ISO/DTS 5354-2

Molecular biomarkers — Detection of DNA in textiles derived from cotton — Part 2: Overview of target sequences for use in polymerase chain reaction (PCR)-based detection methods for cotton genetically modified (GM) events

This document provides a list of target sequences that can be used to screen for the presence of genetically modified material in Cotton and Cotton products. Its use is limited to cottonseed, leaf, cotton fibre and cotton fibre-derived materials from which sufficiently high-quality DNA can be extracted. NOTE 1: This list of target sequences provides guidance for the screening of all currently known GM cotton events and GM cotton events that contain the same DNA sequences. Further guidance on screening of foodstuffs is provided in CEN /TS 16 707 NOTE 2: Sampling is outside of the scope of this document. Information on sampling cotton products may be found in ISO 1130: 1975 and in ASTM Dl441-12.

Biomarqueurs moléculaires — Détection d’ADN dans les textiles dérivés du coton — Partie 2: Présentation des séquences cibles à utiliser dans les méthodes de détection reposant sur une réaction de polymérisation en chaîne (PCR) des événements de coton génétiquement modifié (GM)

General Information

• Status: Not Published
• ICS: 59.080.01 - Textiles in general
• 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

Relations

• Revises: IWA 32:2019 - Screening of genetically modified organisms (GMOs) in cotton and textiles
• Effective Date: 06-Jun-2022

Standards Content (Sample)

ISO/DTS 5354-2:2023(E)
ISO/TC 34/SC 16/JWG 12
Secretariat: ANSI
Date: 2023-02-27
Molecular biomarkerbiomarkers — Detection of DNA in textiles
derived from cotton part —
Part 2:
Overview of target sequences for use in polymerase chain reaction
(PCR-)-based detection methods for cotton genetically modified
(GM) events
DTS stage
© ISO 2023 – All rights reserved
---------------------- Page: 1 ----------------------
ISO/DTS 5354-2:2023(E)
© ISO 2023

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
E-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/DTS 5354-2:2023(E)
Contents

Foreword ............................................................................................................................................................................................ iv

Introduction ........................................................................................................................................................................................ v

1 Scope ....................................................................................................................................................................................... 1

2 Normative references ....................................................................................................................................................... 1

3 Terms and definitions ...................................................................................................................................................... 2

4 GM element screening ...................................................................................................................................................... 2

4.1 Principle ................................................................................................................................................................................. 2

4.2 Procedure .............................................................................................................................................................................. 2

4.3 Primers and probes ........................................................................................................................................................... 2

4.3.1 T-nos ................................................................................................................................................................................... 2

4.3.2 P-35S................................................................................................................................................................................... 3

4.3.3 cry1Ab/Ac ........................................................................................................................................................................ 3

4.3.4 pat ........................................................................................................................................................................................ 3

4.3.5 P-FMV ................................................................................................................................................................................. 3

4.3.6 otp/mepsps ..................................................................................................................................................................... 3

4.3.7 cry1Ac ................................................................................................................................................................................ 3

4.3.8 nptII .................................................................................................................................................................................... 3

4.3.9 cry-1Ab .............................................................................................................................................................................. 4

4.3.10 T-35S .................................................................................................................................................................................. 4

4.3.11 P-ubi1 ................................................................................................................................................................................. 4

4.3.12 cp4-epsps .......................................................................................................................................................................... 4

4.3.13 cry- 2Ab2 .......................................................................................................................................................................... 4

4.3.14 cry1C ................................................................................................................................................................................... 4

4.3.15 T-E9 ..................................................................................................................................................................................... 4

4.3.16 cry1F ................................................................................................................................................................................... 4

4.4 Reference materials .......................................................................................................................................................... 5

4.5 GM cotton events ................................................................................................................................................................ 5

4.6 Validation .............................................................................................................................................................................. 9

4.7 Interpretation and expression of results ................................................................................................................. 9

4.8 Results .................................................................................................................................................................................... 9

4.9 Reporting ............................................................................................................................................................................... 9

5 Test report ............................................................................................................................................................................ 9

Bibliography .................................................................................................................................................................................... 11

© ISO 2023 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/DTS 5354-2:2023(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 23ISO/TC 34, Food Productsproducts,

Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.

