EN ISO 21570:2005
(Main)Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods (ISO 21570:2005)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods (ISO 21570:2005)
ISO 21570:2005 provides the overall framework of quantitative methods for the detection of genetically modified organisms (GMO) in foodstuffs, using the polymerase chain reaction (PCR).
It defines general requirements for the specific amplification of DNA target sequences in order to quantify the relative GMO-derived DNA content and to confirm the identity of the amplified DNA sequence.
Guidelines, minimum requirements and performance criteria laid down in ISO 21570:2005 are intended to ensure that comparable, accurate and reproducible results are obtained in different laboratories.
ISO 21570:2005 has been established for food matrices, but is also applicable to other matrices, e.g. feed and plant samples from the environment.
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und ihren Produkten - Quantitative auf Nukleinsäuren basierende Verfahren (ISO 21570:2005)
Diese Internationale Norm liefert den Gesamtrahmen für die quantitativen Verfahren zum Nachweis von gentechnisch veränderten Organismen (GVO) in Lebensmitteln mit der Polymerase-Kettenreaktion (PCR).
Sie legt allgemeine Anforderungen an die spezifische Amplifikation von DNA Zielsequenzen zur Quantifizierung des relativen DNA-Gehaltes an GVO und zur Bestätigung der Identität der amplifizierten DNA Sequenz fest.
In dieser internationalen Norm angeführte Anleitungen, Mindestanforderungen und Leistungskriterien sind dafür vorgesehen sicherzustellen, dass in verschiedenen Laboratorien vergleichbare, genaue und reproduzierbare Ergebnisse erhalten werden.
Diese Internationale Norm wurde für Lebensmittelmatrices erarbeitet, ist jedoch auch auf andere Matrices anwendbar, z. B. Futtermittel und aus der Umwelt entnommene Pflanzenproben.
In den Anhängen A bis D sind spezifische Beispiele von Verfahren angegeben.
Produits alimentaires - Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés - Méthodes quantitatives basées sur l'utilisation des acides nucléiques (ISO 21570:2005)
L'ISO 21570:2005 fournit un cadre général auquel doit satisfaire toute méthode quantitative de détection des organismes génétiquement modifiés (OGM) dans les produits alimentaires utilisant la PCR (réaction de polymérisation en chaîne).
Elle définit les exigences générales relatives à l'amplification spécifique de séquences cibles d'ADN, afin de quantifier la teneur relative en ADN dérivé d'OGM et de confirmer l'identité de la séquence d'ADN amplifiée.
Les lignes directrices, les exigences minimales et les critères de performance exposés dans l'ISO 21570:2005 ont pour but de garantir l'obtention de résultats comparables, exacts et reproductibles, dans différents laboratoires.
L'ISO 21570:2005 a été élaborée pour les matrices de produits alimentaires, mais elle est aussi applicable à d'autres matrices, par exemple des aliments pour animaux et des échantillons de plantes issus de l'environnement.
Živila – Analitske metode za odkrivanje gensko spremenjenih organizmov in njihovih produktov – Kvantitativne metode na osnovi nukleinske kisline (ISO 21570:2005)
Ta mednarodni standard določa splošni okvir kvantitativnih metod za odkrivanje gensko spremenjenih organizmov (GMO) v živilih s polimerazno verižno reakcijo (PCR). Določa splošne zahteve za posebno povečanje ciljnih sekvenc DNK, da bi se količinsko opredelila relativna vsebnost DNK iz gensko spremenjenih organizmov in potrdila identiteta povečane sekvence DNK. Smernice, minimalne zahteve in merila učinkovitosti iz tega mednarodnega standarda so namenjeni zagotavljanju, da se v različnih laboratorijih pridobijo primerljivi, točni in ponovljivi rezultati. Ta mednarodni standard je bil pripravljen za živilske matrice, a se uporablja tudi za druge matrice, npr. za vzorce krme in rastlin iz okolja. Posebni primeri metod so opisani v dodatkih od A do D.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-marec-2006
Živila – Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov – Kvantitativne metode na osnovi nukleinske kisline (ISO
21570:2005)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - Quantitative nucleic acid based methods (ISO 21570:2005)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Quantitative auf Nukleinsäuren basierende Verfahren (ISO
21570:2005)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Méthodes quantitatives basées sur
l'utilisation des acides nucléiques (ISO 21570:2005)
Ta slovenski standard je istoveten z: EN ISO 21570:2005
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 21570
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2005
ICS 67.050
English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - Quantitative nucleic
acid based methods (ISO 21570:2005)
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 -
produits dérivés - Méthodes quantitatives basées sur Quantitative auf Nukleinsäuren basierende Verfahren (ISO
l'utilisation des acides nucléiques (ISO 21570:2005) 21570:2005)
This European Standard was approved by CEN on 26 October 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 21570:2005: E
worldwide for CEN national Members.
