ISO/DTS 21569-10.2

ISO/DTS 21569-10
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

ISO/DTS 21569-10:2025(en)
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ii
ISO/DTS 21569-10:2025(en)
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
ISO/DTS 21569-10:2025(en)
Foreword
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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
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iv
ISO/DTS 21569-10:2025(en)
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:
ISO/DTS 21569-10:2025(en)
— 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
ISO/DTS 21569-10:2025(en)
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 laboratory equipmentapparatus and, in particular, the following equipment is
required:shall be used.
6.1 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 6Table 6 and 1010 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 21571:2005and2005 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 2Table 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 1× concentrated
polymerase)
Forward and reverse primer see Table 1
Probe see Table 1
Water add to obtain 25 µl
ISO/DTS 21569-10:2025(en)
Reagent Final concentration in 25 µl PCR reaction
TM TM
a 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
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 either 5 µl of sample DNA or 5 µl of
the respective control solution (extraction blank control, positive DNA target control, negative control) into
the respective well and seal the reaction compartments. A PCR inhibition control should be carried out as
described in (ISO 24276:2006 (e.g. using a 1:4 dilution of the extracted DNA).
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 MgCl2, dNTPs and DNA
1× concentrated
polymerase)
Forward and reverse primer see Table 1
Probe see Table 1
Water add to obtain 25 µl
a TM
In the interlaboratory trial for the construct-specific method, the TaqMan Universal PCR Master Mix, no
TM
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. 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
program.
Transfer the reaction set-ups into the real-time PCR device and start the temperature-time programme (see
Table 3program (Table 3).).
7.4 Temperature-time programprogramme
The temperature-time programprogramme as outlined in Table 3Table 3 has been used in the validation
study. The use of different reaction conditions and real-time PCR cyclers maycan require specific optimization.
Depending on the PCR instrument or PCR master mix used, the temperature-time program mayprogramme
can require adjustments.
Table 3 — Temperature-time programprogramme
Method Step Parameter Temperature Time Fluorescence Cycles
Inserted Cells
measurement
Construct-specific detection method
1 Initial denaturation 95 °C 10 min no 1 Inserted Cells
Con
stru
ct-
spe
ISO/DTS 21569-10:2025(en)
Method Step Parameter Temperature Time Fluorescence Cycles
Inserted Cells
measurement
Denaturation 95 °C 15 s no
2 Amplification 45
Annealing and
60 °C 60 s yes
elongation
Event-specific detection method
1 Initial denaturation 95 °C 10 min no 1
Denaturation 95 °C 15 s no Inserted Cells
2 Amplification 45
Annealing and
57 °C 60 s yes
elongation
8 Accept/Rejectreject criteria
8.1 General
8.1.11.1.1 General
The evaluation of PCR amplification results is performed with the respective device-specific data analysis
programprogramme. If the amplification of the target sequence is successful in a sample (positive result), the
cycle number of interest will occur when the accumulated fluorescence signal exceeds a specified threshold
for the first time (C value).
q
8.2 Detection
The target sequence is considered as detected in a sample, if all of the following apply:
— —  a sigmoid-shaped amplification curve and typical increase in the measured fluorescence is detected
with the primers and probe specific for the CS-GHc2 – T-AFP construct or the Atlantic salmon genome – P-
AFP event, and;
— —  no amplification and increase in fluorescence have occurred in the PCR control reactions with no DNA
added (PCR reagent control, extraction blank control), and);
— —  the expected Cq values are achieved in the amplification controls (positive DNA target control for the
CS-GHc2 – T-AFP construct, the Atlantic salmon genome – P-AFP event, PCR inhibition control). In the case
of event-specific detection method, reactions with a C value of < 43 were determined as positive.
q
NOTE In the case of event-specific detection method, reactions with a Cq value of < 43 were determined as
positive.
9 Validation status and performance criteria
9.1 Sensitivity
Within the framework of the single-laboratory validation that involved dilution series with linearized plasmid
DNA containing the target sequence, absolute limits of detection (LOD ) of at least 10 copies (for the
95 %
[1 ] [3 ]
construct-specific detection method) ) and 25 copies (for the event-specific detection method) ) were
determined. For the construct-specific method, an interlaboratory trial was then performed, leading to a
[2]
detection limit of at least 5 copies (for the construct-specific detection method) (see Table 7) (Table 7).).
[5 ]
The determination of the LOD followed the guideline of Reference, , where the LOD is set at the concentration
where 59 out of 60 replicates are positive. This criterion is met at 5 copies per Reactionreaction (113 out of
114 replicates positive).
Event-
specific
detection
ISO/DTS 21569-10:2025(en)
9.2 Specificity
Table 4 — Specificity tests for the construct-specific and event-specific real-time PCR methods
(underlined: construct donor species)
Target Experimental confirmation
Positive: AquAdvantage Salmon
Negative:
Fish:
— Salmoidae: Atlantic salmon, Trout, Rainbow trout, Coho salmon, Sockeye salmon,
Pink salmon, Brook trout, Chinook salmon
— Ammodytidae: Sand eel
— Carangidae: Atlantic horse mackerel
— Cichlidae: Nile tilapia
— Clup
...