ISO/DTS 21569-10

Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 10: Construct- and event-specific detection methods for genetically modified salmon expressing CS-GHc2 growth hormone

This International Standard describes a procedure for the detection of a DNA sequence of a construct used to (genetically) enhance the growth of fish commonly found in aquaculture. The genetically modified AquAdvantage Atlantic salmon (Salmo salar) carries this construct and can be detected based on a real-time PCR targeting the border between the growth hormone coding sequence (CS-GHc2) of Oncorhynchus tshawytscha (Chinook salmon) and the antifreeze terminator (T-AFP) of (Macro-) Zoarces americanus (ocean pout). This method can be applied to identify the GM fish or for screening purposes. This part of the ISO 21569 series 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. The application of these methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.

Méthodes horizontales pour l'analyse moléculaire de biomarqueurs — Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés — Partie 10: Méthodes de détection spécifiques à la construction et à l'événement pour le saumon génétiquement modifié exprimant l'hormone de croissance CS-GHc2

General Information

• Status: Not Published
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16/WG 14 - Genetically engineered content detection and quantification
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 11-Jul-2025
• Completion Date: 11-Jul-2025

Relations

• Consolidates: ISO 4980:2023 - Benefit-risk assessment for sports and recreational facilities, activities and equipment
• Effective Date: 07-Sep-2024
• Revises: ISO 21569:2005 - Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived products — Qualitative nucleic acid based methods
• Effective Date: 31-Aug-2024

Standards Content (Sample)

FINAL DRAFT
Technical
Specification
ISO/TC 34/SC 16
Horizontal methods for molecular
Secretariat: ANSI
biomarker analysis — Methods
Voting begins on:
of analysis for the detection of
2025-07-11
genetically modified organisms and
Voting terminates on:
derived products —
2025-10-03
Part 10:
Construct- and event-specific
detection methods for genetically
modified salmon expressing CS-
GHc2 growth hormone
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
Technical
Specification
ISO/TC 34/SC 16
Horizontal methods for molecular
Secretariat: ANSI
biomarker analysis — Methods
Voting begins on:
of analysis for the detection of
genetically modified organisms and
Voting terminates on:
derived products —
Part 10:
Construct- and event-specific
detection methods for genetically
modified salmon expressing CS-
GHc2 growth hormone
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure . 3
7.1 Preparation of test samples .3
7.2 DNA extraction .3
7.3 PCR setup.3
7.4 Temperature-time programme.4
8 Accept/reject criteria . 4
8.1 General .4
8.2 Detection .4
9 Validation status and performance criteria . 5
9.1 Sensitivity .5
9.2 Specificity .5
9.3 Robustness of the two methods .6
9.4 Interlaboratory trial for the construct-specific detection method .6
9.4.1 General .6
9.4.2 PCR efficiency and limit of detection (LOD ) .8
95 %
9.4.3 False-positive and false-negative rates for DNA samples (blind samples) .9
9.5 Interlaboratory trial for the event-specific detection method .9
9.5.1 General .9
9.5.2 Validation results (blind samples) . .10
9.6 Summary evaluation .10
10 Test report .11
Bibliography .12

iii
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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, in collaboration with the European Committee for
Standardization (CEN) Technical Committee CEN/TC 275, Food analysis - Horizontal methods, in accordance
with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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
FINAL DRAFT Technical Specification ISO/DTS 21569-10:2025(en)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 10:
Construct- and event-specific detection methods for genetically
modified salmon expressing CS-GHc2 growth hormone
1 Scope
This document specifies procedures for the detection of a DNA sequence of a construct used to (genetically)
enhance the growth of fish commonly found in aquaculture. The genetically modified AquAdvantage
Atlantic salmon (Salmo salar) carries this construct and can be detected based on a real-time polymerase
chain reaction (PCR) targeting either the border between the growth hormone coding sequence (CS-GHc2)
of Oncorhynchus tshawytscha (Chinook salmon) and the antifreeze terminator (T-AFP) of (Macro-) Zoarces
americanus (ocean pout), i.e. with the construct-specific method, or the border between the Atlantic salmon
genomic DNA and the antifreeze promoter (P-AFP) of ocean pout, i.e. with the event-specific method. These
methods can be applied to identify the genetically modified (GM) fish or for screening purposes.
This document 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. The application of these methods requires
the extraction of an adequate amount of amplifiable DNA from the relevant matrix.
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 — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/

