SIST EN ISO 21569:2005

SLOVENSKI STANDARD
SIST EN ISO 21569:2005
01-oktober-2005
Živila - Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov - Kvalitativne metode na osnovi nukleinske kisline (ISO
21569:2005
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Qualitative nucleic acid based methods (ISO 21569:2005)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Qualitative auf Nukleinsäuren basierende Verfahren (ISO 21569:2005)
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 (ISO 21569:2005)
Ta slovenski standard je istoveten z: EN ISO 21569:2005
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 21569:2005 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 21569:2005

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SIST EN ISO 21569:2005
EUROPEAN STANDARD
EN ISO 21569
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2005
ICS 67.050
English version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Qualitative nucleic
acid based methods (ISO 21569:2005)
Produits alimentaires - Méthodes d'analyse pour la Lebensmittel - Verfahren zum Nachweis von gentechnisch
détection des organismes génétiquement modifiés et des modifizierten Organismen und irhen Produkten - Qualitative
produits dérivés - Méthodes qualitatives basées sur auf Nukleinsäuren basierende Verfahren (ISO 21569:2005)
l'utilisation des acides nucléiques (ISO 21569:2005)
This European Standard was approved by CEN on 6 June 2005.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the Central Secretariat or to any CEN member.
This European Standard 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia,
Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2005 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21569:2005: E
worldwide for CEN national Members.

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SIST EN ISO 21569:2005
EN ISO 21569:2005 (E)




Foreword



This document (EN ISO 21569:2005) has been prepared by Technical Committee CEN/TC 275 "Food
analysis - Horizontal methods", the secretariat of which is held by DIN, in collaboration with Technical
Committee ISO/TC 34 "Agricultural food products".

This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by December 2005, and conflicting national standards shall be
withdrawn at the latest by December 2005.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Cyprus, Czech Republic,
Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland
and United Kingdom.

2

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SIST EN ISO 21569:2005

INTERNATIONAL ISO
STANDARD 21569
First edition
2005-06-15


Foodstuffs — Methods of analysis for the
detection of genetically modified
organisms and derived products —
Qualitative nucleic acid based methods
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





Reference number
ISO 21569:2005(E)
©
ISO 2005

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SIST EN ISO 21569:2005
ISO 21569:2005(E)
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© ISO 2005
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
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Published in Switzerland

ii © ISO 2005 – All rights reserved

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SIST EN ISO 21569:2005
ISO 21569:2005(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
4 Principle of the method. 2
5 Reagents. 2
6 Apparatus and equipment. 3
7 Procedure. 3
8 Interpretation. 6
9 Expression of results and quality assurance . 6
10 Test report. 7
Annex A (informative) Target-taxon-specific methods. 8
Annex B (informative) Screening methods. 24
Annex C (informative) Construct-specific methods . 40
Annex D (informative) Event-specific methods. 62
Bibliography . 67

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SIST EN ISO 21569:2005
ISO 21569:2005(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.
ISO 21569 was prepared by the European Committee for Standardization (CEN) Technical Committee
CEN/TC 275, Food analysis — Horizontal methods, in collaboration with Technical Committee ISO/TC 34,
Food products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).

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SIST EN ISO 21569:2005
ISO 21569:2005(E)
Introduction
The search for a genetically modified origin of ingredients is performed by means of the following successive
(or simultaneous) steps. After sample collection, nucleic acids are extracted from the test portion. Extracted
nucleic acids can be further purified, simultaneously or after the extraction process. Afterwards, they are
quantified (if necessary), diluted (if necessary) and subjected to analytical procedures (such as PCR). These
steps are detailed in this International Standard and in the following series of International Standards with the
general title Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products:
 Sampling (ISO 21568);
 Quantitative nucleic acid based methods (ISO 21570);
 Nucleic acid extraction (ISO 21571).
Further information about general requirements and definitions involving the steps cited above are collected in
ISO 24276.
The qualitative detection of DNA target sequences is performed in order to obtain a yes or no answer to the
question whether a certain target sequence is detected or not relative to appropriate controls and within the
detection limits of the analytical method used and test portion analysed.
The specificity of the methods, as described in Annexes A to D, ranges from screening methods to detect
common DNA sequences characteristic of GMOs, to specific identification of a genetic construct or a specific
transformation event.
The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that
compliance with this document may involve the use of a patent concerning the PCR technology.
ISO takes no position concerning the evidence, validity and scope of this patent right.
ISO has been informed that Applied Biosystems, Roche Molecular Systems, Inc. and F. Hoffman La Roche
Ltd. hold patent rights concerning the PCR technology. The companies have assured the ISO that they are
willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants
throughout the world. In this respect, the statements of the holders of these patent rights are registered with
ISO. Information may be obtained from:
Licensing Department
Applied Biosystems
850 Lincoln Centre Drive
Foster City, CA 94404
USA
and
Roche Molecular Systems, Inc.
Licensing Department
1145 Atlantic Avenue
Alameda, CA 94501
USA
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent
rights.
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SIST EN ISO 21569:2005
INTERNATIONAL STANDARD ISO 21569:2005(E)

Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Qualitative nucleic acid based methods
1 Scope
This International Standard describes the procedure to qualitatively detect genetically modified organisms
(GMOs) and derived products by analysing the nucleic acids extracted from the sample under study. The main
focus is on polymerase chain reaction (PCR) based amplification methods.
It gives general requirements for the specific detection and identification of target nucleic acid sequences
(DNA) and for the confirmation of the identity of the amplified DNA sequence.
Guidelines, minimum requirements and performance criteria laid down in this International Standard are
intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories.
This International Standard has been established for food matrices, but could also be applied to other
matrices (e.g. feed and plant samples from the environment).
Specific examples of methods are provided in Annexes A to D.
2 Normative references
The following referenced documents are indispensable for the application 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 21571:2005, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — Nucleic acid extraction
1)
ISO 24276:— , Foodstuffs — Nucleic acid based 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 24276 apply.

1) To be published.
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
4 Principle of the method
4.1 General
Qualitative analysis consists of specific detection of target nucleic acid sequences in the test samples. Each
method shall specify the target sequence.
A qualitative result shall clearly demonstrate the presence or absence of the genetic element under study,
relative to appropriate controls and within the detection limits of the analytical method used and test portion
analysed.
4.2 PCR amplification
Amplification of the target sequence occurs in vitro through a reaction catalysed by a DNA polymerase in the
[1], [2]
presence of oligonucleotide primers and deoxyribonucleoside triphosphates in a defined reaction buffer .
An important prerequisite for the amplification of the target sequence is that the reaction mixture contains no
polymerase inhibitors. Amplification of the DNA is a cyclical process consisting of
 denaturation of the double-stranded DNA into single-stranded nucleic acid by means of heating,
 annealing of the primers to the target sequence at a suitable temperature, and
 extension of the primers, which are bound to both single strands, by a DNA polymerase suitable for PCR,
at an appropriate temperature.
4.3 Detection and confirmation of PCR products
PCR products are detected by gel electrophoresis or an appropriate alternative, if necessary, after isolation by
means of a suitable separation procedure.
The identity of any detected target sequence can be verified by an appropriate technique (e.g. by restriction
enzyme analysis, by hybridization or by DNA sequence analysis).
In the case of real-time PCR analysis, amplification and detection occur simultaneously.
5 Reagents
It is generally advisable to store the reaction solutions required for the analytical method at approximately
–20 °C if not specified otherwise.
It may also be appropriate to aliquot the reaction solutions required for the analytical method in order to avoid
subjecting them to repeated freeze-thaw cycles, and/or to reduce chances of cross contamination.
5.1 Target DNA/control
5.2 Water
5.3 Deoxyribonucleoside triphosphate (dNTP) solution, containing dATP, dCTP, dGTP, and dTTP or
dUTP.
NOTE The use of dUTP can interfere with restriction enzyme analyses of PCR products.
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
5.4 PCR buffer solution
The PCR buffer solution is usually delivered with the DNA polymerase, which may or may not include MgCl
2
in a concentration specified by the manufacturer. The final MgCl concentrations are method specific and are
2
therefore listed in each annex. Ready-to-use reagents may be commercially available. The manufacturer’s
instructions for use should be considered.
5.5 Thermostable DNA polymerase
5.6 Forward primer
5.7 Reverse primer
6 Apparatus and equipment
See ISO 24276 and Annexes A to D for details.
7 Procedure
7.1 Quality, integrity and amplifiability of nucleic acid extracts
[3]
The nucleic acid solution shall be pure enough for subsequent analysis . The quality and amount of nucleic
acid extracted using a given method on a given matrix shall be both repeatable and reproducible.
NOTE The quality, integrity and amount of the DNA template influences the outcome of the PCR, and hence the
analytical results obtained. The limit of detection of a specific method may therefore depend on whether the material to be
analysed has been processed or refined, and on the degree of degradation of the DNA therein.
[4]
Nucleic acids for use in PCR should be substantially free of PCR inhibitors . PCR inhibition controls shall be
included as described in ISO 24276.
7.2 Performance criteria
General performance criteria are described in ISO 24276.
The values for the performance characteristics are given for each method as outlined in Annexes A to D and
should take into account the genome sizes; see Reference [5].
The reaction conditions, especially the MgCl concentration and the thermocycling conditions should be
2
optimized for every primer pair and/or system. When any primer system is used for the first time, it is
necessary to demonstrate beforehand that the cycle conditions chosen for the particular matrix to be studied
avoid undesirable competitive products that would otherwise reduce the sensitivity of the PCR detection.