This first edition, along with ISO 5354-1:— , cancels and replaces IWA 32:2019, which has been

technically revised throughout.
A list of all parts in the ISO 5354 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.
Under preparation.
iv © ISO 2023 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DTS 5354-2:2023(E)
Introduction

Detection and identification of genetically modified (GM) cotton materials can be achieved by screening

methods followed by more specific analysis of the materials. The target sequences used for screening

should provide reproducible data using a variety of equipment, chemistries, and reagents.

In this screening analysis, DNA sequences of target sequences common to many GM events are assayed

in order to economically and reliably determine whether GM material is present.

The aim of this standarddocument is to provide an overview of target sequences that can be found in GM

cotton. Detection and identification of these target sequences can be used to determine the presence of

GM events in cotton and some cotton products. ISO 5354-1 describes methods that can be used to extract

DNA from cotton matrices that can subsequently be analysed with target sequences described within this

standarddocument.
This standarddocument provides an overview of known GM cotton events.

Also included are six primer and probe sequences recommended to determine the presence of most of

these GM cottons events. Only those elements for which a detection method is available are listed.

Based on elements that are detected and not detected during screening, the presence or absence of

certain Cottoncotton GM events can be evaluated and confirmed by event-specific methods. A database

[7 ]
is available to aid in this process at the European GMO database. [1].
[8 ]

Event-specific detection methods maycan be found via the CropLife International database [2] and the

[9 ]
EURL GMFF GMOMETHODS database JRC GMO Method Database [3]. .
© ISO 2023 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO/DTS 5354-2:2023(E)
Molecular Biomarkers—biomarkers — Detection of DNA in textiles
derived from cotton part —
Part 2:
Overview of target sequences for use in polymerase chain reaction
(PCR-)-based detection methods for cotton genetically modified
(GM) events
1 Scope

This document providesestablishes a list of target sequences that can be used to screen for the presence

of genetically modified (GM) material in cotton and cotton products. Its use

This document is limitedapplicable to cottonseed, cotton leaf, cotton fibre and cotton fibre-derived

materials from which sufficiently high-quality DNA can be extracted.

Methods describing the extraction of DNA from different cotton samples can be found in ISO 5354-1.

NOTE 1: This The list of target sequences provides guidance for the screening of all currently known GM cotton

events and GM cotton events that contain the same DNA sequences. Further guidance on screening of foodstuffs is

provided in CEN/TS 16707 [4].

NOTE 2 : Sampling is outside of the scope of this document. Information on sampling cotton products maycan be

found in ISO 1130:1975 [5] and in ASTM D1441-12 [6].
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/TS 16393:2019, Molecular biomarker analysis — Determination of the performance characteristics of

qualitative measurement methods and validation of methods

ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived

products — General requirements and definitions

ISO 21569 (all parts), Horizontal methods for molecular biomarker analysis — Methods of analysis for the

detection of genetically modified organisms and derived products

ISO 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Nucleic acid extraction

ISO 21570, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Quantitative nucleic acid based methods

ISO 21569-1, Horizontal methods for molecular biomarker analysis — Methods of analysis for the

detection of genetically modified organisms and derived products — Part 1: Qualitative PCR-based DNA

analysis

ISO/TS 21569-3:2020, Horizontal methods for molecular biomarker analysis — Methods of analysis for

the detection of genetically modified organisms and derived products — Part 3: Construct-specific real-

time PCR method for detection of P35S-pat-sequence for screening for genetically modified organisms

© ISO 2023 – All rights reserved 1
---------------------- Page: 6 ----------------------
ISO/DTS 5354-2:2023(E)

ISO/TS 21569-4:2016, Horizontal methods for molecular biomarker analysis — Methods of analysis for

the detection of genetically modified organisms and derived products — Part 4: Real-time PCR based

screening methods for the detection of the P-nos and P-nos-nptII DNA sequences

ISO/TS 21569-5:2016, Horizontal methods for molecular biomarker analysis — Methods of analysis for

the detection of genetically modified organisms and derived products — Part 5: Real-time PCR based

screening method for the detection of the FMV promoter (P-FMV) DNA sequence

ISO/TS 21569-6:2016, 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
83 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following 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/
3.1
cottonseed
seed from cotton plants
3.2
cotton lint
raw fibre that has gone through the ginning process of the definition
94 GM element screening
9.14.1 Principle

