、P and dUTP). Ready-to-use reagent mixtures or individual components can be used. Reagents and polymerases which lead to equal or better results may also be used. 5.1.3 Oligonucleotides (see Table 1). Table 1 Oligonucleotides Name DNA sequence of the oligonucleotide Final concentration in the PCR Tno
28、s-dfr construct as the target sequence (Reference 1): NOST-Spec F W 5-AgC gCg CAA ACT Agg ATA AA-3 800 nmol/l NOST-Spec RV 5-ACC TTC Cgg CTC gAT gTC TA-3 800 nmol/l NOST-Spec Probe 5-(FAM)-CgC gCg Cgg TgT CAT CTA Tg-(BHQ)-3 a 100 nmol/l aFAM: 6-Carboxyfluorescein, BHQ: black hole quencher. NOTE Equi
29、valent reporter dyes and/or quencher dyes can be used for the probe if they can be shown to yield similar or better results. 5.1.4 Standard DNA for calibration A standard DNA solution of a known concentration (ng/l) is used to calculate the copy numbers of the Tnos-df r target sequence. When using g
30、enomic linseed DNA as the standard DNA, the number of haploid genome equivalents per microlitre, n hgEq , shall be calculated on the basis of the molecular mass of the linseed haploid genome which is approximately 0,7 pg (Reference 2) and by applying Equation (1): n m = (1) where DNA is the DNA conc
31、entration in nanograms per microlitre; m hg is the haploid genome mass, in picograms. In the collaborative trial, a plasmid was used as standard DNA which contains a copy of the 105 bp Tnos-df r fragment and the 68 bp large SAD gene fragment, respectively. Because the exact number of integrations of
32、 the Tnos-dfr construct in event FP967 in linseed is not known at the time of the specification of this document, the calculated GM-content only represents an estimation which is based on the assumption that the target sequence is present as a single copy per haploid genome. 2 ISO 2012 All rights re
33、served ISO/TS 21569-2:2012(E) 6 Apparatus 6.1 General Regarding the apparatus and materials, see ISO 21569. In addition to the usual laboratory equipment the following equipment is required. 6.2 PCR device Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection o
34、f fluorescence signals generated during PCR. 7 Sampling All samples shall be identified unambiguously. 8 Procedure 8.1 Test sample preparation It should be ensured that the test sample used for DNA extraction is representative of the laboratory sample, e.g. by grinding or homogenizing the samples. T
35、ake into consideration the measures and operational steps specified in ISO 21571 and ISO 24276. 8.2 Preparation of the DNA extracts Concerning the preparation of DNA from the test sample, the general instructions and measures described in ISO 21571 should be followed. It is recommended that one of t
36、he DNA extraction methods described in ISO 21571:2005, Annex A be chosen. 8.3 DNA extraction It is recommended that the DNA extraction be performed by means of the CTAB method with a test portion of 1 g of the homogenized sample (see ISO 21571:2005, A.3.1). Due to problems of purity, an additional p
37、urification step (gel filtration, e.g. by means of micro spin columns) may be necessary. As long as comparability is ensured, other extraction and purification methods (e.g. kit systems) can be applied, using lower test portions, if necessary (Reference 1). 8.4 PCR setup The method is described for
38、a total volume of 25 l per PCR. The reagents given in Table 2 should be used. Reagents are completely thawed at room temperature and should be briefly centrifuged before use. Each reagent should be carefully mixed immediately before pipetting. A reagent mixture is prepared which contains all compone
39、nts 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. A volume of 5 l of sample DNA is used. ISO 2012 All rights reserved 3 ISO/TS 21569-2:2012(E) Table 2 Ad
40、dition of reagents 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 Primer see Table 1 Probe see Table 1 Water add to obtain 25 l a In the collaborative study, TaqMan Universal Mastermix (Applied
41、Biosystems) was used as the PCR buffer solution. This information is given for the 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 resu
42、lts. If necessary, adapt the amounts of the reagents and the temperature-time programme. Mix the reagent mixture, centrifuge briefly and pipette 20 l into each reaction vial. For the PCR reagent control, add 5 l water into the respective reaction set-up. Pipette either 5 l of sample DNA or 5 l of th
43、e 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. 8.5 Temperaturetime programme The temperatu
44、re-time programme, as outlined in Table 3, has been used in the validation study. It was used in combination with the TaqMan Universal Mastermix. The use of different reaction conditions and real-time PCR cyclers may require specific optimization. The time for initial denaturation depends on the mas
45、ter mix used. Table 3 Temperature-time programme Step Parameter Temperature Time Fluorescence measurement Cycles 1 Initial denaturation 95 C 10 min no 1 2 Amplification Denaturation 95 C 15 s no 45 Annealing and elongation 60 C 60 s yes 9 Accept/reject criteria 9.1 General A corresponding real-time
46、PCR device-specific data analysis programme is used for the identification of PCR products. The amplification results may be given in a different manner, depending on the device used. In the absence of detectable PCR products (negative result), e.g. “undetermined”, “no amp”, or the maximum number of
47、 possible cycles is given in the report. If the amplification of the DNA target sequence occurred in a sample (positive result), a sigmoid shaped amplification curve can be observed and the cycle number is calculated at which a predetermined fluorescence threshold value was exceeded (C tvalue or C p
48、value). 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 interpreting the data. In this case, the device-specific instructions given in the manual regarding
49、 the use of the interpretation software shall be applied. 4 ISO 2012 All rights reserved ISO/TS 21569-2:2012(E) 9.2 Identification The target sequence is considered as detected, if by using the Tnos-df r specific primers NOST-Spec FW and NOST-Spec RV and the probe NOST-Spec- Probe, a sigmoid shaped amplification curve can be observed and a predetermined fluorescence threshold value was exceeded by using a linseed specific real-time PCR (Reference