4 Principle
DNA is extracted from the test sample by applying a suitable method with performance characteristics in
accordance with ISO 21571:2005.
The DNA analysis consists of the following two parts:
a) verification of the amount, quality
...

ISO/TC 34/SC 16
ISO/CD TS 21569-10(en)
Secretariat: ANSI
Date: 2025-06-26
Horizontal methods for molecularMolecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 10:
Construct- and event-specific detection methods for genetically
modified salmon expressing CS-GHc2 growth hormone

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
E-mail: [e-mail]
Website: www.iso.org
Field Code Changed
Published in Switzerland
ii
Contents
Foreword . iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure . 3
7.1 Preparation of test samples . 3
7.2 DNA extraction . 3
7.3 PCR setup . 3
7.4 Temperature-time programme . 4
8 Accept/reject criteria . 5
8.1 General . 5
8.2 Detection . 5
9 Validation status and performance criteria. 5
9.1 Sensitivity . 5
9.2 Specificity . 6
9.3 Robustness of the two methods . 10
9.4 Interlaboratory trial for the construct-specific detection method . 10
9.5 Interlaboratory trial for the event-specific detection method . 13
9.6 Summary evaluation . 15
10 Test report . 15
Bibliography . 16

iii
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 documentsdocument 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).
Field Code Changed
Attention is drawnISO draws attention to the possibility that some of the elementsimplementation of this
document may beinvolve the subjectuse of (a) patent(s). ISO takes no position concerning the evidence,
validity or applicability of any claimed patent rights in respect thereof. As of the date of publication of this
document, ISO had not received notice of (a) patent(s) which may be required to implement this document.
However, implementers are cautioned that this may not represent the latest information, which may be
obtained from the patent database available at www.iso.org/patents. 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 ).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation onof 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.htmlthe following URL: .
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis., in collaboration with the European Committee for
Standardization (CEN) Technical Committee CEN/TC 275, Food analysis - Horizontal methods, in accordance
with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
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.
Field Code Changed
iv
MolecularHorizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 10:
Construct- and event-specific detection methods for genetically
modified salmon expressing CS-GHc2 growth hormone
1 Scope
This International Standard describesdocument specifies procedures for the detection of a DNA sequence of a
construct used to (genetically) enhance the growth of fish commonly found in aquaculture. The genetically
modified AquAdvantage Atlantic salmon (Salmo salar) carries this construct and can be detected based on a
real-time polymerase chain reaction (PCR) targeting either the border between the growth hormone coding
sequence (CS-GHc2) of Oncorhynchus tshawytscha (Chinook salmon) and the antifreeze terminator (T-AFP) of
(Macro-) Zoarces americanus (ocean pout), i.e.,. with the construct-specific method, or the border between the
Atlantic salmon genomic DNA and the antifreeze promoter (P-AFP) of ocean pout, i.e.,. with the event-specific
method. These methods can be applied to identify the genetically modified (GM) fish or for screening purposes.
This part of the ISO 21569 seriesdocument is applicable for the analysis of DNA extracted from foodstuffs. It
maycan also be suitable for the analysis of DNA extracted from other products such as feedstuffs. The
