ISO/TS 21569-7:2022
(Main)Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 7: Real-time PCR based methods for the detection of CaMV and Agrobacterium Ti-plasmid derived DNA sequences
Horizontal methods for molecular biomarker analysis — Methods of analysis for the detection of genetically modified organisms and derived products — Part 7: Real-time PCR based methods for the detection of CaMV and Agrobacterium Ti-plasmid derived DNA sequences
This document specifies a procedure for the detection of a DNA sequence of the open reading frame five (ORF V) from cauliflower mosaic virus (CaMV) and a procedure for the detection of the DNA sequence of the nopaline synthase (nos) gene from tumour-inducing (Ti) plasmids of phytopathogenic Rhizobium radiobacter (formerly named Agrobacterium tumefaciens). The procedures can be used in the context of screening for genetically modified crop/plants and their derived products to further clarify a positive PCR result for a specific promoter or terminator of CaMV (P-35S, T-35S), or both, and the nos gene (P-nos, T-nos), respectively. The methods specified in this document will detect and identify naturally occurring CaMV or Rhizobium radiobacter (Ti plasmid) DNA, or both, if present in the sample in the absence of a genetically modified plant event containing the specified target sequences. Both methods are based on the real-time polymerase chain reaction (PCR) and are applicable for the analysis of DNA extracted from foodstuffs and other products such as feedstuffs and seeds/grains. The application of the methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix. With appropriate calibration material, the CaMV ORF V or nos copy number, or both, can be estimated and compared, respectively, with the estimated copy number for the promoter (P-35S, P-nos) or the terminator (T-35S, T-nos) sequences, or both. Thereby, conclusions are possible about the presence of an unknown genetically modified organism (GMO) in addition to any detected CaMV DNA or Rhizobium radiobacter Ti plasmid DNA, or both, in a test sample.
Méthodes horizontales d'analyse moléculaire de biomarqueurs — Méthodes d'analyse pour la détection des organismes génétiquement modifiés et des produits dérivés — Partie 7: Méthodes par PCR en temps réel pour la détection des séquences ADN dérivées du plasmide Ti de la bactérie Agrobacterium et du CaMV (virus de la mosaïque du chou-fleur)
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TECHNICAL ISO/TS
SPECIFICATION 21569-7
First edition
2022-12
Horizontal methods for molecular
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 7:
Real-time PCR based methods for the
detection of CaMV and Agrobacterium
Ti-plasmid derived DNA sequences
Reference number
ISO/TS 21569-7:2022(E)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO/TS 21569-7:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21569-7:2022(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure .3
7.1 Preparation of test samples . 3
7.2 Preparation of DNA extracts . 3
7.3 PCR setup. 3
7.4 Temperature-time programme. 4
8 Accept/reject criteria . 5
8.1 General . 5
8.2 Identification of nos . 5
8.3 Identification of CaMV ORF V . 5
9 Validation status and performance criteria . 6
9.1 Specificity . 6
9.2 Sensitivity . 7
9.3 Robustness . 7
9.4 Interlaboratory trials . 8
9.4.1 General . 8
9.4.2 False-positive rate/false-negative rate . 9
9.4.3 PCR efficiency and detection limit . 10
9.4.4 Summary evaluation of the interlaboratory trials . . 11
10 Test report .11
Bibliography .12
iii
© ISO 2022 – All rights reserved
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ISO/TS 21569-7:2022(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 21569 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
© ISO 2022 – All rights reserved
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TECHNICAL SPECIFICATION ISO/TS 21569-7:2022(E)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically
modified organisms and derived products —
Part 7:
Real-time PCR based methods for the detection of CaMV
and Agrobacterium Ti-plasmid derived DNA sequences
1 Scope
This document specifies a procedure for the detection of a DNA sequence of the open reading frame five
(ORF V) from cauliflower mosaic virus (CaMV) and a procedure for the detection of the DNA sequence
of the nopaline synthase (nos) gene from tumour-inducing (Ti) plasmids of phytopathogenic Rhizobium
radiobacter (formerly named Agrobacterium tumefaciens). The procedures can be used in the context of
screening for genetically modified crop/plants and their derived products to further clarify a positive
PCR result for a specific promoter or terminator of CaMV (P-35S, T-35S), or both, and the nos gene
(P-nos, T-nos), respectively.
