prCEN ISO/TS 21569-10

SLOVENSKI STANDARD
01-september-2025
Horizontalne metode za analizo molekularnih biomarkerjev - Analitske metode za
odkrivanje gensko spremenjenih organizmov in njihovih proizvodov - 10. del:
Konstruktno- in dogodkovno-specifične metode za odkrivanje gensko
spremenjenega lososa, ki izraža rastni hormon CS-GHc2 (ISO/DTS 21569-10:2025)
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 (ISO/DTS 21569-10:2025)
Untersuchung auf molekulare Biomarker - Verfahren zum Nachweis von gentechnisch
modifizierten Organismen und ihren Produkten - Teil 10: Konstrukt- und
ereignisspezifische Nachweisverfahren für gentechnisch veränderte AquAdvantage-
Lachse
Titre manque — Partie 10: Titre manque
Ta slovenski standard je istoveten z: prCEN ISO/TS 21569-10
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
67.120.30 Ribe in ribji proizvodi Fish and fishery products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

FINAL DRAFT
Technical
Specification
ISO/DTS 21569-10
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.
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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
ISO/DTS 21569-10:2025(en) © ISO 2025

FINAL DRAFT
ISO/DTS 21569-10:2025(en)
Technical
Specification
ISO/DTS 21569-10
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.
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Published in Switzerland Reference number
ISO/DTS 21569-10:2025(en) © ISO 2025

ii
ISO/DTS 21569-10:2025(en)
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
ISO/DTS 21569-10:2025(en)
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/

ISO/DTS 21569-10:2025(en)
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 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
the AquAdvantage salmon genome (construct-specific method, Accession AY594644, 75 bp amplicon)
[1][2]
or targeting the border between Atlantic salmon genome and P-AFP event in the AquAdvantage
[3]
salmon 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” ).
5 Reagents and materials
Chemicals of recognized analytical grade, appropriate for molecular biology, shall be used. 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 should be used.
5.1 Interlaboratory trial and control material.
5.1.1 DNA extracted from different salmon (Salmo salar) samples, taken in the context of official
control by the Institute for Hygiene and Environment in Hamburg, Germany for the construct-specific
method, or the National Institute for Health Sciences in Japan for the event-specific method.
5.1.2 Plasmid DNA, containing the target sequences of the AquAdvantage salmon construct-specific PCR
[1][2] [3]
methods, or event-specific PCR methods .
5.2 PCR reagents.
5.2.1 PCR master mix, contains thermostable DNA polymerase (for hot-start PCR), MgCl and
deoxyribonucleoside triphosphates (dNTPs).
5.2.2 TE buffer, c(Tris-HCl) = 1 mmol/l, c(EDTA) = 0,1 mmol/l (pH = 8,0).
5.2.3 Oligonucleotides (see Table 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
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
Equivalent reporter dyes and/or (internal) quencher can be used for the probe if they can be shown to yield similar or better
results.
ISO/DTS 21569-10:2025(en)
TTaabblle 1 e 1 ((ccoonnttiinnueuedd))
Final concentration
Name DNA sequence of oligonucleotide
in PCR
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 in accordance with ISO 21569. The usual
laboratory apparatus and, in particular, the following shall be used.
6.1 Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of
fluorescence signals generated during PCR. In the interlaboratory trial, the real-time PCR devices listed in
Table 6 and 10 were used.
7 Procedure
7.1 Preparation of test samples
It should be ensured that the test sample used for DNA extraction is representative of the laboratory
sample (e.g. by grinding or homogenizing of the samples). Measures and operational steps to be taken into
consideration shall be as described in ISO 21571:2005 and ISO 24276.
7.2 DNA extraction
For the extraction of DNA from the test portion, the general instructions and requirements described in
ISO 21571 shall be followed.
7.3 PCR setup
The methods are described for a total volume of 25 μl per reaction mixture with the set-up given in Table 2.
Table 2 — Reaction set-up for PCR
Reagent Final concentration in 25 µl PCR reaction
Sample DNA (up to 200 ng for construct-specific, and 5 µl diluted DNA
50 ng for event-specific detection method) or controls
a
PCR master mix (including MgCl , dNTPs and DNA poly- 1× concentrated
merase)
Forward and reverse primer see Table 1
Probe see Table 1
Water add to obtain 25 µl
a TM TM
In the interlaboratory trial for the construct-specific method, the TaqMan Universal PCR Master Mix, no AmpErase UNG
(Thermo Fisher Scientific) was used. In the interlaboratory trial for the event-specific method, the FastStart Universal Probe
Master (Roche) was used. This information is given for the convenience of users of this document and does not constitute an
endorsement by ISO of the product named. Equivalent products from other manufacturers may be used if they yield similar or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
Completely thaw reagents at suitable conditions. Each reagent should be carefully mixed and briefly
centrifuged immediately before pipetting. Prepare a PCR reagent mixture which contains all components
except for the sample DNA. The required amount of the PCR reagent mixture depends on the number of

ISO/DTS 21569-10:2025(en)
reactions to be performed, including at least two additional reactions as a pipetting reserve. Add 5 µl of the
respective sample DNA to each reaction.
Mix the PCR reagent mixture, centrifuge briefly and pipette 20 µl into each reaction vial. For the amplification
reagent control, add 5 µl water into the respective reaction set-up. Pipette eithe
...