application of these methods requires the extraction of an adequate amount of amplifiable DNA from the
relevant matrix.
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 — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 21569:2005, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — Qualitative nucleic acid based methods
ISO 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 16577apply 16577 apply.
ISO and IEC maintain terminologicalterminology databases for use in standardization at the following
addresses:
— IEC Electropedia: available at ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
DNA is extracted from the test sample by applying a suitable method with performance characteristics
according toin accordance with ISO 21571:2005.
The DNA analysis consists of the following two parts:
a) verification of the amount, quality and amplifiability of the extracted DNA (not part of this method);
b) analysis using the real-time PCR detection methods targeting the CS-GHc2 - T-AFP construct inserted in
[1][2
the AquAdvantage salmon genome (construct-specific method, Accession AY594644, 75 bp amplicon) )
]
or targeting the border between Atlantic salmon genome and P-AFP event in the AquAdvantage salmon
[3 ]
genome (event-specific method, Accession AY687640.1, 156 bp amplicon) ) . .
Detection of PCR products is done using a specific hydrolysis probe labelled with fluorescent dyes (FAM as
reporter dye and a quencher) that binds the respective target sequence between the two primers (commonly
[4 ]
called “TaqMan chemistry” 'TaqMan chemistry' ). ).
5 Reagents and materials
5.11.1 General
Chemicals of recognized analytical grade, appropriate for molecular biology, shall be used, as a rule. The water
used shall be double distilled or PCR grade water (i.e. nuclease and nucleic acid free). For all operations in
which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected pipette
tips as protection against cross contamination is recommendedshould be used.
5.25.1 Interlaboratory trial and control material.
5.2.15.1.1 5.2.1 DNA extracted from different salmon (Salmo salar) samples, taken in the context of
official control by the Institute for Hygiene and Environment in Hamburg, HamburgGermany for the
construct-specific method, or the National Institute for Health Sciences in Japan for the event-specific method.
5.2.25.1.2 5.2.2 Plasmid DNA, containing the target sequences of the AquAdvantage salmon construct-
[1][2 ] [3 ]
specific PCR methods, , or event-specific PCR methods . .
5.35.2 PCR reagents.
5.3.15.2.1 5.3.1 PCR master mix, contains thermostable DNA polymerase (for hot-start PCR), MgCl2 and
deoxyribonucleoside triphosphates (dNTPs).
5.3.25.2.2 5.3.2 TE buffer, c(Tris-HCl) = = 1 mmol/l, c(EDTA) = = 0,1 mmol/l (pH = = 8,0).
5.3.35.2.3 5.3.3 Oligonucleotides (see Table 1Table 1).).
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of oligonucleotide
in PCR
[1][2]
CS-GHc2 - T-AFP construct as the target sequence
GH-tFreeze-F 5' - CTC CAC Agg TTT TgA CAT gTT CA - 3' 340 nmol/l
Final concentration
Name DNA sequence of oligonucleotide
in PCR
GH-tFreeze-R 5' - gCC AgC AAg AgC CCA TCT C - 3' 340 nmol/l
GH-tFreeze-P 5' –(6-FAM)- TTC CTA ATC TgT ATC Tgg gAA ACC gAA CCC T –(TAMRA)- 3' 540 nmol/l
[3]
Atlantic salmon genome - P-AFP event as the target sequence
AquAd-F 5' – TgC TgA TgC CTC TgA TAC CAC - 3' 800 nmol/l
AquAd-R 5' – ATg CCT CTA gTg CAA gTT CAg TC - 3' 800 nmol/l
AquAd-P 5' –(6-FAM)– CAg TAg TAC AAC gTT ggC AgA TgT ATg AgA ACT –(BHQ1)- 3' 100 nmol/l
FAM: 6-Carboxyfluorescein; TAMRA: 5-Carboxytetramethylrhodamin; BHQ1: black hole quencher 1
Equivalent reporter dyes and/or (internal) quencher can be used for the probe if they can be shown to yield similar or better results.
6 Apparatus
Requirements concerning apparatus and materials shall be according toin accordance with ISO 21569. In
addition to theThe usual l
...