The methods specified in this document will detect and identify naturally occurring CaMV or Rhizobium
radiobacter (Ti plasmid) DNA, or both, if present in the sample in the absence of a genetically modified
plant event containing the specified target sequences.
Both methods are based on the real-time polymerase chain reaction (PCR) and are applicable for the
analysis of DNA extracted from foodstuffs and other products such as feedstuffs and seeds/grains. The
application of the methods requires the extraction of an adequate amount of amplifiable DNA from the
relevant matrix.
With appropriate calibration material, the CaMV ORF V or nos copy number, or both, can be estimated
and compared, respectively, with the estimated copy number for the promoter (P-35S, P-nos) or the
terminator (T-35S, T-nos) sequences, or both. Thereby, conclusions are possible about the presence of
an unknown genetically modified organism (GMO) in addition to any detected CaMV DNA or Rhizobium
radiobacter Ti plasmid DNA, or both, in a test sample.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 21569:2005, 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
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
1
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ISO/TS 21569-7:2022(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
DNA extracts of test portions are used that showed a positive PCR result in screening tests for specific
promoter/terminator sequences derived from CaMV and/or from Ti plasmid of Rhizobium radiobacter.
The tests consist of two parts, namely:
a) detection of the CaMV ORF V and/or the nos DNA sequence in a real-time PCR;
b) estimation of the copy numbers on basis of the measured Cq values compared to a standard curve
using reference materials, if the CaMV ORF V and/or the nos gene target sequences are amplified.
For further confirmation, in case of positive results in the nos PCR tests, it is recommended to perform
[1]
a further test for the detection of chromosomal Rhizobium radiobacter DNA.
5 Reagents and materials
Chemicals of recognized analytical grade, appropriate for molecular biology shall be used. The water
used shall be double-distilled or PCR-grade water (i.e. nuclease and nucleic acid free). For all operations
in which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected
pipette tips as protection against cross-contamination is recommended.
5.1 Thermostable DNA polymerase, (for hot-start PCR). PCR buffer solution, which contains
magnesium chloride and deoxyribonucleoside triphosphates (dNTPs). Ready-to-use reagent mixtures
or mixes of individual components can be used. Reagents and polymerases which lead to equal or better
results may also be used.
5.2 Positive control materials. DNA extracted from Rhizobium radiobacter strains with Ti plasmid
(DSM-5172 or ATCC 33970D-5) and from CaMV isolates (DSMZ PV-0226; DSMZ PV-0227; DSMZ PV-
0228; DSMZ PV-0229).
5.3 Oligonucleotides. See Tables 1 and 2.
Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield
similar or better results.
Table 1 — Oligonucleotides for detection of nos
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
nos gene sequence from Rhizobium radiobacter Ti plasmids as target
At-nop-f2 5′-CCA gCC RTS TAC TgA TTA TTg TMA C-3′ 300 nmol/l
At-nop-r2 5′-TgC gAg TTC RCC gTT gAA g-3′ 300 nmol/l
a
At-nop-s1 5′-(FAM)-CCg TgC ggA CgT TCA CgA CAg-(BHQ1)-3′ 150 nmol/l
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
2
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ISO/TS 21569-7:2022(E)
Table 2 — Oligonucleotides for detection of CaMV ORF V
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
ORF V sequence from CaMV as target
CaMV-ORFV-fd2 5′-ATY AAg CCC AgY AAA AgC CC-3′ 300 nmol/l
CaMV-ORFV-rd2 5′-CTY CgC TTC TCg gCT TCR TT-3′ 300 nmol/l
a
CaMV-ORFV-P2 5′-(FAM)-CAT ggC ACC AgC CTT CTT ggT CAA C-(BHQ1)-3′ 150 nmol/l
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
5.4 Standard DNA for calibration. DNA solution of a known concentration (ng/µl) to estimate the
copy number of the target sequence.
When using genomic CaMV or Rhizobium radiobacter DNA as the standard DNA, the number of genome
equivalents should be calculated on the basis of the molecular mass of the genome according to
Formula (1):
()δ ×1000
γ = (1)
E
γ
M
where
γ is the number of genome equivalents per microliter (µl);
E
δ is the DNA concentration in nanograms per microliter (ng/µl);
γ is the genome mass in femtograms (fg).
M
The respective copy number for the target sequence can be calculated based on the genome equivalents.
6 Apparatus
Requirements concerning apparatus and materials shall be in accordance with ISO 21569. In addition
to the usual laboratory equipment, the following equipment shall be used.
