ISO/TS 21569-2:2012
(Main)Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 2: Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products
Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 2: Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products
This method describes a procedure for the detection of a DNA sequence present in a genetically modified linseed (Linum usitatissimum) line (event FP967, also named as "CDC Triffid"). For this purpose, extracted DNA is used in a real-time PCR and the genetic modification (GM) is specifically detected by amplification of a 105 bp DNA sequence representing the transition between the nopalin synthase gene terminator (Tnos) from Agrobacterium tumefaciens and the dihydrofolate reductase gene (dfrA1) from a Class 1 integron of Escherichia coli. The method described is applicable for the analysis of DNA extracted from foodstuffs. It may 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 for the purpose of analysis.
Méthodes horizontales d'analyse moléculaire de biomarqueurs — Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés — Partie 2: Méthode PCR en temps réel spécifique de la construction pour la détection d'un évènement FP967 dans les graines de lin et les produits à base de graines de lin
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Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 21569-2
First edition
2012-09-01
Horizontal methods for molecular
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 2:
Construct-specific real-time PCR
method for detection of event FP967
in linseed and linseed products
Méthodes horizontales d’analyse moléculaire de biomarqueurs —
Méthodes d’analyse pour la détection des organismes génétiquement
modifiés et des produits dérivés —
Partie 2: Méthode PCR en temps réel spécifique de la construction
pour la détection d’un évènement FP967 dans les graines de lin et les
produits à base de graines de lin
Reference number
ISO/TS 21569-2:2012(E)
ISO 2012
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ISO/TS 21569-2:2012(E)
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© ISO 2012
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ii © ISO 2012 – All rights reserved
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ISO/TS 21569-2:2012(E)
Contents Page
Foreword ........................................................................................................................................................................................................................................iv
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Principle ........................................................................................................................................................................................................................ 1
5 Reagents and materials ................................................................................................................................................................................. 2
5.1 PCR reagents ............................................................................................................................................................................................. 2
6 Apparatus ..................................................................................................................................................................................................................... 3
6.1 General ........................................................................................................................................................................................................... 3
6.2 PCR device ................................................................................................................................................................................................... 3
7 Sampling ........................................................................................................................................................................................................................ 3
8 Procedure..................................................................................................................................................................................................................... 3
8.1 Test sample preparation ................................................................................................................................................................. 3
8.2 Preparation of the DNA extracts .............................................................................................................................................. 3
8.3 DNA extraction ........................................................................................................................................................................................ 3
8.4 PCR setup ..................................................................................................................................................................................................... 3
8.5 Temperature ..............................................................................................................................................................
time programme .................................................................................................................................................................................... 4
9 Accept/reject criteria ...................................................................................................................................................................................... 4
9.1 General ........................................................................................................................................................................................................... 4
9.2 Identification ............................................................................................................................................................................................ 5
10 Validation status and performance criteria ............................................................................................................................. 5
10.1 Robustness of the method ............................................................................................................................................................. 5
10.2 Intralaboratory trial ........................................................................................................................................................................... 5
10.3 Collaborative trial ................................................................................................................................................................................. 5
10.4 Sensitivity .................................................................................................................................................................................................... 7
10.5 Specificity .................................................................................................................................................................................................... 7
11 Test report ................................................................................................................................................................................................................... 8
Bibliography ................................................................................................................................................................................................................................ 9
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ISO/TS 21569-2:2012(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.In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for
a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or
ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be
transformed into an International Standard or be withdrawn.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/TS 21569-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.ISO/TS 21569 consists of the following parts, under the general title Horizontal methods for molecular
biomarker analysis — Methods of analysis for the detection of genetically modified organisms and
derived products:— Part 2: Construct-specific real-time PCR method for detection of event FP967 in linseed and linseed products
iv © ISO 2012 – All rights reserved---------------------- Page: 4 ----------------------
TECHNICAL SPECIFICATION ISO/TS 21569-2:2012(E)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically
modified organisms and derived products —
Part 2:
Construct-specific real-time PCR method for detection of
event FP967 in linseed and linseed products
1 Scope
This method describes a procedure for the detection of a DNA sequence present in a genetically
modified linseed (Linum usitatissimum) line (event FP967, also named as “CDC Triffid”). For this purpose,
extracted DNA is used in a real-time PCR and the genetic modification (GM) is specifically detected by
amplification of a 105 bp DNA sequence representing the transition between the nopalin synthase gene
terminator (Tnos) from Agrobacterium tumefaciens and the dihydrofolate reductase gene (dfrA1) from a
Class 1 integron of Escherichia coli.The method described is applicable for the analysis of DNA extracted from foodstuffs. It may 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 for the purpose of analysis.2 Normative references
ISO 21569, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Qualitative nucleic acid based methodsISO 21571:2005, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — Nucleic acid extractionISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 24276 apply.