Foreword
This document (EN ISO 21570:2005) 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 May 2006, and conflicting national standards shall be withdrawn at
the latest by May 2006.
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.
INTERNATIONAL ISO
STANDARD 21570
First edition
2005-11-01
Foodstuffs — Methods of analysis for the
detection of genetically modified
organisms and derived products —
Quantitative nucleic acid based methods
Produits alimentaires — Méthodes d'analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Méthodes quantitatives basées sur l'utilisation des acides nucléiques
Reference number
ISO 21570:2005(E)
©
ISO 2005
ISO 21570:2005(E)
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ii © ISO 2005 – All rights reserved
ISO 21570:2005(E)
Contents Page
Foreword. v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 2
4.1 General. 2
4.2 Amplification, detection and confirmation of PCR products . 2
4.3 Quantitation of PCR products . 2
5 Reagents. 2
6 Apparatus and equipment . 2
7 Guidelines concerning the procedure. 3
7.1 General. 3
7.2 Target sequence stability. 3
7.3 Calibration of the analysis . 3
7.4 Quantitation considerations . 3
7.5 Quality assurance requirements . 3
8 Interpretation. 4
9 Expression of results . 4
10 Test report . 5
Annex A (informative) Target taxon-specific methods. 6
A.1 Target taxon-specific method for the absolute quantitation of the adh1 gene DNA of
maize using real-time PCR. 6
Annex B (informative) Screening methods. 12
B.1 Screening method for the relative quantitation of the 35S-promoter DNA of soya bean line
GTS 40-3-2 using real-time PCR. 12
Annex C (informative) Construct-specific methods . 20
C.1 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using
real-time PCR (Method 1) . 20
C.2 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using
real-time PCR (Method 2) . 27
C.3 Construct-specific method for the quantitation of Event176 maize DNA using real-time
PCR. 34
C.4 Construct-specific method for the quantitation of soya bean line GTS 40-3-2 DNA using
real-time PCR . 41
C.5 Construct-specific method for the quantitation of maize line MON 810 DNA using
real-time PCR . 49
C.6 Construct-specific method for the quantitation of maize line Event176 DNA using
real-time PCR . 56
C.7 Construct-specific method for the quantitation of maize line Bt11 DNA using real-time
PCR. 63
ISO 21570:2005(E)
C.8 Construct-specific method for the quantitation of maize line GA21 DNA using real-time
PCR. 71
C.9 Construct-specific method for the quantitation of maize line T25 DNA using real-time PCR . 78
Annex D (informative) Event-specific methods . 87
D.1 Event-specific method for the absolute and relative quantitation of maize line Bt11 DNA
based on real-time PCR. 87
D.2 Event-specific method for the relative quantitation of maize line MON 810 DNA using
real-time PCR. 93
Bibliography . 100
iv © ISO 2005 – All rights reserved
ISO 21570:2005(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.
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.
ISO 21570 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).
ISO 21570:2005(E)
Introduction
The search for ingredients of genetically modified origin 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 the present and in the following International Standards:
ISO 21569, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Qualitative nucleic acid based methods
ISO 21570, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Quantitative nucleic acid based methods
Further information about definitions and general items involving the steps cited above are collected in:
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions.
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 these patent rights.
ISO has been informed that Applied Biosystems, Roche Molecular Systems, Inc. and Hoffman-La Roche hold
patent rights concerning 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
Attention is drawn to the possibility that some of the elements of this document may be subject of patent rights
other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights.
vi © ISO 2005 – All rights reserved
INTERNATIONAL STANDARD ISO 21570:2005(E)
Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
Quantitative nucleic acid based methods
1 Scope
This International Standard provides the overall framework of quantitative methods for the detection of
genetically modified organisms (GMOs) in foodstuffs, using the polymerase chain reaction (PCR).