A screening strategy is used whereby a minimum number of tests are conducted to identify two or more

of all possible GM events that are currently known to occur in cotton. Transgene target sequences for

analysis can be chosen from Table 1 by the user based on the expected events or as a strategy to provide

the most information with the least use of resources. Common genetic sequences, or elements, present

across multiple GM constructs are initially targeted. The combined presence or absence of individual

elements can be used to infer the presence of one or more GM events.
9.24.2 Procedure

A procedure using a limited number of target sequences is chosen and may be followed up by adding

further target sequences to the analysis. If detected, no further testing is required. If not-detected, further

testing is necessary for up to all six elements. A minimum of two detection methods (targeting two of T-

nos, P-35S, cry1Ab/Ac, pat otp/mepsps or P-FMV) are applied based on expectations of which GM event

can be present, as a first screen. Screening with the six target sequences T-nos, P-35S, cry1Ab/Ac, pat,

otp/mepsps, and P-FMV is likely to detect most events if they are present (see Table 1). Internationally

recognized methods should be applied, if possible.
9.34.3 Primers and Probesprobes
9.3.14.3.1 T-nos

PCR screening for the biomarker T-nos using the following primers and probes is described in

ISO/TS 21569-4. (See also ISO 21569:2005/Amd 1:2013).
180-F CATGTAATGCATGACGTTATTTATG
2 © ISO 2023 – All rights reserved
---------------------- Page: 7 ----------------------
ISO/DTS 5354-2:2023(E)
180-R TTGTTTTCTATCGCGTATTAAATGT
Tm-180 FAM-ATGGGTTTTTATGATTAGAGTCCCGCAA-TAMRA
9.3.24.3.2 P-35S
...

FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 5354-2
ISO/TC 34/SC 16
Molecular biomarkers — Detection of
Secretariat: ANSI
DNA in textiles derived from cotton —
Voting begins on:
2023-03-13
Part 2:
Voting terminates on:
Overview of target sequences for use
2023-05-08
in polymerase chain reaction (PCR)-
based detection methods for cotton
genetically modified (GM) events
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTS 5354-2:2023(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2023
---------------------- Page: 1 ----------------------
ISO/DTS 5354-2:2023(E)
FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 5354-2
ISO/TC 34/SC 16
Molecular biomarkers — Detection of
Secretariat: ANSI
DNA in textiles derived from cotton —
Voting begins on:
Part 2:
Voting terminates on:
Overview of target sequences for use
in polymerase chain reaction (PCR)-
based detection methods for cotton
genetically modified (GM) events
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023

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.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
CH-1214 Vernier, Geneva
DOCUMENTATION.
Phone: [phone]
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: [e-mail]
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTS 5354-2:2023(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
© ISO 2023 – All rights reserved
NATIONAL REGULATIONS. © ISO 2023
---------------------- Page: 2 ----------------------
ISO/DTS 5354-2:2023(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 GM element screening ....................................................................................................................................................................................2

4.1 Principle ....................................................................................................................................................................................................... 2

4.2 Procedure .................................................................................................................................................................................................... 2

4.3 Primers and probes ............................................................................................................................................................................ 2

4.3.1 T-nos .............................................................................................................................................................................................. 2

4.3.2 P-35S .............................................................................................................................................................................................. 2

4.3.3 cry1Ab/Ac ................................................................................................................................................................................. 2

4.3.4 pat .................................................................................................................................................................................................... 3

4.3.5 P-FMV ........................................................................................................................................................................................... 3

4.3.6 otp/mepsps ............................................................................................................................................................................. 3

4.3.7 cry1Ac .......................................................................................................................................................................................... 3

4.3.8 nptII ................................................................................................................................................................................................ 3

4.3.9 cry-1Ab ........................................................................................................................................................................................ 3

4.3.10 T-35S .............................................................................................................................................................................................. 3

