ISO/TS 21569-8:2025
(Main)Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 8: DNA extraction from alfalfa seeds and real-time PCR based detection methods for genetically modified alfalfa events J101, J163 and KK179
Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 8: DNA extraction from alfalfa seeds and real-time PCR based detection methods for genetically modified alfalfa events J101, J163 and KK179
This document specifies procedures for DNA extraction from alfalfa (Medicago sativa) seeds and for the specific detection of the herbicide-tolerant alfalfa events J101 and J163 and the lignin-modified alfalfa event KK179 in crop/plant/seed/grain test samples. The detection methods are based on real-time PCR and are targeting the DNA transition sequences between the alfalfa genome and the respective integrated gene construct. The methods can be applied for direct event-specific identification or as a follow-up analysis, if sequences encoding the promoter of the Figwort mosaic virus (P-FMV), the terminator of the nopaline synthase gene from Rhizobium radiobacter (T-nos), or the construct CTP2/CP4-EPSPS (herbicide tolerance) were detected by screening analyses of test samples. In this document, the methods were validated using ground alfalfa seeds and DNA extracted thereof. The PCR methods are also applicable for the analysis of other matrices such as feed and foodstuffs. The application of these PCR 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 8: Extraction d'ADN à partir de graines de luzerne et méthodes de détection spécifiques à l'événement basées sur la PCR en temps réel pour les lignées de luzerne génétiquement modifiées J101, J163 et KK179
General Information
Buy Standard
Standards Content (Sample)
Technical
Specification
ISO/TS 21569-8
First edition
Horizontal methods for molecular
2025-04
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 8:
DNA extraction from alfalfa seeds
and real-time PCR based detection
methods for genetically modified
alfalfa events J101, J163 and KK179
Méthodes horizontales pour l'analyse moléculaire de
biomarqueurs — Méthodes d'analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Partie 8: Extraction d'ADN à partir de graines de luzerne et
méthodes de détection spécifiques à l'événement basées sur la
PCR en temps réel pour les lignées de luzerne génétiquement
modifiées J101, J163 et KK179
Reference number
© ISO 2025
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
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 Interlaboratory trial and control material .2
5.3 DNA extraction reagents and solutions .2
5.4 PCR reagents .3
6 Apparatus . 4
7 Procedure . 4
7.1 Preparation of test samples .4
7.2 Preparation of DNA extracts .4
7.2.1 General .4
7.2.2 DNA extraction from ground alfalfa seeds .5
7.3 PCR set-up .5
7.4 Temperature-time programme.6
8 Accept/reject criteria . 6
8.1 General .6
8.2 Identification .6
9 Validation status and performance criteria . 7
9.1 Sensitivity .7
9.2 Specificity .7
9.3 Robustness .7
9.4 Interlaboratory trial .8
9.4.1 General .8
9.4.2 Results of DNA extractions from ground alfalfa seed samples .9
9.4.3 False-positive rate/false negative rate .9
9.4.4 PCR efficiency and detection limit .10
9.5 Summary evaluation .11
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.
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 21569-8:2025(en)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 8:
DNA extraction from alfalfa seeds and real-time PCR based
detection methods for genetically modified alfalfa events
J101, J163 and KK179
1 Scope
This document specifies procedures for DNA extraction from alfalfa (Medicago sativa) seeds and for the
specific detection of the herbicide-tolerant alfalfa events J101 and J163 and the lignin-modified alfalfa event
KK179 in crop/plant/seed/grain test samples.
The detection methods are based on real-time PCR and are targeting the DNA transition sequences between
the alfalfa genome and the respective integrated gene construct. The methods can be applied for direct
event-specific identification or as a follow-up analysis, if sequences encoding the promoter of the Figwort
mosaic virus (P-FMV), the terminator of the nopaline synthase gene from Rhizobium radiobacter (T-nos), or
the construct CTP2/CP4-EPSPS (herbicide tolerance) were detected by screening analyses of test samples.
In this document, the methods were validated using ground alfalfa seeds and DNA extracted thereof.
The PCR methods are also applicable for the analysis of other matrices such as feed and foodstuffs. The
application of these PCR 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, 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 as described in this document or by applying another suitable method
with comparable performance characteristics (see ISO 21571). The DNA analysis consists of two parts:
a) verification of the amount, quality and amplifiability of the extracted DNA, by means of the real-time
[1]
PCR for the alfalfa-specific reference gene acc1;
b) analysis using the real-time PCR detection methods specific for the alfalfa events J101, J163 and KK179,
[2][3][4]
respectively.
Detection of PCR products is done using specific hydrolysis probes labelled with fluorescent dyes (FAM as
reporter dye and one or two quenchers) that bind the respective target sequences between the two primers
[5]
(so-called
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.