ISO/TS 21569-7:2022

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)

General Information

• Status: Published
• Publication Date: 04-Dec-2022
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Current Stage: 6060 - International Standard published
• Start Date: 05-Dec-2022
• Due Date: 05-Apr-2025
• Completion Date: 05-Dec-2022

Standards Content (Sample)

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 2022
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ii
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
Foreword
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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
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iv
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
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.
Table 2 — Ol
...