6.1 Real-time PCR device, which shall be suitable for the excitation of fluorescent molecules and the
detection of fluorescence signals generated during PCR.
7 Procedure
7.1 Preparation of test samples
The test portion used for DNA extraction should be representative of the laboratory sample, e.g. by
grinding or homogenizing of the samples. Measures and operational steps that should be considered
are described in ISO 21571 and ISO 24276.
7.2 Preparation of DNA extracts
For the extraction of DNA from the test portion, the general instructions and requirements specified in
ISO 21571 shall be followed.
7.3 PCR setup
7.3.1 The method description applies to a total volume of 25 µl per reaction mixture with the setup
given in Tables 3 and 4. The PCR reagent mixture is prepared containing all components except for the
3
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ISO/TS 21569-7:2022(E)
sample DNA. The required amount of PCR reagent mixture depends on the number of reactions to be
performed.
Table 3 — Reaction setup for the amplification of the nos target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl , dNTPs and hot-start DNA polymerase) 12,5 µl
2
Primers At-nop-f2 and At-nop-r2 see Table 1
Probe At-nop-s1 see Table 1
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
Table 4 — Reaction setup for amplification of the CaMV ORF V target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl , dNTPs and hot-start DNA polymerase) 12,5 µl
2
Primers CaMV-ORFV-fd2 and CaMV-ORFV-rd2 see Table 2
Probe CaMV-ORFV-P2 see Table 2
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
7.3.2 Mix the PCR reagent mixture, centrifuge briefly and pipette 20 µl into each reaction vial.
7.3.3 For the amplification reagent control, add 5 µl of water into the respective reaction setup.
7.3.4 Pipette either 5 µl of sample DNA or 5 µl of the respective control solution (extraction blank
control, positive DNA target control).
7.3.5 If necessary, prepare a PCR inhibition control as specified in ISO 24276.
7.3.6 Carefully seal the reaction setup, transfer them into the real-time PCR device and start the
temperature-time program (Table 5).
7.4 Temperature-time programme
The temperature-time programme as outlined in Table 5 was used in the validation study. The use of
different reaction conditions and real-time PCR devices may require specific optimization. The time for
initial denaturation depends on the master mix used.
4
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ISO/TS 21569-7:2022(E)
Table 5 — Temperature-time programme
Step Parameter Temperature Time Cycles
1 Initial denaturation 95 °C 2 min 1
Denaturation 95 °C 15 s
2 Amplification 45
Annealing and 60 °C 90 s
Elongation
8 Accept/reject criteria
8.1 General
The evaluation of PCR amplification results is performed with the respective device-specific data
analysis programme. If the amplification of the target sequence was successful in a sample (positive
result), the cycle number is calculated at which a specified fluorescence threshold was exceeded for the
first time (Cq value).
8.2 Identification of nos
The nos gene target sequence is considered as detected, if:
— a sigmoid-shaped amplification curve and typical increase in the measured fluorescence is detected
using the specific primers At-nop-f2 and At-nop-r2 and the probe At-nop-s1;
— no amplification and increase in fluorescence have occurred in the PCR control reactions with no
DNA added (PCR reagent control, extraction blank control).
In the case of a negative PCR result, the expected Cq values are achieved in the amplification controls
(positive DNA target control, PCR inhibition control).
The Cq value obtained for nos with the test sample DNA should be compared with the Cq values
obtained in positive PCR tests for the genetic elements P-nos or T-nos. If
...
© ISO 2022 – All rights reserved
ISO/PRF TS 21569-7:2022(E)
Date: 2022-10-18
ISO TC 34/SC 16/WG 14
Secretariat: ANSI
Horizontal methods for molecular biomarker analysis — Methods
of analysis for the detection of genetically modified organisms and
derived products — Part 7: Real-time PCR based methods for the
detection of CaMV and Agrobacterium Ti-plasmid derived DNA
sequences
---------------------- Page: 1 ----------------------
ISO/PRF TS 21569-7:2022(E)
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of
this publication may be reproduced or utilized otherwise in any form or by any means, electronic or
mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO at the address below or ISO’s member body in the
country of the requester.
