Molecular biomarker analysis -- Methods of analysis for the detection of genetically modified organisms and derived products

This document specifies 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 nopaline 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 can 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.

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

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Published
Publication Date
04-Jul-2021
Current Stage
5060 - Close of voting Proof returned by Secretariat
Start Date
20-Apr-2021
Completion Date
20-Apr-2021
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TECHNICAL ISO/TS
SPECIFICATION 21569-2
Second edition
2021-07
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
Analyse moléculaire de biomarqueurs —
Partie 2: Méthode PCR en temps réel construit-spécifique pour
la détection d'un événement FP 967 dans les graines de lin et les
produits à base de graines de lin
Reference number
ISO/TS 21569-2:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 21569-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced 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 ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21569-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

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 Intra-laboratory trial ......................................................................................................................................................................... 5

10.3 Collaborative trial ................................................................................................................................................................................. 5

10.4 Sensitivity .................................................................................................................................................................................................... 7

10.5 Specificity .................................................................................................................................................................................................... 7

11 Test report ................................................................................................................................................................................................................... 8

Bibliography .............................................................................................................................................................................................................................10

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/TS 21569-2:2021(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.

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 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 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 .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 of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food Products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.

This second edition cancels and replaces the first edition (ISO/TS 21569-2:2012), which has been

technically revised.
The main changes compared to the previous edition are as follows:
— the single target copy integration into the genome has been updated;

— an explanation of dfr A/Spectinomycin resistance cassette juxtaposition has been added;

— minor typographical improvements have been made.
A list of all parts in the ISO 21569 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 21569-2:2021(E)
Introduction

Flaxseed (Linum usitatissimum L.) FP967 (CDC Triffid Flax) is the only GMO linseed flax listed in

[1]

the International Service for the Acquisition of Agro-biotech Applications (ISAAA) . FP967 was

regenerated from a single Norlin Flax hypocotyl (regenerant number 12115) transformed with an

agrobacterium/Ti plasmid system containing the NPT-11 gene encoding kanamycin resistance and a

[2][3]

modified Arabidopsis acetolate synthase gene with reduced enzyme affinity for chlorosulfuron

[4][5][6][7]

. The in planta T-DNA construct includes a repeat and re-arrangement of the T-DNA forming

an inverted-repeat structure of the right border, as confirmed by next generation sequencing and PCR

cloning. The FP967 GM construct is stable within the recombinant plant genome and demonstrates

[8]

functional resistance to the sufonylurea herbicides chlorsufuron, metsulfuron, and triasulfuron .

[8][9]

Published event-specific assays for FP967 have been described . One generates two products from

[8]

the recombinant and one product from the non-recombinant . The other generates a single product

but requires an internal control PCR test for linseed-specific (Linum usitatissimum) stearoyl-acyl carrier

[9]

protein desaturase 2 gene (SAD) . Event-specific assays are most useful for proprietary and breeding

uses when exact identity or copy number of a transgene is required.
[10]

The FP967 PCR assay described in this document is construct-specific . It generates a 105 bp product

spanning the junction between the T-nos and dfrA1 elements of the transgene construct. Construct-

specific assays are usually used as generic GM screening tools able to cross-detect different GM events

carrying the same gene fusion. Because FP967 is the only flaxseed construct to carry a spectinomycin

selectable marker and the only listed GM flax event, the described assay is conclusive for genetically

modified identification among approved GMOs. It has been widely accepted and deployed and has been

effective identifying and eliminating unwanted adventitious presence from unrelated breeding lines

and commercial stocks. It is also more sensitive than reported for the available event-specific test

because there are two copies of the target in the recombinant (see Figure 1). Adding event-specific

testing options to the testing portfolio would require considerable effort (especially experimental

comparison and validation to recommend one of the available event-specific assays) with no ultimate

benefit to the final purpose.

Next generation sequencing and PCR cloning of the T-DNA of FP967 revealed a repeat and rearrangement

of an internal T-DNA fragment forming an inverted-repeat structure of the right border of the T-DNA in

the flax genome. Although, there is only a single copy of the FP967 T-DNA, the order and arrangement

of the NOS gene, the Arabidopsis acetolactate synthase (NP_001189794.1), pBR322 (J01749.1), neomycin

phosphotransferase II (AY909580.1), and the Escherichia coli spectinomycin resistance/dihydrofolate

reductase (SpecR/DHFR) region are no longer consistent with the original plasmids used to transform

[8]

FP967 . This rearrangement was not anticipated in the development of the construct specific assay.

