EN ISO 21569:2005/A1:2013

SLOVENSKI STANDARD
01-junij-2013
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Kvalitativne metode na osnovi nukleinske kisline - Dopolnilo
A1 (ISO 21569:2005/Amd 1:2013)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Qualitative nucleic acid based methods - Amendment 1 (ISO
21569:2005/Amd 1:2013)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Qualitative auf Nukleinsäuren basierende Verfahren - Änderung 1 (ISO
21569:2005/Amd 1:2013)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Méthodes qualitatives basées sur
l'utilisation des acides nucléiques - Amendement 1 (ISO 21569:2005/Amd 1:2013)
Ta slovenski standard je istoveten z: EN ISO 21569:2005/A1:2013
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 21569:2005/A1
NORME EUROPÉENNE
EUROPÄISCHE NORM
April 2013
ICS 67.050
English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Qualitative nucleic
acid based methods - Amendment 1 (ISO 21569:2005/Amd
1:2013)
Produits alimentaires - Méthodes d'analyse pour la Lebensmittel - Verfahren zum Nachweis von gentechnisch
détection des organismes génétiquement modifiés et des modifizierten Organismen und ihren Produkten - Qualitative
produits dérivés - Méthodes qualitatives basées sur auf Nukleinsäuren basierende Verfahren - Änderung 1 (ISO
l'utilisation des acides nucléiques - Amendement 1 (ISO 21569:2005/Amd 1:2013)
21569:2005/Amd 1:2013)
This amendment A1 modifies the European Standard EN ISO 21569:2005; it was approved by CEN on 14 March 2013.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for inclusion of this
amendment into the relevant national standard without any alteration. Up-to-date lists and bibliographical references concerning such
national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.

This amendment exists in three official versions (English, French, German). A version in any other language made by translation under the
responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21569:2005/A1:2013: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (EN ISO 21569:2005/A1:2013) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
This Amendment to the European Standard EN ISO 21569:2005 shall be given the status of a national
standard, either by publication of an identical text or by endorsement, at the latest by October 2013, and
conflicting national standards shall be withdrawn at the latest by October 2013.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech
Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece,
Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO 21569:2005/Amd 1:2013 has been approved by CEN as EN ISO 21569:2005/A1:2013 without
any modification.
INTERNATIONAL ISO
STANDARD 21569
First edition
2005-06-15
AMENDMENT 1
2013-04-01
Foodstuffs — Methods of analysis for
the detection of genetically modified
organisms and derived products —
Qualitative nucleic acid based methods
AMENDMENT 1
Produits alimentaires — Méthodes d’analyse pour la détection
des organismes génétiquement modifiés et des produits dérivés —
Méthodes qualitatives basées sur l’utilisation des acides nucléiques
AMENDEMENT 1
Reference number
ISO 21569:2005/Amd.1:2013(E)
©
ISO 2013
ISO 21569:2005/Amd.1:2013(E)
© ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
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Published in Switzerland
ii © ISO 2013 – All rights reserved

ISO 21569:2005/Amd.1:2013(E)
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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
Amendment 1 to ISO 21569:2005was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.
ISO 21569:2005/Amd.1:2013(E)
Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Qualitative nucleic acid based methods
AMENDMENT 1
No attempt has been made in this amendment to update the footnote numbering to fit in with the scheme
adopted in ISO 21569:2005. The footnote numbers given are for use refer solely within this amendment.
Page v, Introduction, paragraph 1
Delete “— Sampling (ISO 21568)”.
Page 2, Clause 2, ISO 24276
Delete the footnote and update the entry to read:
ISO 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms
and derived products — General requirements and definitions
Page 2, 4.1, paragraph 2
Delete the existing text and insert the following.
A qualitative result shall clearly demonstrate the presence or absence of the genetic element under
study, relative to appropriate controls.
NOTE Detection limits and size of the test portion are critical aspects of a method.