ISO Copyright Office
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland.
ii © ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/PRF TS 21569-7:2022(E)
Contents
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 4
7 Procedure . 4
7.1 Preparation of test samples . 4
7.2 Preparation of DNA extracts . 4
7.3 PCR setup . 4
7.4 Temperature-time programme . 5
8 Accept/reject criteria . 5
8.1 General . 5
8.2 Identification of nos . 5
8.3 Identification of CaMV ORF V . 6
9 Validation status and performance criteria . 6
9.1 Specificity . 6
9.2 Sensitivity . 8
9.3 Robustness . 8
9.4 Interlaboratory trials . 8
9.4.1 General . 8
9.4.2 False-positive rate/false-negative rate . 10
9.4.3 PCR efficiency and detection limit . 10
9.4.4 Summary evaluation of the interlaboratory trials . 12
10 Test report . 12
Bibliography . 13
© ISO 2022 – All rights reserved iii
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ISO/PRF TS 21569-7:2022(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee 16ISO/TC 34, Food Productsproducts,
Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 21569 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv © ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
TECHNICAL SPECIFICATION ISO/PRF TS 21569-7:2022(E)
Horizontal methods for molecular biomarker analysis — Methods
of analysis for the detection of genetically modified organisms and
derived products — Part 7: Real-time PCR based methods for the
detection of CaMV and Agrobacterium Ti-plasmid derived DNA
sequences
1 Scope
This document specifies a procedure for the detection of a DNA sequence of the open reading frame five
(ORF V) from Cauliflower Mosaic Viruscauliflower mosaic virus (CaMV) and a procedure for the detection
of the DNA sequence of the nopaline synthase (nos) gene from tumour-inducing (Ti) plasmids of
phytopathogenic Rhizobium radiobacter (formerly named Agrobacterium tumefaciens). The procedures
can be used in the context of screening for genetically modified crop/plants and their derived products
to further clarify a positive PCR result for a specific promoter or terminator of CaMV (P-35S, T-35S), or
both, and the nos gene (P-nos, T-nos), respectively.
The methods specified in this document will detect and identify naturally occurring CaMV or Rhizobium
radiobacter (Ti plasmid) DNA, or both, if present in the sample in the absence of a genetically modified
plant event containing the specified target sequences.
Both methods are based on the real-time polymerase chain reaction (PCR) and are applicable for the
analysis of DNA extracted from foodstuffs and other products such as feedstuffs and seeds/grains. The
application of the methods requires the extraction of an adequate amount of amplifiable DNA from the
relevant matrix.
With appropriate calibration material, the CaMV ORF V or nos copy number, or both, can be estimated
and compared, respectively, with the estimated copy number for the promoter (P-35S, P-nos) or the
terminator (T-35S, T-nos) sequences, or both. Thereby, conclusions are possible about the presence of an
unknown genetically modified organism (GMO) in addition to any detected CaMV DNA or Rhizobium
radiobacter Ti plasmid DNA, or both, in a test sample.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 21569:2005, 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
© ISO 2022 – All rights reserved 1
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ISO/PRF TS 21569-7:2022(E)
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions.
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 Molecular biomarker
analysis — Vocabulary for molecular biomarker analytical methods in agriculture and food production
apply.
ISO and IEC maintain terminologicalterminology databases for use in standardization at the following
addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
4 Principle
DNA extracts of test portions are used that showed a positive PCR result in screening tests for specific
promoter/terminator sequences derived from CaMV and/or from Ti plasmid of Rhizobium radiobacter.
The tests consist of two parts, namely,:
a) detection of the CaMV ORF V and/or the nos DNA sequence in a real-time PCR;
b) estimation of the copy numbers on basis of the measured Cq values compared to a standard curve
using reference materials, if the CaMV ORF V and/or the nos gene target sequences are amplified.
For further confirmation, in case of positive results in the nos PCR tests, it is recommended to perform a
[1]
further test for the detection of chromosomal Rhizobium radiobacter DNA.
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 PCR-grade water (i.e. nuclease and nucleic acid free). For all
operations in which gloves are used, it should be ensured that these are powder-free. The use of aerosol-
protected pipette tips as protection against cross-contamination is recommended.
5.1 PCR reagents
5.1.1 5.1 Thermostable DNA polymerase, (for hot-start PCR). PCR buffer solution
Contains, which contains magnesium chloride and deoxyribonucleoside triphosphates (dNTPs). Ready-
to-use reagent mixtures or mixes of individual components can be used. Reagents and polymerases which
lead to equal or better results may also be used.
5.2 Positive control materials. DNA extracted from Rhizobium radiobacter strains with Ti plasmid
(DSM-5172 or ATCC 33970D-5) and from CaMV isolates (DSMZ PV-0226; DSMZ PV-0227; DSMZ PV-
0228; DSMZ PV-0229).
