ISO/TS 21569-6:2026

Technical
Specification
ISO/TS 21569-6
Second edition
Horizontal methods for molecular
2026-01
biomarker analysis — Methods
of analysis for the detection of
genetically modified organisms and
derived products —
Part 6:
Real-time PCR based screening
methods for the detection of
cry1Ab/Ac and Pubi-cry DNA
sequences
Méthodes horizontales d’analyse de biomarqueurs
moléculaires — Méthodes d’analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Partie 6: Méthodes de criblage par PCR en temps réel pour la
détection des séquences ADN cry1Ab/Ac et Pubi-cry
Reference number
© ISO 2026
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ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Reagents and materials . 2
5.1 General .2
5.2 PCR reagents .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 . 4
8.1 General .4
8.2 Identification .5
9 Validation status and performance criteria . 5
9.1 General .5
9.2 Robustness .5
9.3 Collaborative trial .5
9.4 Sensitivity .7
9.5 Specificity .8
10 Test report . 10
Bibliography .11

iii
Foreword
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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.
This second edition cancels and replaces the first edition (ISO 21569-6:2016), which has been technically
revised.
The main changes are as follows:
— in 8.2 and Table 5 , “C ” and “C ” have been replaced by "C ";
t p q
— the Bibliography has been revised to correct a bibliographic reference.
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
Technical Specification ISO/TS 21569-6:2026(en)
Horizontal methods for molecular biomarker analysis —
Methods of analysis for the detection of genetically modified
organisms and derived products —
Part 6:
Real-time PCR based screening methods for the detection of
cry1Ab/Ac and Pubi-cry DNA sequences
1 Scope
This document specifies a procedure for the detection of a DNA sequence of the modified cry1Ab/Ac gene
and a procedure for the detection of the DNA transition sequence between the maize ubiquitin promoter
(Pubi) and the cry1Ab/Ac gene. The modified cry1Ab/Ac gene and the Pubi-cry construct are frequently found
in genetically modified Bt (Bacillus thuringiensis) plants. Both detection methods are based on real-time
PCR and can be used for qualitative screening purposes. For identification and quantification of a specific
genetically modified plant (event) a follow-up analysis has to be carried out.
This document is applicable to the analysis of DNA extracted from foodstuffs. It can also be suitable for
the analysis of DNA extracted from other products such as feedstuffs and seeds. The application of these
methods requires the extraction of an adequate amount of amplifiable DNA from the relevant matrix.
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 24276:2006, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions
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
[1]
DNA is extracted from the test portion applying a suitable method (see ISO 21571:2005 ). The DNA analysis
consists of two parts:
a) verification of the amount and amplifiability of the extracted DNA, e.g. by means of a target taxon-
[2] [3]
specific real-time PCR (according to ISO 21570:2005 , see also Reference );
[4]
b) detection of the cry1Ab/Ac and/or the Pubi-cry DNA sequence in a real-time PCR (see References and
[5]
).
5 Reagents and materials
5.1 General
For the purpose of this document, only chemicals and water of recognized analytical grade, appropriate
for molecular biology shall be used. Unless stated otherwise, solutions should be prepared by dissolving
the corresponding reagents in water and be autoclaved. For all operations requiring gloves, it should be
ensured that these are powder-free. The use of aerosol-protected pipette tips as protection against cross
contamination is recommended.
5.2 PCR reagents
5.2.1 Thermostable DNA polymerase (for hot-start PCR).
5.2.2 PCR buffer solution, containing 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.
5.2.3 Oligonucleotides (see Table 1 and Table 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 cry1Ab/Ac
Final concentration
Name DNA sequence of the oligonucleotide
in the PCR
[4]
cry1Ab/Ac as target sequence :
Bt-F1(mod) 5ʹ – gAg gAA ATg CgT ATT CAA TTC AAC – 3ʹ 400 nmol/l
Bt-R 5ʹ – TTC Tgg ACT gCg AAC AAT gg – 3ʹ 400 nmol/l
Bt-P 5ʹ – (FAM) – ACA TgA ACA gCg CCT TgA CCA CAg C – (NFQ) – 3ʹ 100 nmol/l
Key
FAM: 6-Carboxyfluorescein
NFQ: non-fluorescent quencher (dark quencher)

Table 2 — Oligonucleotides for detection of Pubi-cry
Final concentration
Name DNA sequence of the oligonucleotide
in the PCR
[4]
Pubi-cry as target sequence :
Pubi-F2 5ʹ-ATT TgC TTg gTA CTg TTT CTT TTg TC-3ʹ 300 nmol/l
Cry-rr-R 5ʹ-TTg TTg TCC ATg gAT CCT CTA gAg T-3ʹ 600 nmol/l
a
Pubi-T2 5ʹ- (FAM)- ACC CTg TTg TTT ggT gTT ACT TCT gCA-(NFQ)-3ʹ 100 nmol/l
Key
FAM: 6-Carboxyfluorescein
NFQ: non-fluorescent quencher (dark quencher)
a [5]
The Pubi-T2 probe is three bases shorter than the probe described in Reference but identical specificity and sensitivity is
achieved.
6 Apparatus
Requirements concerning apparatus and materials shall be in accordance with ISO 21569:2005. In addition
to the usual laboratory equipment, the following equipment shall be used.
6.1 Real-time PCR device, suitable for the excitation of fluorescent molecules and the detection of
fluorescence signals generated during PCR.
7 Procedure
7.1 Preparation of test samples
It should be ensured that the test portion used for DNA extraction is representative of the laboratory
sample (e.g. by grinding or homogenizing of the samples). Measures and operational steps to be taken into
[1]
consideration should be as described in ISO 21571:2005 and ISO 24276:2006.
7.2 Preparation of DNA extracts
Concerning the preparation of DNA from the test portion, the general instructions and measures described
in ISO 21571 shall be followed. It is recommended to choose one of the DNA extraction methods described in
[1]
ISO 21571:2005 , Annex A.
7.3 PCR setup
The method is described for a total volume of 25 μl per PCR. The reaction setup is given in Table 3.
Completely thaw reagents at room temperature. Each reagent should be carefully mixed and briefly
centrifuged immediately before pipetting. A PCR reagent mixture is prepared which contains all components
except for the sample DNA. The required amount of the PCR reagent mixture depends on the number of
reactions to be performed, including at least one additional reaction as a pipetting reserve. Add 5 µl of
sample DNA to each reaction.
Table 3 — Reaction setup for the amplification
Description Set up
Total reaction volume 25 µl
Sample DNA (up to 200 ng) or controls 5 µl
a
PCR buffer solution (including MgCl , dNTP’s and hot-start DNA polymerase) 12,5 µl
Primers Bt-F1(mod) and Bt-R or primers Pubi-F2 and Cry-rr-R see Table 1 or Table 2
Probe Bt-P or probe Pubi-T2 see Table 1 or Table 2
Water add to obtain 25 µl
a TM
In the interlaboratory trial, TaqMan Universal PCR Mastermix (Life Technologies) was used as PCR buffer solution. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the product
named. 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.
Mix the PCR reagent mixture, centrifuge briefly and pipette 20 µl into each reaction vial. For the amplification
reagent control, add 5 µl of water into the respective reaction setup. Pipette either 5 µl of sample DNA or 5
µl of the respective control solution (extraction blank control, positive DNA target control). If necessary,
prepare a PCR inhibition control as described in ISO 24276.
Transfer the reaction setups into the thermal cycler and start the temperature-time programme.
7.4 Temperature-time programme
The temperature-time programme as outlined in Table 4 was used in the validation study. The use of
different reaction conditions and real-time PCR cyclers can require sp
...