ISO/TS 21569-9:2025

Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 9: Construct-specific real-time PCR based screening method for the detection of the P35S-nptII DNA-sequence

This document specifies a procedure for the detection of the DNA transition sequence between the 35S promoter region from cauliflower mosaic virus (P35S) and the neomycin-phosphotransferase gene (nptII) from the Tn5 transposon of Escherichia coli. The P35S-nptII segment is part of a construct which confers resistance to neomycin/kanamycin antibiotics frequently found in genetically modified (GM) plants. The detection method is based on real-time PCR and can be used for qualitative screening purposes. For identification and quantification of a specific GM plant (event) a follow-up analysis has to be carried out. This method 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.

Méthodes horizontales pour l'analyse moléculaire de biomarqueurs — Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés — Partie 9: Méthode de criblage construit spécifique basée sur la PCR en temps réel pour la détection de la séquence ADN P35S-nptII

General Information

• Status: Published
• Publication Date: 02-Apr-2025
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Current Stage: 6060 - International Standard published
• Start Date: 03-Apr-2025
• Due Date: 03-Apr-2025
• Completion Date: 03-Apr-2025

Standards Content (Sample)

Technical
Specification
ISO/TS 21569-9
First edition
Horizontal methods for molecular
2025-04
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 9:
Construct-specific real-time PCR
based screening method for the
detection of the P35S-nptII DNA-
sequence
Méthodes horizontales pour l'analyse moléculaire de
biomarqueurs — Méthodes d'analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Partie 9: Méthode de criblage construit spécifique basée sur la PCR
en temps réel pour la détection de la séquence ADN P35S-nptII
Reference number
© ISO 2025
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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
5.1 General .2
5.2 PCR reagents .2
6 Apparatus . 3
7 Procedure . 3
7.1 Preparation of test samples .3
7.2 Preparation of DNA extracts .3
7.3 PCR set-up .3
7.4 Temperature-time programme.4
8 Accept/reject criteria . 4
8.1 General .4
8.2 Identification .4
9 Validation status and performance criteria . 5
9.1 Specificity .5
9.2 Sensitivity .6
9.3 Robustness .6
9.4 Interlaboratory trial .7
9.4.1 General .7
9.4.2 False positive rate/false negative rate .7
9.4.3 PCR efficiency and detection limit .8
10 Test report . 9
Bibliography .10

iii
Foreword
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bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
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The procedures used to develop this document and those intended for its further maintenance are described
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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.
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
Technical Specification ISO/TS 21569-9:2025(en)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 9:
Construct-specific real-time PCR based screening method for
the detection of the P35S-nptII DNA-sequence
1 Scope
This document specifies a procedure for the detection of the DNA transition sequence between the 35S
promoter region from cauliflower mosaic virus (P35S) and the neomycin-phosphotransferase gene (nptII)
from the Tn5 transposon of Escherichia coli. The P35S-nptII segment is part of a construct which confers
resistance to neomycin/kanamycin antibiotics frequently found in genetically modified (GM) plants.
The detection method is based on real-time PCR and can be used for qualitative screening purposes. For
identification and quantification of a specific GM plant (event) a follow-up analysis has to be carried out.
This method 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.
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, 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 (see ISO 21571). The DNA analysis
consists of two parts:
a) verification of the amount and amplifiability of the extracted DNA, e.g. by means of a simplex real-time
[1][2]
PCR specific for the universal plant actin gene or another target taxon (see ISO 21570);
[4][5]
b) detection of the P35S-nptII DNA sequence in a simplex real-time PCR.
NOTE In the case of a positive result, further analyses, if possible, with event-specific PCR methods, are carried
out for the identification of the GM plant (event).
5 Reagents and materials
5.1 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 recommended.
5.2 PCR reagents
5.2.1 Thermostable DNA polymerase (for hot-start PCR).
5.2.2 PCR buffer solution, containing magnesium chloride and deoxyribonucleoside triphosphates
(dNTPs).
Ready-to-use reagent mixtures or mixes of individual components can be used. Reagents and DNA
polymerases that lead to equal or better results may also be used.
5.2.3 Oligonucleotides (see Table 1).
Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield
similar or better results.
Table 1 — Oligonucleotides
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
[1][2]
Plant actin as the target sequence :
Act-f 5′- CAA gCA gCA TgA AgA TCA Agg T −3′ 900 nmol/l
Act-r 5′- CAC ATC TgT Tgg AAA gTg CTg Ag -3′ 900 nmol/l
Act-probe 5′-(FAM)- CCT CCA ATC CAg ACA CTg TAC TTY CTC TC -(BHQ1)-3′ 200 nmol/l
[4][5]
P35S-nptII construct as the target sequence :
Primer 35S-F 5′- TAT CCT TCg CAA gAC CCT TCC -3′ 400 nmol/l
Primer nptII-R 5′- gAT TgT CTg TTg TgC CCA gTC A -3′ 400 nmol/l
a
Probe nptII-Tm2 5′-(FAM)- AgC CgA ATA gCC TCT CCA CCC AAg C -(BHQ1)-3′ 100 nmol/l
Key ®
FAM: 6-
...