5.1.2 5.3 Oligonucleotides
(. See Tables 1 and 2).
2 © ISO 2022 – All rights reserved
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ISO/PRF TS 21569-7:2022(E)
Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield
similar or better results.
Table 1 — Oligonucleotides for detection of nos
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
nos gene sequence from Rhizobium radiobacter Ti plasmids as target
At-nop-f2 5’-5′-CCA gCC RTS TAC TgA TTA TTg TMA C-3’3′ 300 nmol/l
At-nop-r2 5’-5′-TgC gAg TTC RCC gTT gAA g-3’ 3′ 300 nmol/l
a a
At-nop-s1 5’-(5′-(FAM)-CCg TgC ggA CgT TCA CgA CAg-(BHQ1)-3’ 3′ 150 nmol/l
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
Table 2 — Oligonucleotides for detection of CaMV ORF V
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
ORF V sequence from CaMV as target
CaMV-ORFV-fd2 5’-5′-ATY AAg CCC AgY AAA AgC CC-3’ 3′ 300 nmol/l
CaMV-ORFV-rd2 5’-5′-CTY CgC TTC TCg gCT TCR TT-3’ 3′ 300 nmol/l
CaMV-ORFV-P2 5’-(5′-(FAM)-CAT ggC ACC AgC CTT CTT ggT CAA C-(BHQ1)- 150 nmol/l
a
3‘′
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield
similar or better results.
5.4 Standard DNA for calibration. DNA solution of a known concentration (ng/µl) to estimate the
copy number of the target sequence.
When using genomic CaMV or Rhizobium radiobacter DNA as the standard DNA, the number of genome
equivalents should be calculated on the basis of the molecular mass of the genome according to
Formula (1).:
δ×1000
( )
γ = ng
E
DNA concentration � �∗1000
γ µl
M
Number of genome equivalents per µl = (1)
genome mass [fg]
(1)
where
γ is the number of genome equivalents per microliter (µl);
E
δ is the DNA concentration in nanograms per microliter (ng/µl);
γ is the genome mass in femtograms (fg).
M
The respective copy number for the target sequence can be calculated based on the genome equivalents.
© ISO 2022 – All rights reserved 3
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ISO/PRF TS 21569-7:2022(E)
6 Apparatus
Requirements concerning apparatus and materials shall be according toin accordance with ISO 21569. In
addition to the usual laboratory equipment, the following equipment is requiredshall be used.
6.1 Real-time PCR device
Shall, which shall be suitable for the excitation of fluorescent molecules and the detection of fluorescence
signals generated during PCR.
7 Procedure
7.1 Preparation of test samples
The test portion used for DNA extraction should be representative of the laboratory sample, e.g.,. by
grinding or homogenizing of the samples. Measures and operational steps that should be considered are
described in ISO 21571 and ISO 24276.
7.2 Preparation of DNA extracts
For the extraction of DNA from the test portion, the general instructions and requirements specified in
ISO 21571 shall be followed.
7.3 PCR setup
7.3.1 The method description applies to a total volume of 25 µl per reaction mixture with the setup
given in Table Tables 3 and Table 4, respectively. The PCR reagent mixture is prepared containing all
components except for the sample DNA. The required amount of PCR reagent mixture depends on the
number of reactions to be performed.
Table 3 — Reaction setup for the amplification of the nos target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl , dNTPs and hot-start DNA polymerase) 12,5 µl
2
Primers At-nop-f2 and At-nop-r2 see Table 1
Probe At-nop-s1 see Table 1
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programprogramme.
Table 4 — Reaction setup for amplification of the CaMV ORF V target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl2, dNTPs and hot-start DNA polymerase) 12,5 µl
Primers CaMV-ORFV-fd2 and CaMV-ORFV-rd2 see Table 2
4 © ISO 2022 – All rights reserved
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ISO/PRF TS 21569-7:2022(E)
Probe CaMV-ORFV-P2 see Table 2
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programprogramme.
7.3.12 Mix the PCR reagent mixture, centrifuge briefly and pipette 20 µl into each reaction vial.
7.3.23 For the amplification reagent control, add 5 µl of water into the respective reaction setup.
7.3.34 Pipette either 5 µl of sample DNA or 5 µl of the respective control solution (extraction blank
control, positive DNA target control).