It defines general requirements for the specific amplification of DNA target sequences, in order to quantify the
relative GMO-derived DNA content and to confirm 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 is also applicable 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 21569:2005, 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
1)
ISO 24276:— , Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
ISO Guide 32, Calibration in analytical chemistry and use of certified reference materials
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 24276 apply.
1) To be published.
ISO 21570:2005(E)
4 Principle
4.1 General
Quantitative analysis consists of the quantitation of target DNA sequences in the test samples. Each method
specifies the target sequences(s).
[1],[2] [3],[4]
Quantitation may be performed using competitive or real-time PCR .
A quantitative analysis should clearly express the quantity of the target genetic element, relative to the
quantity of a specific reference, appropriate calibrants and controls, and be within the dynamic range of the
analytical method used and the test portion analysed.
The analysis generally consists of
⎯ amplification of one or more specific target sequences,
⎯ detection and confirmation of the specificity of the PCR product(s), and
⎯ quantitation of the amplified fragments relative to calibrants.
NOTE In the case of real-time PCR analysis, amplification, detection and confirmation occur simultaneously.
4.2 Amplification, detection and confirmation of PCR products
See ISO 21569 for the principles of amplification, detection and confirmation of the DNA sequences.
4.3 Quantitation of PCR products
The principle of quantitation is usually to determine the ratio (expressed as a percent) of two DNA target
sequences; i.e. a sequence representing the genetically modified organism of interest and an (endogenous)
target taxon-specific sequence. However, in some cases, quantitation can also be carried out relative to a
specified amount of food matrix (e.g. when detecting GM microorganisms in foods).
Calibrants (calibration materials) used for quantitation should be traceable to certified reference materials
(CRMs), if available. If not available, other suitable reference material should be used. Example guidance is
provided in Reference [5]. Information on validation studies and measurement uncertainty has been gathered
[6],[7],[8],[9]
from international studies .
5 Reagents
All reagents and materials used in the analysis should be identical, or equivalent, to those specified in the
method. Otherwise, all reagents and materials should be of molecular biology grade. These reagents shall be
stored and used as recommended by the supplier or according to the laboratory quality assurance
specifications. For a list of reagents, see the specific annex.
6 Apparatus and equipment
See Annexes A to D and ISO 24276.
2 © ISO 2005 – All rights reserved
ISO 21570:2005(E)
7 Guidelines concerning the procedure
7.1 General
General considerations relevant to PCR amplification for the detection of GMOs are described in ISO 21569.
Annexes A to D specify PCR detection methods together with details of their scope of application. The
demonstrated performance characteristics for each method are detailed.
The concentration of the DNA sequence of interest should be within the dynamic range of the method.
NOTE A target taxon specific monitor run can be undertaken to determine whether the template DNA is of sufficient
quality (length and structural integrity), purity and quantity to allow the detection and quantitation of a GMO belonging to
the target taxon. This may be of particular relevance when DNA is extracted from composite or highly processed matrices.
The DNA extracted from each test portion should be analysed at least in duplicate.
Appropriate controls shall be included (see ISO 24276:—, Table 1).
7.2 Target sequence stability
The allelic and copy number stability of the target sequence should be considered for cultivars of different
geographic and phylogenic origins.
7.3 Calibration of the analysis
An appropriate number of calibration points and replicates covering the range of quantitation shall be applied
[e.g. four calibration points with two replicates (altogether 4 × 2 values) or six calibration points with one
measurement at each point (altogether 6 values)]. The quality of the calibration influences the measurement
[9]
uncertainty .
As an alternative to genomic DNA calibration reference materials, for example, a dilution series of a plasmid
or synthetic dsDNA containing the target sequence may be used, provided that it is demonstrated to perform
in an equivalent way to the genomic DNA reference material and the genomic DNA extracted from the sample.
7.4 Quantitation considerations
PCR methods should be appropriately designed to minimize the variability.
NOTE Depending on the method used and/or the material analysed, the presence of stacked genes can lead to
overestimation of the true GMO content.
For the determination of the limit of quantitation (LOQ), see ISO 24276.
Calculation of the GMO content based on copy numbers of target sequences per haploid genome is
influenced by the homo- and heterozygosity of the species under investigation. For details, see Annexes A to
D.
Use of the ∆∆C (cycle of threshold) method is only valid if the amplification efficiencies of the target
t
taxon-specific assay and the GMO-specific assay are very similar.