4 PrincipleDNA is extracted from the test sample applying a suitable method. The DNA analysis consists of two parts:
a) Verification of the amount, quality and amplifiability of the extracted DNA, e.g. by means of a target
taxon specific real-time PCR with primers amplifying a 68 bp long fragment from the linseed-specific
(Linum usitatissimum) stearoyl-acyl carrier protein desaturase 2 gene (SAD) (Reference [1]).
b) Detection of the Tnos-dfr construct in a real-time PCR (Reference [1]).© ISO 2012 – All rights reserved 1
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ISO/TS 21569-2:2012(E)
5 Reagents and materials
Chemicals of recognized analytical grade, appropriate for molecular biology shall be used, as a rule. The
water used shall be double distilled or of an adequate quality. Unless stated otherwise, solutions should
be prepared by dissolving the corresponding reagents in water and autoclaved. For all operations in
which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected
pipette tips serves as protection against cross-contamination.5.1 PCR reagents
5.1.1 Thermostable DNA polymerase (for hot-start PCR).
5.1.2 PCR buffer solution (contains magnesium chloride and deoxyribonucleoside triphosphate dATP,
dCTP, dGTP and dUTP).Ready-to-use reagent mixtures or individual components can be used. Reagents and polymerases which
lead to equal or better results may also be used.5.1.3 Oligonucleotides (see Table 1).
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in the PCR
Tnos-dfr construct as the target sequence (Reference [1]):
NOST-Spec FW 5’-AgC gCg CAA ACT Agg ATA AA-3’ 800 nmol/l
NOST-Spec RV 5’-ACC TTC Cgg CTC gAT gTC TA-3’ 800 nmol/l
NOST-Spec Probe 5’-(FAM)-CgC gCg Cgg TgT CAT CTA Tg-(BHQ)-3’ 100 nmol/l
FAM: 6-Carboxyfluorescein, BHQ: black hole quencher.
NOTE Equivalent reporter dyes and/or quencher dyes can be used for the probe if they can be shown to yield
similar or better results.5.1.4 Standard DNA for calibration
A standard DNA solution of a known concentration (ng/µl) is used to calculate the copy numbers of the
Tnos-dfr target sequence.When using genomic linseed DNA as the standard DNA, the number of haploid genome equivalents per
microlitre, n , shall be calculated on the basis of the molecular mass of the linseed haploid genome
hgEqwhich is approximately 0,7 pg (Reference [2]) and by applying Equation (1):
[DNA]×1000
n = (1)
hgEq
where
[DNA] is the DNA concentration in nanograms per microlitre;
m is the haploid genome mass, in picograms.
In the collaborative trial, a plasmid was used as standard DNA which contains a copy of the 105 bp Tnos-dfr
fragment and the 68 bp large SAD gene fragment, respectively. Because the exact number of integrations
of the Tnos-dfr construct in event FP967 in linseed is not known at the time of the specification of this
document, the calculated GM-content only represents an estimation which is based on the assumption
that the target sequence is present as a single copy per haploid genome.2 © ISO 2012 – All rights reserved
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ISO/TS 21569-2:2012(E)
6 Apparatus
6.1 General
Regarding the apparatus and materials, see ISO 21569. In addition to the usual laboratory equipment
the following equipment is required.6.2 PCR device
Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of fluorescence
signals generated during PCR.7 Sampling
All samples shall be identified unambiguously.
8 Procedure
8.1 Test sample preparation
It should be ensured that the test sample used for DNA extraction is representative of the laboratory
sample, e.g. by grinding or homogenizing the samples. Take into consideration the measures and
operational steps specified in ISO 21571 and ISO 24276.8.2 Preparation of the DNA extracts
Concerning the preparation of DNA from the test sample, the general instructions and measures
described in ISO 21571 should be followed. It is recommended that one of the DNA extraction methods
described in ISO 21571:2005, Annex A be chosen.8.3 DNA extraction
It is recommended that the DNA extraction be performed by means of the CTAB method with a test
portion of 1 g of the homogenized sample (see ISO 21571:2005, A.3.1).Due to problems of purity, an additional purification step (gel filtration, e.g. by means of micro spin
columns) may be necessary.As long as comparability is ensured, other extraction and purification methods (e.g. kit systems) can be
applied, using lower test portions, if necessary (Reference [1]).8.4 PCR setup
The method is described for a total volume of 25 μl per PCR. The reagents given in Table 2 should be used.
Reagents are completely thawed at room temperature and should be briefly centrifuged before use.
Each reagent should be carefully mixed immediat...
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