7.3.45 If necessary, prepare a PCR inhibition control as specified in ISO 24276.
7.3.56 Carefully seal the reaction setup, transfer them into the real-time PCR device and start the
temperature-time program (Table 5).
7.4 Temperature-time programprogramme
The temperature-time programprogramme as outlined in Table 5 has beenwas used in the validation
study. The use of different reaction conditions and real-time PCR devices may require specific
optimization. The time for initial denaturation depends on the master mix used.
Table 5 — Temperature-time programprogramme
Step Parameter Temperature Time Cycles
1 Initial denaturation 95 °C 2 min 1
Denaturation 95 °C 15 s
2 Amplification 45
Annealing and 60 °C 90 s
Elongation
8 Accept/Rejectreject criteria
8.1 General
The evaluation of PCR amplification results is performed with the respective device-specific data analysis
programprogramme. If the amplification of the target sequence was successful in a sample (positive
result), the cycle number is calculated at which a specified fluorescence threshold was exceeded for the
first time (Cq value).
8.2 Identification of nos
The nos gene target sequence is considered as detected, if:
•— a sigmoid-shaped amplification curve and typical increase in the measured fluorescence is detected
using the specific primers At-nop-f2 and At-nop-r2 and the probe At-nop-s1, ;
•— no amplification and increase in fluorescence have occurred in the PCR control reactions with no
DNA added (PCR reagent control, extraction blank control).
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ISO/PRF TS 21569-7:2022(E)
In the case of a negative PCR result, the expected Cq values are achieved in the amplification controls
(positive DNA target control, PCR inhibition control).
NOTE: The Cq value obtained for nos with the test sample DNA should be compared with the Cq values
obtained in positive PCR tests for the genetic elements P-nos or T-nos. If the values are approximately the
same, it is plausible to interpret that the Cq values for P-nos or T-nos or both can be explained solely by a
Ti plasmid from wild-type Rhizobium radiobacter detected via the nos gene. If the Cq values for P-nos or
T-nos or both are significantly smaller, it can be suspected that the sample contains DNA from an
unknown GMO. In any case, the absence of genetically modified (GM) linseed FP967 mustshall be clarified
(see section 9.1). To clarify positive results for nos, an examination for the presence of chromosomal
[1]
Rhizobium radiobacter DNA is useful.
8.3 Identification of CaMV ORF V
The target sequence is considered to be detected in the samples if, in the real-time PCR assay:
•—
...
TECHNICAL ISO/TS
SPECIFICATION 21569-7
First edition
Horizontal methods for molecular
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 7:
Real-time PCR based methods for the
detection of CaMV and Agrobacterium
Ti-plasmid derived DNA sequences
PROOF/ÉPREUVE
Reference number
ISO/TS 21569-7:2022(E)
© ISO/TS 2022
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ISO/TS 21569-7:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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ISO/TS 21569-7:2022(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure .3
7.1 Preparation of test samples . 3
7.2 Preparation of DNA extracts . 3
7.3 PCR setup. 3
7.4 Temperature-time programme. 4
8 Accept/reject criteria . 5
8.1 General . 5
8.2 Identification of nos . 5
8.3 Identification of CaMV ORF V . 5
9 Validation status and performance criteria . 6
9.1 Specificity . 6
9.2 Sensitivity . 7
9.3 Robustness . 7
9.4 Interlaboratory trials . 8
9.4.1 General . 8
9.4.2 False-positive rate/false-negative rate . 9
9.4.3 PCR efficiency and detection limit . 10
9.4.4 Summary evaluation of the interlaboratory trials . . 11
10 Test report .11
Bibliography .12
iii
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ISO/TS 21569-7:2022(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.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 21569 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
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TECHNICAL SPECIFICATION ISO/TS 21569-7:2022(E)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically
modified organisms and derived products —
Part 7:
Real-time PCR based methods for the detection of CaMV
and Agrobacterium Ti-plasmid derived DNA sequences
1 Scope
This document specifies a procedure for the detection of a DNA sequence of the open reading frame five
(ORF V) from cauliflower mosaic virus (CaMV) and a procedure for the detection of the DNA sequence
of the nopaline synthase (nos) gene from tumour-inducing (Ti) plasmids of phytopathogenic Rhizobium
radiobacter (formerly named Agrobacterium tumefaciens). The procedures can be used in the context of
screening for genetically modified crop/plants and their derived products to further clarify a positive
PCR result for a specific promoter or terminator of CaMV (P-35S, T-35S), or both, and the nos gene
(P-nos, T-nos), respectively.