In an optimal reaction, less than 40 cycles are required to amplify W 10 target molecules to produce a product
that is readily detectable by standard methods. As the cycle number increases, non-specific products could
accumulate. The optimized PCR should be able to amplify in 40 PCR cycles from a pure reference sample of
100 copies of template DNA enough copies of the PCR product to be detectable. The characteristic
temperature/time profile for each primer system and the reaction mixture appropriate for the apparatus used
and the number of cycles shall be strictly adhered to.
In general, the specificity of the reaction should be enhanced as much as possible (e.g. by using hot-start
PCR). Hot-start PCR is recommended as a means of reducing side reactions such as the amplification of non-
target sequences in background DNA (mispriming) and primer-oligomerization (it thus increases specificity).
The values derived from the validation study may not be applicable to analyte concentration ranges and
matrices other than given in the respective annexes.
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
7.3 Aspects of PCR design
7.3.1 General
Because the performance of each specific PCR should be comparable with other specific PCRs, the following
aspects of PCR design shall be taken into account.
7.3.2 Size of PCR products
The size of the target sequence shall be selected to match the range of molecular mass available in the
nucleic acid extract being analysed.
EXAMPLE For highly degraded DNA from processed foodstuffs, the size of the PCR product should ideally be in the
range of 60 bp to 150 bp. For raw materials, a broader range of PCR products up to, for example, 250 bp is applicable.
However, if prior experimental studies are carried out to determine the validity of primer sets yielding different
sized PCR products, these may be used on the matrix for which they have been validated.
7.3.3 Primers
7.3.3.1 General
Primer sequence information is included in Annexes A to D.
7.3.3.2 Primer design
The primer sequences should preferably have the following characteristics wherever practicable:
 length of each primer: 18 to 30 nucleotides;
 optimal annealing temperature ≈ 60 °C (should be established experimentally), i.e. estimated melting
temperature u 65 °C;
 GC:AT ratio = 50:50 if possible, or else as close to this ratio as possible;
 high internal stability (avoid concentration of Gs and Cs in short segments of primers);
 minimal 3' end complementarity to avoid primer-dimer formation;
 minimal secondary structure;
 minimal dimer formation with specific detection probe(s) designed for the PCR.
Software packages are available to help with primer design.
7.3.3.3 Validation of primers
7.3.3.3.1 General
The ability of the primers to detect the target sequence shall be validated.
Primer validation should be carried out in two steps: a first theoretical evaluation, and a second experimental
evaluation.
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
7.3.3.3.2 Theoretical evaluation of the specificity
Theoretical evaluation shall as a minimum be carried out by performing a sequence similarity search (e.g.
® 2) ® 2)
FastA, Blast ) against one of the major nucleic acid sequence databases (e.g. EMBL, GenBank ).
Homologous gene sequences may be retrieved from the sequence databases and aligned to obtain an
estimate of the chance of finding similar sequences in the target taxon or other organisms.
7.3.3.3.3 Experimental evaluation of the specificity
Irrespective of the design criteria used, the specificity of primers shall always be experimentally evaluated to
confirm the primers’ ability to discriminate between the target and closely related non-target sequences.
Primers designed to detect taxon-specific target sequences should be shown to detect these sequences
reliably in a representative number of different members of the taxon.
7.4 PCR target descriptions
For the qualitative detection and identification of GMOs, various PCR tests may be performed, depending on
the type of matrices under study and/or the requirements of the analysis. These analyses may be directed at
sequences specific for target taxa, genetic constructs and transformation events, as well as elements suitable
for screening purposes.
7.5 Controls
Because of the risk of obtaining false positive and/or false negative results, appropriate controls shall be
included in each diagnostic PCR assay (see ISO 24276).
If available and appropriate, certified reference materials should be used as positive and negative controls.
7.6 PCR set-up, detection and confirmation of PCR products
Annexes A to D give details on the specific PCR procedure steps.
NOTE In the case of detection of the PCR products by gel electrophoresis, the size of the PCR products can be
estimated using a suitable DNA size marker of known length to run in parallel with the PCR products under test.
It may be desirable in some cases to confirm a positive or negative result for a particular genetic modification.
This may be achieved by employing primers to an alternative target sequence; this is particularly suitable for
confirmation of screening test results.
A positive identification of the specific target DNA sequence may be confirmed by an appropriate method
other than size determination of the PCR product, for example
 by hybridization of the PCR product with specific probes, or
 by carrying out restriction analyses of the PCR product; the length of the resulting fragments has to
correspond to the expected length of the target DNA sequence after restriction, or
 by sequencing of the PCR product, or
 other equivalent confirmation.