Figure 1 provides a graphic depicting the genomic position of the insert, the anticipated recombinant

structure and the deduced recombinant structure based on DNA sequencing. It also shows the location

of the event and construct-specific PCR assays on each of these.
© ISO 2021 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO/TS 21569-2:2021(E)
Key
A insertion site of flax genome 5 pBR322 12 FP 967 insertion site
B anticipated recombinant 6 left inside homology Non-recombinant PCR.
T-DNA
C deduced recombinant T-DNA 7 nopaline synthase Left side event specific PCR.

1 flaxseed genomic region 1 8 spectinomycin resistance gene Right side event specific PCR.

2 right border 9 chimeric neomycin Construct specific PCR.
phosphotransferase
3 undetermined sequence 10 Arabidopsis acetolactate synthase
4 left border 11 flaxseed genomic region 2

NOTE As a result of the rearrangement of the T-DNA in the recombinant two copies of the target amplicon

were formed. This increases the sensitivity of the construct specific assay.
Figure 1 — FP967 insertion into the flax genome
vi © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 21569-2:2021(E)
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 document specifies 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 nopaline 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 can 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

The following documents are referred 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 16577, Molecular biomarker analysis — Terms and definitions

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 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 16577 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
© ISO 2021 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO/TS 21569-2:2021(E)
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-

[10]

specific (Linum usitatissimum) stearoyl-acyl carrier protein desaturase 2 gene (SAD) ;

[10]
b) detection of the Tnos-dfr construct in a real-time PCR .
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 triphosphates:

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
Name DNA sequence of the oligonucleotide Final concentration in the PCR
[10]
Tnos-dfr construct as the target sequence :
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
[11]
which is approximately 0,7 pg and by applying Formula (1):
C ×1000
DNA
n = (1)
hgEq
where
2 © ISO 2021 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TS 21569-2:2021(E)
C is the DNA concentration in nanograms per microlitre;
DNA
m is the haploid genome mass, in picograms.

In the collaborative trial, a plasmid was used as standard DNA that contained a single copy of the

105 bp Tnos-dfr fragment and the 68 bp large SAD gene fragment, respectively. There is a single copy

[7]

the Tnos-dfr construct in event FP967 in linseed . The calculated GM-content is based on the single

copy presence of the target sequence per haploid genome.
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. Samples should be representative of the lot.

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, for example, 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 be chosen. DNA extraction from flaxseed for GMO analysis has been describ

...

TECHNICAL ISO/TS
SPECIFICATION 21569-2
Second edition
Molecular biomarker analysis —
Qualitative PCR-based DNA analytical
methods —
Part 2:
Construct-specific real-time PCR
method for detection of event FP967
in linseed and linseed products
PROOF/ÉPREUVE
Reference number
ISO/TS 21569-2:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 21569-2:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced 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 ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21569-2:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

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 Intra-laboratory trial ......................................................................................................................................................................... 5

10.3 Collaborative trial ................................................................................................................................................................................. 5

10.4 Sensitivity .................................................................................................................................................................................................... 7

10.5 Specificity .................................................................................................................................................................................................... 7

11 Test report ................................................................................................................................................................................................................... 8

Bibliography ................................................................................................................................................................................................................................ 9

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 21569-2:2021(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.

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 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 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 .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 of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food Products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.

This second edition cancels and replaces the first edition (ISO 21569-2: 2012), which has been

technically revised.
The main changes compared to the previous edition are as follows:
— the single target copy integration into the genome has been updated;

— an explanation of dfr A/Spectinomycin resistance cassette juxtaposition has been added;

— minor typographical improvements have been made.
A list of all parts in the ISO 21569 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 21569-2:2021(E)
Introduction

Flaxseed (Linum usitatissimum L.) FP967 (CDC Triffid Flax) is the only GMO linseed flax listed in

[1]

the International Service for the Acquisition of Agro-biotech Applications (ISAAA) . FP967 was

regenerated from a single Norlin Flax hypocotyl (regenerant number 12115) transformed with an

agrobacterium/Ti plasmid system containing the NPT-11 gene encoding kanamycin resistance and a

[2][3]

modified Arabidopsis acetolate synthase gene with reduced enzyme affinity for chlorosulfuron

[4][5][6][7]

. The in planta T-DNA construct includes a repeat and re-arrangement of the T-DNA forming

an inverted-repeat structure of the right border, as confirmed by next generation sequencing and PCR

cloning. The FP967 GM construct is stable within the recombinant plant genome and demonstrates

[8]

functional resistance to the sufonylurea herbicides chlorsufuron, metsulfruon, and triasulfuron .