7.5 Quality assurance requirements
Consistency between measurements is desirable to obtain reliable estimates of target sequence quantities.
However, knowledge of the relative standard deviation of repeatability of the method is required to establish
whether the measurements are consistent (see the ISO 5725 series for details). To calculate the relative
standard deviation of repeatability, the number of separate measurements per laboratory sample may exceed
what is feasible in practice in terms of acceptable costs. Consequently, if a specified GMO-derived DNA is to
be reported (in percent), a feasible solution should require the following as a minimum:
ISO 21570:2005(E)
a) within test portion consistency:
⎯ through rejection of measurements
⎯ through maximum deviation observed between dilutions and individual measurements equals the
value expected from the corresponding dilution factor ± 33 %;
b) between test portion consistency:
⎯ estimated relative GMO-derived DNA concentrations obtained under a) for each test portion should
not differ by values greater than −50 % to +100 % of the estimated quantity value (equal to a ∆C of 1
t
in real-time PCR) (i.e. for two test portions, measurements of 1,0 % and 2,0 % are acceptable,
measurements of 0,9 % and 2,1 % are not).
In order to guarantee accuracy of the measurements, a reference material (RM), preferably certified (CRM),
for the quantity of the event concerned, with an appropriate level of metrological reliability and with reasonable
similarity of matrix shall be selected and analysed. In the absence of a CRM, in-house RM may be prepared
by a procedure demonstrating stability, homogeneity and traceability, and ensuring the absence of bias. The
quantified uncertainty shall fulfil the required uncertainty for the calibration (see ISO Guide 32).
8 Interpretation
The PCR result will be either
a) fit for quantitation of the target sequence provided
⎯ the result is positive according to ISO 21569:2005, 8.1,
⎯ the observable inhibition of the reaction is negligible,
⎯ the analysis produces an unambiguous measurement value,
⎯ the target sequence content is within the dynamic range of the method, and
⎯ the analysis is calibrated in an acceptable way (see 7.3), or
b) not fit for quantitation of the target sequence if any of the conditions listed above are not fulfilled.
The measurement uncertainty shall be sufficiently small to enable the laboratory to draw the relevant
conclusions.
Annexes A to D describe the measurement of the target DNA quantities. These quantities can be used to
calculate the GMO content. These calculations usually take into consideration relevant biological factors, such
as the homo- or heterozygosity of the target sequences.
If the GM target sequence content or the taxon-specific target sequence content is below the limit of
quantitation, the result shall only be expressed qualitatively.
NOTE Stating that the GMO-derived DNA content is below the practical LOQ accompanied by a specification of that
LOQ is considered to be a qualitative expression of the result.
9 Expression of results
The results shall clearly state the quantity of the GM target sequence relative to the target taxon-specific
sequence. The results should also provide values for the measurement uncertainty, such as the standard
4 © ISO 2005 – All rights reserved
ISO 21570:2005(E)
deviation or relative standard deviation. Furthermore, the LOD and LOQ of the method and the practical LOD
and LOQ should be reported.
The target sequences may or may not be detected, or the quantity of at least one of them may be below the
limit of quantitation. Table 1 describes the four alternative cases and the corresponding expression of the
result to be included into the test report.
Table 1 — Expression of results
Result Expression of the result
Target taxon-specific sequence is not See ISO 21569.
detected.
“For species x, DNA was not detected.”
Target taxon-specific sequence is According to ISO 21569.
detected but GM target sequence is not
“For species x, GMO-derived DNA was not detected.”
detected.
In addition, if applicable, add: “The practical limit of detection is X %.”
(Specify unit used.)
The target taxon-specific sequence and For each GMO, state:
the GM target sequence are both detected
“GMO (specify the GMO) derived DNA as determined by detection of
but the quantity is below the LOQ of at
(specify target sequence) derived from (specify species) was detected.”
least one of the target sequences.
In addition, if applicable: “The practical limit of quantitation is X %.” (Specify
unit used.)
The target taxon-specific sequence and For each GMO, state:
the GM target sequence are both detected
“The content of GMO (specify the GMO) derived DNA as determined by
and the quantity is above the LOQ for both
detection of (specify target sequence) derived from (specify species) is
target sequences.
X ± uncertainty %.” (Specify unit used.)
The GMO-derived DNA content may also be reported as being above or below a specific value, taking into
account the measurement uncertainty.