2) Blast and GenBank are examples of suitable products available commercially. This information is given for the
convenience of users of this International Standard and does not constitute an endorsement by ISO of these products.
Equivalent products may be used if they can be shown to give the same results.
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
If the primers used are designed to detect sequences derived from infectious organisms (a naturally occurring
non-genetically modified organism such as a virus or a bacterium), then it is highly recommended that it be
verified that the detected DNA is indeed derived from a GMO. This can be done by checking for the absence
of other DNA derived from the infectious organism.
EXAMPLE The 35S promoter is derived from cauliflower mosaic virus (CaMV), and consequently detection of the
[6]
CaMV 35S promoter could be due to the presence of either GMO-derived and/or CaMV-derived DNA . By checking for
presence of the other CaMV-derived DNA, it may be possible to confirm the GMO origin of the CaMV 35S promoter if no
other CaMV-derived DNA is detected.
8 Interpretation
8.1 General
The PCR result will be either
a) positive if a specific PCR product has been detected, and all the controls give results as specified in
ISO 24276:—, Table 2, or
b) negative if a specific PCR product has not been detected, and all the controls give results as specified in
ISO 24276:—, Table 2.
NOTE Event-specific target sequences are sometimes present together with other event-specific sequences in a
[7]
single GMO (e.g. due to gene stacking ).
If the results are ambiguous, the procedure shall be repeated; see ISO 24276.
8.2 Verification
Verification of positive or negative results for target sequences may be achieved as described in 7.6.
9 Expression of results and quality assurance
9.1 General
The results shall be expressed unambiguously, i.e. not as “±”.
A negative result shall never be expressed as “GMO not present”.
Ideally, the limit of detection (LOD) should be provided with reference to the test sample. However, this
requires particular materials, DNA of exceptionally high quality, and/or use of sophisticated laboratory
equipment that is not available to all laboratories. Consequently, the analysis can become very labour
intensive and/or expensive, and therefore not applicable in practice for routine purposes.
As a minimum, the LOD shall be provided with reference to a reference material, and a relative value based
on a specified matrix (preferably a given amount of genomic DNA solution, e.g. 100 ng of 0,01 % GTS 40-3-2
DNA).
9.2 Expression of a negative result
The following text shall appear in the test report:
“For sample X, target sequence Y was not detected.
The LOD of the method is x % determined with ABC (identify the reference material).”
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SIST EN ISO 21569:2005
ISO 21569:2005(E)
If it cannot be demonstrated that the amount of target DNA included in the PCR is sufficient for the LOD to be
applicable, then the following sentence shall be added:
“However, the amount of the target DNA extracted from species X may be/was insufficient for the LOD to
be applicable for this sample.”
NOTE The LOD of the sample is determined by the quantity of DNA of the species included in the analytical reaction
[7]
(copy number), and the ratio relative to the absolute LOD of the GM target (copy number) .
9.3 Expression of a positive result
The following text shall appear in the test report:
“For sample X, target sequence Y was detected.”
The identity of the GMO may be included, if available.
9.4 Quality assurance requirements
Results from both test portions shall be c
...