The methods specified in this document will detect and identify naturally occurring CaMV or Rhizobium
radiobacter (Ti plasmid) DNA, or both, if present in the sample in the absence of a genetically modified
plant event containing the specified target sequences.
Both methods are based on the real-time polymerase chain reaction (PCR) and are applicable for the
analysis of DNA extracted from foodstuffs and other products such as feedstuffs and seeds/grains. The
application of the methods requires the extraction of an adequate amount of amplifiable DNA from the
relevant matrix.
With appropriate calibration material, the CaMV ORF V or nos copy number, or both, can be estimated
and compared, respectively, with the estimated copy number for the promoter (P-35S, P-nos) or the
terminator (T-35S, T-nos) sequences, or both. Thereby, conclusions are possible about the presence of
an unknown genetically modified organism (GMO) in addition to any detected CaMV DNA or Rhizobium
radiobacter Ti plasmid DNA, or both, in a test sample.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
ISO 21569:2005, 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
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
1
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ISO/TS 21569-7:2022(E)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Principle
DNA extracts of test portions are used that showed a positive PCR result in screening tests for specific
promoter/terminator sequences derived from CaMV and/or from Ti plasmid of Rhizobium radiobacter.
The tests consist of two parts, namely:
a) detection of the CaMV ORF V and/or the nos DNA sequence in a real-time PCR;
b) estimation of the copy numbers on basis of the measured Cq values compared to a standard curve
using reference materials, if the CaMV ORF V and/or the nos gene target sequences are amplified.
For further confirmation, in case of positive results in the nos PCR tests, it is recommended to perform
[1]
a further test for the detection of chromosomal Rhizobium radiobacter DNA.
5 Reagents and materials
Chemicals of recognized analytical grade, appropriate for molecular biology shall be used. The water
used shall be double-distilled or PCR-grade water (i.e. nuclease and nucleic acid free). For all operations
in which gloves are used, it should be ensured that these are powder-free. The use of aerosol-protected
pipette tips as protection against cross-contamination is recommended.
5.1 Thermostable DNA polymerase, (for hot-start PCR). PCR buffer solution, which contains
magnesium chloride and deoxyribonucleoside triphosphates (dNTPs). Ready-to-use reagent mixtures
or mixes of individual components can be used. Reagents and polymerases which lead to equal or better
results may also be used.
5.2 Positive control materials. DNA extracted from Rhizobium radiobacter strains with Ti plasmid
(DSM-5172 or ATCC 33970D-5) and from CaMV isolates (DSMZ PV-0226; DSMZ PV-0227; DSMZ PV-
0228; DSMZ PV-0229).
5.3 Oligonucleotides. See Tables 1 and 2.
Equivalent reporter dyes and/or quencher dyes may be used for the probe if they can be shown to yield
similar or better results.
Table 1 — Oligonucleotides for detection of nos
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
nos gene sequence from Rhizobium radiobacter Ti plasmids as target
At-nop-f2 5′-CCA gCC RTS TAC TgA TTA TTg TMA C-3′ 300 nmol/l
At-nop-r2 5′-TgC gAg TTC RCC gTT gAA g-3′ 300 nmol/l
a
At-nop-s1 5′-(FAM)-CCg TgC ggA CgT TCA CgA CAg-(BHQ1)-3′ 150 nmol/l
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
2
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ISO/TS 21569-7:2022(E)
Table 2 — Oligonucleotides for detection of CaMV ORF V
Name DNA sequence of the oligonucleotide Final concentration
in the PCR
ORF V sequence from CaMV as target
CaMV-ORFV-fd2 5′-ATY AAg CCC AgY AAA AgC CC-3′ 300 nmol/l
CaMV-ORFV-rd2 5′-CTY CgC TTC TCg gCT TCR TT-3′ 300 nmol/l
a
CaMV-ORFV-P2 5′-(FAM)-CAT ggC ACC AgC CTT CTT ggT CAA C-(BHQ1)-3′ 150 nmol/l
a
FAM: 6-Carboxyfluorescein, BHQ1: black hole quencher 1.
5.4 Standard DNA for calibration. DNA solution of a known concentration (ng/µl) to estimate the
copy number of the target sequence.
