Horizontal methods for molecular biomarker analysis -- Methods of analysis for the detection of genetically modified organisms and derived products

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 detected 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 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 amount of amplifiable DNA from the relevant matrix for the purpose of analysis.

Méthodes horizontales d'analyse moléculaire de biomarqueurs -- Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés

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Status
Replaced
Publication Date
02-Sep-2012
Withdrawal Date
02-Sep-2012
Current Stage
9599 - Withdrawal of International Standard
Completion Date
03-Sep-2012
Ref Project

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TECHNICAL ISO/TS
SPECIFICATION 21569-2
First edition
2012-09-01
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
Méthodes horizontales d’analyse moléculaire de biomarqueurs —
Méthodes d’analyse pour la détection des organismes génétiquement
modifiés et des produits dérivés —
Partie 2: Méthode PCR en temps réel spécifique de la construction
pour la détection d’un évènement FP967 dans les graines de lin et les
produits à base de graines de lin
Reference number
ISO/TS 21569-2:2012(E)
ISO 2012
---------------------- Page: 1 ----------------------
ISO/TS 21569-2:2012(E)
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

means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the

address below or ISO’s member body in the country of the requester.
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Published in Switzerland
ii © ISO 2012 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21569-2:2012(E)
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

6.2 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

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 2012 – All rights reserved iii
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ISO/TS 21569-2:2012(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 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 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 in 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 % 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

experts 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 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 reviewed 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 identifying 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 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

iv © ISO 2012 – All rights reserved
---------------------- Page: 4 ----------------------
TECHNICAL 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 Scope

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 detected 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 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 amount of amplifiable DNA from the

relevant matrix for the purpose of analysis.
2 Normative 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 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 extracted 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

(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
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ISO/TS 21569-2:2012(E)
5 Reagents and materials

Chemicals of recognized analytical grade, appropriate for 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 that 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, dGTP 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
Final concentration
Name DNA sequence of the oligonucleotide
in the PCR
Tnos-dfr construct as the target sequence (Reference [1]):
NOST-Spec FW 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’ 100 nmol/l
FAM: 6-Carboxyfluorescein, BHQ: black hole quencher.

NOTE Equivalent reporter dyes and/or quencher dyes can be used for the probe if they can be shown to yield

similar or better results.
5.1.4 Standard DNA for calibration

A standard DNA solution of a known concentration (ng/µl) is used to calculate the copy numbers of the

Tnos-dfr target sequence.

When using genomic linseed DNA as the standard DNA, the number of haploid genome equivalents per

microlitre, n , shall be calculated on the basis of the molecular mass of the linseed haploid genome

hgEq
which is approximately 0,7 pg (Reference [2]) and by applying Equation (1):
[DNA]×1000
n = (1)
hgEq
where
[DNA] is the DNA concentration in nanograms per microlitre;
m 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-dfr

fragment and the 68 bp large SAD gene fragment, respectively. Because the exact number of integrations

of 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 reserved
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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 of 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. Take 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 the 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 purification 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 a total volume of 25 μl 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 immediat
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