Page 2, 7.3.3.3.3, paragraph 2
Delete “a representative” and insert “an appropriate” so that the text reads as follows.
Primers designed to detect taxon-specific target sequences should be shown to detect these
sequences reliably in an appropriate number of different members of the taxon.
Page 6, 8.1 a) and b)
In both cases, delete “ISO 24276:—”, and insert “ISO 24276:2006.
Page 7, 9.4
ISO 21569:2005/Amd.1:2013(E)
Delete the existing text and insert the following.
Results within the same test portion shall be consistent. In case of +/− results for the two replicates,
repeat the two PCR for the respective test portion. If the two novel replicates are tested +/− or −/−,
the test portion is considered as negative.
Results from all test portions shall be consistent. When at least one test portion gives a positive
result and at least one gives a negative result, the analysis shall be repeated.
If at least one repetition of the procedure, beginning with the nucleic acid extraction, gives ambiguous
results such as a positive and a negative result, the report should state that the sample is negative
at the limit of detection (LOD).
Page 7, Clause 10, list item 2
Delete the existing text and insert the following.
— the specificity of the analytical method (event specific, construct specific, or screening method);
Page 23, Annex A
Insert A.5 and A.6 after the existing text.
A.5 Target taxon-specific method for the detection of DNAs derived from rice
A.5.1 Purpose, relevance and scientific basis
The GMO Detection Laboratory of Shanghai Jiao Tong University (GMDL-SJTU) organized a collaborative
study for validation of the applicability of a target taxon-specific method using the rice sucrose phosphate
synthase (SPS) gene as an endogenous gene for qualitative analysis of genetically modified (GM) or non-
GM rice. This study involved 12 laboratories from Spain, Korea, Lithuania, Slovenia, Japan, Italy, and China.
The operational procedure of the collaborative study comprised the following modules:
— qualitative PCR for validation of the heterogeneity of the SPS gene among rice cultivars for different
geographic and phylogenetic origins;
— qualitative PCR for validation of the species specificity of SPS gene for rice;
— qualitative PCR for evaluation of the LOD of the established SPS qualitative PCR assay.
The collaborative study was carried out in accordance with Reference [44].
The results of the collaborative study as well as the related protocol are given in A.5.3.
A.5.2 Principle
The method has been optimized for rice seeds and other processed products such as seed powder.
Applicability of the SPS gene was evaluated in this collaborative study using DNA samples extracted
from rice seeds and other plant materials.
The collaborative study organizer provided method-specific reagents (primers, probes, reaction master
mix), and the test DNA samples extracted from rice materials to collaborative study participants.
2 © ISO 2013 – All rights reserved

ISO 21569:2005/Amd.1:2013(E)
A.5.3 Validation status and performance criteria
A.5.3.1 Robustness of the method
Robustness has been tested on the SPS gene qualitative PCR system for three different annealing
temperatures (i.e. 56 °C, 58 °C, and 60 °C), on three different DNA samples containing known amounts
of rice DNA (10 ng, 1 ng, 0,1 ng rice genome DNA samples) and with three repetitions per sample.
The qualitative PCR systems demonstrated the expected robustness and performed well at all three
annealing temperatures and three concentrations of the rice DNA samples.
1)
The SPS gene qualitative PCR system was also tested on different thermal cyclers (PTC-100, MJ
Research and instruments from Bio-Rad and Applied Biosystems), on three different reaction volumes
(25 μl, 30 μl, and 50 μl) and three repetitions per volume. The qualitative PCR systems had the expected
robustness and performed well on different thermal cyclers and with different reaction volumes.
A.5.3.2 Intralaboratory trial
The rice SPS gene has been described as being suitable for use as an endogenous reference gene in rice
identification and quantification (Reference [44]). The detailed technical information was modified
from Reference [44].
For sample preparation in the collaboration study, all the DNA samples were extracted by the GMDL-
SJTU using the CTAB method adopted from ISO 21571:2005, A.3. Spectrophotometric quantification
of DNA extracted was performed using a method adopted from ISO 21571:2005, B.1. After the DNA
quantification, a qualitative PCR using an 18S PCR system (Reference [45]) was carried out to provide
data about possible PCR inhibition.
The SPS gene PCR system was tested using rice genomic DNA by three researchers at the GMDL-SJTU.
The results were satisfactory; in particular, for qualitative PCR, the results show that the SPS gene is
specific for rice, and the LOD is about 0,1 %.
A.5.3.3 Collaborative trial
For the collaborative study, each participant received 12 rice DNA samples for he
...