10 Test report
The test report shall be written in accordance with ISO 24276 and ISO 21569 and shall contain at least the
following additional information:
a) LOQ of the method and the matrix used to establish it;
b) the practical LOQ;
c) a reference to the method which has been used for the extraction of DNA;
d) the reference material used;
e) the results expressed according to Clause 9.
ISO 21570:2005(E)
Annex A
(informative)
Target taxon-specific methods
A.1 Target taxon-specific method for the absolute quantitation of the adh1 gene DNA
of maize using real-time PCR
A.1.1 Introduction
This annex describes a method for the specific amplification and quantitation of the taxon-specific
(housekeeping) maize (Zea mays) adh1 gene (coding for alcohol dehydrogenase 1) for determination of the
content of maize DNA, or for testing for the presence/absence of detectable PCR inhibitors in DNA solutions
extracted from products containing maize-derived DNA, e.g. foods.
For limitations, see A.1.8.
A.1.2 Validation status and performance characteristics
A.1.2.1 General
The method has been optimized for DNA extracted from pure ground maize kernels, maize leaves and
[10] [11]
certified reference materials (IRMM-411, IRMM-412, IRMM-413 series ) .
The reproducibility of the described method has been tested in a collaborative trial using unknown samples
(U1 to U6) consisting of wild type maize DNA at different corresponding copy number of the target sequence
(see A.1.2.2), and in other collaborative trials in combination with methods specific for GM maize events, e.g.
Bt11 (see D.1).
[11]
The copy number of the target sequence per haploid genome is estimated to be 1 .
[11]
The allelic stability of the target sequence has been established .
A.1.2.2 Collaborative trial
The method has been validated in a collaborative trial organized by the European Commission's Joint
Research Centre (EC-JRC), Institute for Health and Consumer Protection (IHCP), in agreement with the
[12]
international harmonized protocol .
[13]
Six samples (S1-S6) of wild type maize DNA (extracted from leaf material containing known absolute copy
numbers (183 486, 61 162, 20 387, 6 796, 2 265, 755) of haploid maize genomes were used to establish a
calibration curve for absolute quantitation of haploid maize genomes in unknown samples. The absolute copy
numbers in the known samples were determined by dividing the sample DNA mass (determined by
fluorometric quantitation of dsDNA with PicoGreen, Molecular Probes, Cat. Number P-7589) by the published
[14]
average 1C value for maize genomes (2,725 pg) .
[13]
Six samples (U1-U6) of wild type maize DNA (extracted from leaf material ) were used as unknown
samples. The expected copy numbers in the unknown samples were determined in the same way as those of
the known samples.
The results of the collaborative trial validation are summarized in Table A.1
6 © ISO 2005 – All rights reserved
ISO 21570:2005(E)
The method has also been validated in combination with event-specific methods for several maize GMO, e.g.
for Bt11 sweet maize. See References [15] and [16] and D.1 for details on the combined (relative quantitation)
trial.
Table A.1 — Validation data
Sample
U1 U2 U3 U4 U5 U6
Number of participating laboratories 12 12 12 12 12 12
Number of laboratories having returned results 10 10 10 10 10 10
Number of invalid laboratories 1 1 1 1 1 1
Number of retained laboratories 9 9 9 9 9 9
Number of samples per laboratory 4 4 4 4 4 4
Number of Cochran outliers 1 1 1 1 — —
Number of Grubbs outliers — 1 1 1 1 1
Number of accepted samples 35 34 34 34 35 35
Expected copy number value 7 339 18 349 36 697 55 046 91 743 146 788
Mean copy number value 9 985 23 885 46 918 75 161 100 541 122 080
Bias of true value (%) 36,1 30,2 27,9 36,5 9,6 −16,8
a
1 318,59 1 463,60 5 796,58 4 539,57 11 306,89 14 843,41
Repeatability standard deviation s
r
b
13,21 6,13 12,35 6,04 11,25 12,16
Repeatability relative standard deviation (%)
a
2 013,12 2 083,57 6 145,39 6 806,85 14 592,04 17 777,70
Reproducibility standard deviation s
R
b
20,16 8,72 13,10 9,06 14,51 14,56
Reproducibility relative standard deviation (%)
a
Expressed as copy number value.
b
Expressed as percentage of the mean value.