[8][9]

Published event-specific assays for FP967 have been described . One generates two products from

[8]

the recombinant and one product from the non-recombinant . The other generates a single product

but requires an internal control PCR test for linseed-specific (Linum usitatissimum) stearoyl-acyl carrier

[9]

protein desaturase 2 gene (SAD) . Event-specific assays are most useful for proprietary and breeding

uses when exact identity or copy number of a transgene is required.
[10]

The FP967 PCR assay described in this document is construct-specific . It generates a 105 bp product

spanning the junction between the T-nos and dfrA1 elements of the transgene construct. Construct-

specific assays are usually used as generic GM screening tools able to cross-detect different GM events

carrying the same gene fusion. Because FP967 is the only flaxseed construct to carry a spectinomycin

selectable marker and the only listed GM flax event, the described assay is conclusive for genetically

modified identification among approved GMOs. It has been widely accepted and deployed and has been

effective identifying and eliminating unwanted adventitious presence from unrelated breeding lines

and commercial stocks. It is also more sensitive than reported for the available event-specific test

because there are two copies of the target in the recombinant (see Figure 1). Adding event-specific

testing options to the testing portfolio would require considerable effort (especially experimental

comparison and validation to recommend one of the available event-specific assays) with no ultimate

benefit to the final purpose.

Next generation sequencing and PCR cloning of the T-DNA of FP967 revealed a repeat and rearrangement

of an internal T-DNA fragment forming an inverted-repeat structure of the right border of the T-DNA in

the flax genome. Although, there is only a single copy of the FP967 T-DNA, the order and arrangement

of the NOS gene, the Arabidopsis acetolactate synthase (NP_001189794.1), pBR322 (J01749.1), neomycin

phosphotransferase II (AY909580.1), and the Escherichia coli spectinomycin resistance/dihydrofolate

reductase (SpecR/DHFR) region are no longer consistent with the original plasmids used to transform

[8]

FP967 . This rearrangement was not anticipated in the development of the construct specific assay.

Figure 1 provides a graphic depicting the genomic position of the insert, the anticipated recombinant

structure and the deduced recombinant structure based on DNA sequencing. It also shows the location

of the event and construct-specific PCR assays on each of these.
© ISO 2021 – All rights reserved PROOF/ÉPREUVE v
---------------------- Page: 5 ----------------------
ISO/TS 21569-2:2021(E)
Key

A insertion site of flax genome 4 left border 10 Arabidopsis acetolactate synthase

Banticipated recombinant 5 pBR322 11 flaxseed genomic region 2
T-DNA
C deduced recombinant T-DNA 6 left inside homology 12 FP 967 insertion site
1 flaxseed genomic region 1 7 nopaline synthase Non-recombinant PCR.
2 right border 8 spectinomycin resistance gene Left side event specific PCR.
3 undetermined sequence 9 chimeric neomycin Right side event specific PCR.
phosphotransferase
Construct specific PCR.

NOTE As a result of the rearrangement of the T-DNA in the recombinant two copies of the target amplicon

were formed. This increases the sensitivity of the construct specific assay.
Figure 1 — FP967 insertion into the flax genome
vi PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/TS 21569-2:2021(E)
Molecular biomarker analysis — Qualitative PCR-based
DNA analytical methods —
Part 2:
Construct-specific real-time PCR method for detection of
event FP967 in linseed and linseed products
1 Scope

This document specifies 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 nopaline 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 can 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

The following documents are referred 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 16577, Molecular biomarker analysis — Terms and definitions

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 16577 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
© ISO 2021 – All rights reserved PROOF/ÉPREUVE 1
---------------------- Page: 7 ----------------------
ISO/TS 21569-2:2021(E)
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-

[10]

specific (Linum usitatissimum) stearoyl-acyl carrier protein desaturase 2 gene (SAD) ;

[10]
b) detection of the Tnos-dfr construct in a real-time PCR .
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 triphosphates:

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
Name DNA sequence of the oligonucleotide Final concentration in the PCR
[10]
Tnos-dfr construct as the target sequence :
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
[11]
which is approximately 0,7 pg and by applying Formula (1):
C ×1000
DNA
n = (1)
hgEq
where
2 PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 8 ----------------------
ISO/TS 21569-2:2021(E)
C is the DNA concentration in nanograms per microlitre;
DNA
m is the haploid genome mass, in picograms.

In the collaborative trial, a plasmid was used as standard DNA that contained a single copy of the

105 bp Tnos-dfr fragment and the 68 bp large SAD gene fragment, respectively. There is a single copy

[7]

the Tnos-dfr construct in event FP967 in linseed . The calculated GM-content is based on the single

copy presence of the target sequence per haploid genome.
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. Samples should be representative of the lot.

8 Procedure
8.1 Test sample preparation

It should be ensured that the test sample used for DNA extraction is representative of the laboratory

...

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