When using genomic CaMV or Rhizobium radiobacter DNA as the standard DNA, the number of genome
equivalents should be calculated on the basis of the molecular mass of the genome according to
Formula (1):
()δ ×1000
γ = (1)
E
γ
M
where
γ is the number of genome equivalents per microliter (µl);
E
δ is the DNA concentration in nanograms per microliter (ng/µl);
γ is the genome mass in femtograms (fg).
M
The respective copy number for the target sequence can be calculated based on the genome equivalents.
6 Apparatus
Requirements concerning apparatus and materials shall be in accordance with ISO 21569. In addition
to the usual laboratory equipment, the following equipment shall be used.
6.1 Real-time PCR device, which shall be suitable for the excitation of fluorescent molecules and the
detection of fluorescence signals generated during PCR.
7 Procedure
7.1 Preparation of test samples
The test portion used for DNA extraction should be representative of the laboratory sample, e.g. by
grinding or homogenizing of the samples. Measures and operational steps that should be considered
are described in ISO 21571 and ISO 24276.
7.2 Preparation of DNA extracts
For the extraction of DNA from the test portion, the general instructions and requirements specified in
ISO 21571 shall be followed.
7.3 PCR setup
7.3.1 The method description applies to a total volume of 25 µl per reaction mixture with the setup
given in Tables 3 and 4. The PCR reagent mixture is prepared containing all components except for the
3
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ISO/TS 21569-7:2022(E)
sample DNA. The required amount of PCR reagent mixture depends on the number of reactions to be
performed.
Table 3 — Reaction setup for the amplification of the nos target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl , dNTPs and hot-start DNA polymerase) 12,5 µl
2
Primers At-nop-f2 and At-nop-r2 see Table 1
Probe At-nop-s1 see Table 1
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
Table 4 — Reaction setup for amplification of the CaMV ORF V target DNA sequence
Reagent Volume
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (contains MgCl , dNTPs and hot-start DNA polymerase) 12,5 µl
2
Primers CaMV-ORFV-fd2 and CaMV-ORFV-rd2 see Table 2
Probe CaMV-ORFV-P2 see Table 2
Water add to obtain 25 µl
a
In the interlaboratory trial, the PerfeCTa qPCR ToughMix (Quanta BioSciences) was used as PCR buffer solution. This
information is given for convenience of users of this document and does not constitute an endorsement by ISO of the
product names. Equivalent products from other manufacturers may be used if they can be shown to give equivalent or
better results. If necessary, adapt the amounts of the reagents and the temperature-time programme.
7.3.2 Mix the PCR reagent mixture, centrifuge briefly and pipette 20 µl into each reaction vial.
7.3.3 For the amplification reagent control, add 5 µl of water into the respective reaction setup.
7.3.4 Pipette either 5 µl of sample DNA or 5 µl of the respective control solution (extraction blank
control, positive DNA target control).
7.3.5 If necessary, prepare a PCR inhibition control as specified in ISO 24276.
7.3.6 Carefully seal the reaction setup, transfer them into the real-time PCR device and start the
temperature-time program (Table 5).
7.4 Temperature-time programme
The temperature-time programme as outlined in Table 5 was used in the validation study. The use of
different reaction conditions and real-time PCR devices may require specific optimization. The time for
initial denaturation depends on the master mix used.
4
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ISO/TS 21569-7:2022(E)
Table 5 — Temperature-time programme
Step Parameter Temperature Time Cycles
1 Initial denaturation 95 °C 2 min 1
Denaturation 95 °C 15 s
2 Amplification 45
Annealing and 60 °C 90 s
Elongation
8 Accept/reject criteria
8.1 General
The evaluation of PCR amplification results is performed with the respective device-specific data
analysis programme. If the amplification of the target sequence was successful in a sample (positive
result), the cycle number is calculated at which a specified fluorescence threshold was exceeded for the
first time (Cq value).
8.2 Identification of nos
The nos gene target sequence is considered as detected, if:
— a sigmoid-shaped amplification curve and typical increase in the measured fluorescence is detected
using the specific primers At-nop-f2 and At-nop-r2 and the probe At-nop-s1;
— no amplification and increase in fluorescence have occurred in the PCR control reactions with no
DNA added (PCR reagent control, extraction blank control).
In the case of a negative PCR result, the expected Cq values are achieved in the amplification controls
(positive DNA target control, PCR inhibition control).
The Cq value obtained for nos with the test sample DNA should be compared with the Cq values
obtained in positive PCR tests for the genetic elements P-nos or T-nos. If the value
...
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