A.1.2.3 Molecular specificity
A.1.2.3.1 General
2)
The method has been designed to target a part of the sequence described in EMBL/GenBank/DDBJ
accession number X04050. This sequence is unique to Zea mays (maize/corn) and Zea mays subsp.
[11]
diploperennis (teosinte) .
A.1.2.3.2 Theoretical specificities
The theoretical specificities of the primers and probes were assessed through a search of the
2)
GenBank/EMBL/DDBJ databases using the nucleotide sequences as query sequences with the BLASTN
2)
programme at http://www.ncbi.nlm.nih.gov/blast/ [October 9, 2003]. The result of the search confirmed a
complete identity only with the expected target sequences.
2) These 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 the product named. Equivalent
products may be used if they can be shown to lead to the same results.
ISO 21570:2005(E)
A.1.2.3.3 Experimental determination of specificity
The specificity of the method was tested against a wide range of non-target taxa and 20 different maize lines
[11]
representing geographically and phylogenetically diverse samples . No cross-reactivity was observed with
the non-target taxa (except with teosinte Zea mays subsp. diploperennis, the wild ancestor of cultivated
[11], [17]
maize) . The copy number and allelic stability of the target sequence across different maize lines has
[11]
been established .
A.1.2.4 Optimization
® 3) ® 3)
This was carried out for the ABI PRISM 7700 sequence detection system (SDS) and TaqMan chemistry .
® 3)
Primer and probe design were done with Primer Express software (Applied Biosystems) .
A.1.2.5 Limit of detection (LOD)
[11]
According to method developer, the absolute LOD is 10 copies of the target sequence .
The lowest number of copies of the target sequence included in the collaborative trial was 7 399 copies of the
target sequence.
A.1.2.6 Limit of quantitation (LOQ)
[11]
According to the method developer, the absolute LOQ is 100 copies of the target sequence .
The lowest number of copies of the target sequence included in the collaborative trial was 7 399.
A.1.3 Adaptation
No specific information is available.
A.1.4 Principle
A 134 bp fragment of the adh1 gene is amplified using two maize adh1-specific primers (see Table A.2).
Accumulation of PCR products is measured at the end of each PCR cycle (real-time) by means of a maize
adh1-specific oligonucleotide probe (ADH1-MDO, see Table A.2) labelled with two fluorescent dyes: FAM as
® 3)
reporter dye and TAMRA as quencher. For that purpose, TaqMan chemistry was employed.
The measured fluorescence signal crosses a user-defined threshold value after a certain number of cycles.
This number is called the C -value. For quantitation of the amount of maize adh1-DNA in an unknown sample,
t
the C -value is converted into a corresponding copy number value by comparison with a calibration curve
t
whose C -values are directly linked with known copy numbers (regression analysis).
t
A.1.5 Reagents
A.1.5.1 General
For quality of reagents to be used, see ISO 24276:—, 6.6.
A.1.5.2 Water.
A.1.5.3 PCR buffer (without MgCl ), 10-fold.
3) These 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 the product named. Equivalent
products may be used if they can be shown to lead to the same results.
8 © ISO 2005 – All rights reserved
ISO 21570:2005(E)
A.1.5.4 MgCl solution, c(MgCl ) = 25 mmol/l.
2 2
A.1.5.5 dNTP solution, c(dNTP) = 2,5 mmol/l (each).
A.1.5.6 Oligonucleotides
Details of the oligonucleotides are listed in Table A.2.
Table A.2 — Oligonucleotides
Name Oligonucleotide DNA sequence Final concentration in PCR
ADH-FF3 5'-CgT CgT TTC CCA TCT CTT CCT CC-3' 300 nmol/l
ADH-RR4 5'-CCA CTC CgA gAC CCT CAg TC-3' 300 nmol/l
a
ADH1-MDO 200 nmol/l
5'-FAM-AAT CAg ggC TCA TTT TCT CgC TCC TCA-TAMRA-3'
a
FAM: 6-carboxyfluorescein; TAMRA: 6-carboxytetramethylrhodamine.
The length of the adh1 amplicon is 134 bp.
A.1.5.7 Thermostable DNA polymerase
® 4)
AmpliTaq Gold DNA polymerase .
A.1.5.8 Uracil N-glycosylase (optional).
A.1.5.9 Amplification reaction mixture
Details of the amplification reaction mixture are listed in Table A.3.
