ISO/TS 21569-2:2021
(Main)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
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
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 — 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
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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 2021
© 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
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
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
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.
Key
A insertion site of flax genome 5 pBR322 12 FP 967 insertion site
a
B anticipated recombinant 6 left inside homology Non-recombinant PCR.
T-DNA
b
C deduced recombinant T-DNA 7 nopaline synthase Left side event specific PCR.
c
1 flaxseed genomic region 1 8 spectinomycin resistance gene Right side event specific PCR.
d
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
...
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 2021
© 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
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
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
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.
Key
A insertion site of flax genome 5 pBR322 12 FP 967 insertion site
a
B anticipated recombinant 6 left inside homology Non-recombinant PCR.
T-DNA
b
C deduced recombinant T-DNA 7 nopaline synthase Left side event specific PCR.
c
1 flaxseed genomic region 1 8 spectinomycin resistance gene Right side event specific PCR.
d
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
...
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