4.3.11 P-ubi1 ............................................................................................................................................................................................ 4

4.3.12 cp4-epsps .................................................................................................................................................................................. 4

4.3.13 cry- 2Ab2 ................................................................................................................................................................................... 4

4.3.14 cry1C ............................................................................................................................................................................................. 4

4.3.15 T-E9................................................................................................................................................................................................. 4

4.3.16 cry1F ............................................................................................................................................................................................. 4

4.4 Reference materials ........................................................................................................................................................................... 5

4.5 GM cotton events .................................................................................................................................................................................. 5

4.6 Validation .................................................................................................................................................................................................... 8

4.7 Interpretation and expression of results ........................................................................................................................ 8

4.8 Results ........................................................................................................................................... ................................................................. 8

4.9 Reporting ..................................................................................................................................................................................................... 8

5 Test report .................................................................................................................................................................................................................. 8

Bibliography .............................................................................................................................................................................................................................10

iii
© ISO 2023 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/DTS 5354-2:2023(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.

This first edition, along with ISO 5354-1:— , cancels and replaces IWA 32:2019, which has been

technically revised throughout.
A list of all parts in the ISO 5354 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.
1) Under preparation.
© ISO 2023 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/DTS 5354-2:2023(E)
Introduction

Detection and identification of genetically modified (GM) cotton materials can be achieved by screening

methods followed by more specific analysis of the materials. The target sequences used for screening

should provide reproducible data using a variety of equipment, chemistries and reagents.

In this screening analysis, DNA sequences of target sequences common to many GM events are assayed

in order to economically and reliably determine whether GM material is present.

The aim of this document is to provide an overview of target sequences that can be found in GM cotton.

Detection and identification of these target sequences can be used to determine the presence of GM

events in cotton and some cotton products. ISO 5354-1 describes methods that can be used to extract

DNA from cotton matrices that can subsequently be analysed with target sequences described within

this document.
This document provides an overview of known GM cotton events.

Also included are six primer and probe sequences recommended to determine the presence of most of

these GM cottons events. Only those elements for which a detection method is available are listed.

Based on elements that are detected and not detected during screening, the presence or absence of

certain cotton GM events can be evaluated and confirmed by event-specific methods. A database is

[7]
available to aid in this process at the European GMO database.
[8]

Event-specific detection methods can be found via the CropLife International database and the EURL

[9]
GMFF GMOMETHODS database .
© ISO 2023 – All rights reserved
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/DTS 5354-2:2023(E)
Molecular biomarkers — Detection of DNA in textiles
derived from cotton —
Part 2:
Overview of target sequences for use in polymerase
chain reaction (PCR)-based detection methods for cotton
genetically modified (GM) events
1 Scope

This document establishes a list of target sequences that can be used to screen for the presence of

genetically modified (GM) material in cotton and cotton products.

This document is applicable to cottonseed, cotton leaf, cotton fibre and cotton fibre-derived materials

from which sufficiently high-quality DNA can be extracted.

Methods describing the extraction of DNA from different cotton samples can be found in ISO 5354-1.

NOTE 1 The list of target sequences provides guidance for the screening of all currently known GM cotton

events and GM cotton events that contain the same DNA sequences. Further guidance on screening of foodstuffs

is provided in CEN/TS 16707.

NOTE 2 Sampling is outside of the scope of this document. Information on sampling cotton products can be

found in ISO 1130:1975 and in ASTM D1441-12.
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 (all parts), Horizontal methods for molecular biomarker analysis — Methods of analysis for the

detection of genetically modified organisms and derived products

ISO 21570, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Quantitative nucleic acid based methods
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following 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/
3.1
cottonseed
seed from cotton plants
© ISO 2023 – All rights reserved
---------------------- Page: 6 ----------------------
ISO/DTS 5354-2:2023(E)
3.2
cotton lint
raw fibre that has gone through the ginning process
4 GM element screening
4.1 Principle

A screening strategy is used whereby a minimum number of tests are conducted to identify two or

more of all possible GM events that are currently known to occur in cotton. Transgene target sequences

for analysis can be chosen from Table 1 by the user based on the expected events or as a strategy to

provide the most information with the least use of resources. Commo
...