Table A.3 — Amplification reaction mixture in the final volume/concentration per reaction vial
Total reaction volume 25 µl
Template DNA (maximum 250 ng) 5 µl
Taq-DNA-polymerase
Decontamination system (dUTP included uracil N-glycosylase) ®
TaqMan Universal Master Mix 2X 12,5 µl (1 X)
a
Reaction buffer (containing passive reference ROX)
dNTP mix
Primers see Table A.2 see A.1.5.6
Probe see Table A.2 see A.1.5.6
a
ROX = carboxy-X-rhodamine.
4) These 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 the product named. Equivalent
products may be used if they can be shown to lead to the same results.
ISO 21570:2005(E)
A.1.6 Apparatus
A.1.6.1 General
Standard laboratory apparatus should be used throughout unless otherwise specified.
A.1.6.2 Thermal cycler ®
The indicated temperature-time profile was originally tested with the ABI PRISM 7700 SDS (Applied
5)
Biosystems) . Other real-time PCR detection systems may be used after adaptation of the reaction
conditions.
A.1.6.3 Reaction vials
The reaction vials shall be suitable for PCR amplification on a real-time thermal cycler, e.g.
® ®
ABI PRISM 96-Well Optical Reaction Plate, or MicroAmp Optical Caps (8 caps/strip, flat) (Applied
5)
Biosystems) .
A.1.7 Procedure: PCR set-up
A.1.7.1 General
The PCR set-up for the reference gene target sequence and for the GMO target sequence should be carried
out in separate vials unless otherwise stated in the relevant GM target-specific annex.
The method is described for a total PCR volume of 25 µl per reaction mixture with the reagents as listed in
Table A.3.
A.1.7.2 PCR controls
If the controls do not yield the expected results, the test results shall be rejected and the analysis shall be
repeated.
As a positive control/calibration reference material, high-quality, pure genomic DNA extracted from maize (e.g.
5) [13]
a CRM from the JRC, IRMM ) may be used . Any other appropriate controls should be included as
described in ISO 24276.
A.1.7.3 Temperature-time programme ®
The temperature-time programme as outlined in Table A.4 was optimized for the ABI PRISM 7700 sequence
5)
detection system (SDS) (Applied Biosystems) . In the validation study, it was used in combination with the
® 5)
AmpliTaq Gold DNA polymerase . The use of other thermal cyclers may require specific adaptation. The
temperature and time required for enzyme activation depend on the particular polymerase used. Table A.4
describes the reaction conditions.
5) These 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 the product named. Equivalent
products may be used if they can be shown to lead to the same results.
10 © ISO 2005 – All rights reserved
ISO 21570:2005(E)
Table A.4 — Procedure: Reaction conditions
Time Temperature
s °C
Pre-PCR: decontamination 120 50
Pre-PCR: activation of DNA polymerase and denaturation of template DNA 600 95
PCR (50 cycles)
Step 1 Denaturation 15 95
Step 2 Annealing elongation 60 60
A.1.8 Limitations and interpretation of the results
The presence of PCR inhibitors may have a strong impact on the accuracy of the estimated copy number of
the adh1 in the analysed samples. Therefore the absence of detectable PCR inhibitors should be verified (see
also ISO 21571:2005, Annex A), for example by setting up serial dilutions of the template DNA and examining
the correspondence between dilutions and differences in C (cycle of threshold) values (i.e. one C corresponds
t t
to a doubling of template concentration).
For the use of this method in combination with a method for the quantitation of GMO-derived DNA, it is
important that the absolute amount of template DNA (ng) be the same for both the adh1 PCR and the
GMO-specific PCR. If not, the absolute copy numbers of the reactions cannot be compared directly, and an
adjustment of the corresponding copy numbers is required. Otherwise a relative GMO concentration cannot
be calculated.
A.1.9 Calibration and calculation of results
Calibration points are produced with DNA containing defined amounts in absolute copy numbers of haploid
genomic maize DNA containing the target sequence.
A calibration curve is produced by plotting C -values against the logarithm of the target copy number for the
t
calibration points. This can be carried out, for example, by use of spread-sheet software such as Microsoft
6)
Excel , or directly by options available with the sequence detection system software.
The calibration curve is used to determine the absolute haploid genomic maize DNA copy numbers of the
unknown samples. Although the sample DNA can be degraded due to food processing or may contain
ingredients other than maize, this does not influence the calculated number of haploid genome copies of
unknown samples.
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