IWA 32:2019

Screening of genetically modified organisms (GMOs) in cotton and textiles

This document provides requirements and recommendations to laboratories that perform genetically modified organism (GMO) analyses in cottonseed, leaf, cotton fibre and cotton fibre-derived materials. The following are within the scope of this document: a) identifying the materials to be assessed, based on the probability of obtaining good quality, fit for purpose DNA from the materials in subsequent steps in the cotton cloth production process; b) specifying a method for efficient DNA isolation from cotton and cotton-derived materials described under point a); c) specifying the cotton-specific method(s) to be used as control for amplifiable DNA; d) specifying the screening procedure that provides optimal chances to detect GMOs as a result of the performance of the lowest number of genetically modified (GM) element screening assays. NOTE 1 The protocol allows for the screening of all currently known GM cotton events and is set up in a way that optimizes the probability of also detecting unknown GM cotton events that possibly contain similar DNA sequences. Further information is given in CEN/TS 16707. Sampling is outside of the scope of this document. NOTE 2 A recommended sampling method is given in ISO 6497. General guidance for the sampling of bulk materials or for cotton-based products is available in standards such as ASTM D1441‑12 and CEN/TS 15568.

Criblage pour la détection des organismes génétiquement modifiés (OGM) dans le coton et les textiles

General Information

• Status: Published
• Publication Date: 11-Apr-2019
• ICS: 59.020 - Processes of the textile industry
• 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
• Parallel Committee: ISO/TC 34 - Food products
• Current Stage: 9092 - International Standard to be revised
• Completion Date: 15-Jun-2020

Relations

• Revised: 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
• Effective Date: 06-Jun-2022

Standards Content (Sample)

INTERNATIONAL IWA
WORKSHOP 32
AGREEMENT
First edition
2019-04
Screening of genetically modified
organisms (GMOs) in cotton and
textiles
Criblage pour la détection des organismes génétiquement modifiés
(OGM) dans le coton et les textiles
Reference number
IWA 32:2019(E)
ISO 2019
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IWA 32:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
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IWA 32:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

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

4 Principle ........................................................................................................................................................................................................................ 2

5 Sample preparation ........................................................................................................................................................................................... 2

6 DNA isolation ............................................................................................................................................................................................................ 3

6.1 General ........................................................................................................................................................................................................... 3

6.2 Principle ........................................................................................................................................................................................................ 3

6.3 Chemicals, reagents and equipment..................................................................................................................................... 4

6.3.1 Reagents .................................................................................................................................................................................. 4

6.3.2 Apparatus and equipment ....................................................................................................................................... 4

6.4 Procedure .................................................................................................................................................................................................... 5

6.4.1 General...................................................................................................................................................................................... 5

6.4.2 Protocol .................................................................................................................................................................................... 5

6.5 Results ............................................................................................................................................................................................................ 6

6.5.1 Analysis .................................................................................................................................................................................... 6

7 DNA quality control ........................................................................................................................................................................................... 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Principle ........................................................................................................................................................................................................ 6

7.3 Chemicals, reagents and equipment, including reference materials........................................................ 6

7.3.1 Reagents .................................................................................................................................................................................. 6

7.3.2 Apparatus and equipment ....................................................................................................................................... 7

7.4 Procedure .................................................................................................................................................................................................... 8

7.4.1 General...................................................................................................................................................................................... 8

7.4.2 Safety precautions .......................................................................................................................................................... 8

7.4.3 Pre-treatment ..................................................................................................................................................................... 8

7.4.4 Amount of sample ........................................................................................................................................................... 8

7.4.5 Procedure ............................................................................................................................................................................... 8

7.5 Results ............................................................................................................................................................................................................ 8

7.5.1 Calculations .......................................................................................................................................................................... 8

7.5.2 Interpretation and expression of results .................................................................................................... 8

7.5.3 Results ....................................................................................................................................................................................... 8

8 GM element screening..................................................................................................................................................................................... 8

8.1 Principle ........................................................................................................................................................................................................ 8

8.2 Chemicals, reagents and equipment, including reference materials........................................................ 9

8.2.1 Reagents and materials .............................................................................................................................................. 9

8.2.2 Apparatus and equipment ....................................................................................................................................10

8.3 Procedure .................................................................................................................................................................................................10

8.3.1 General...................................................................................................................................................................................10

8.3.2 Safety precautions .......................................................................................................................................................10

8.3.3 Pre-treatment ..................................................................................................................................................................10

8.3.4 Amount of sample ........................................................................................................................................................10

8.3.5 Procedure ............................................................................................................................................................................10

8.4 Interpretation and expression of results .......................................................................................................................10

8.5 Results .........................................................................................................................................................................................................11

8.6 Reporting of data collection......................................................................................................................................................11

9 Test report ................................................................................................................................................................................................................11

Annex A (informative) Overview of known GM cotton events .................................................................................................12

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Annex B (informative) Overview of detection methods applied by RIKILT ...............................................................16

Annex C (informative) In-house validation RIKILT .............................................................................................................................18

Annex D (informative) Workshop contributors .....................................................................................................................................27

Bibliography .............................................................................................................................................................................................................................30

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IWA 32:2019(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.

International Workshop Agreement IWA 32 was approved at a workshop hosted by the Netherlands

Standardization Institute (NEN), in association with the Organic Cotton Accelerator, held in New Delhi,

India, in January 2019.

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.
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IWA 32:2019(E)
Introduction
0.1 General

This purpose of this document is to provide guidance to laboratories worldwide to assess, in a

standardized way, whether cotton, cotton fibre and/or cotton-derived materials are produced from, or

contain materials from, genetically modified (GM) cotton plants. This document is intended for non-GM

cotton and textiles production lines, but it can be applied to any production line that wants to check the

presence of GM cotton.
0.2 Protocol

The GM screening protocol described in this document is based on Polymerase Chain Reaction (PCR)-

based methods, as these methods are the minimal set of DNA-based methods to cover all known GM-

cotton events. The protocol is written for and tested to work on all four of the major commercial cotton

species: Gossypium hirsutum, G. barbadense, G. arboreum G. herbaceum.

Cotton (Gossypium spp.) has been cultivated for lint for over 8 000 years. There are over 50 species in

the Gossypium genus (Wendel et al., 2009). The Gossypium genome is complex, containing 2,25 to 2,43

gigabase (Arumuganathan and Earle, 1991). While GM-cotton cultivation covers a large part of global

cotton production today, there are countries where the cultivation of GM cotton is not allowed by law

as well as voluntary private and/or public standards that do not allow the intentional use of genetically

modified organisms (GMOs) in the cotton and textile production process. This creates a need for an

adequate and harmonized protocol on the screening of cotton and cotton-derived materials for the

potential presence of GM-cotton related sequences.

This document describes a procedure to screen seed, leaf and (processed) fibre samples in the cotton

production chain for the potential presence of GM-related DNA elements. The protocol describes three

major steps:
a) an effective way to isolate DNA from cotton materials;

b) a method to confirm that the isolated DNA consists of amplifiable cotton DNA, i.e. suitable for PCR,

preferably a low copy nuclear target;

c) A screening method consisting of a minimum set of detection methods covering all the currently

known GM cotton events, to be performed on the cotton DNA isolate.

If the results of the screening methods described in this protocol are ‘not detected’, the likelihood that the

cotton sample is (at least partly) derived from GM cotton is minimal, based on the ability of the screening

methods to detect elements and constructs of the GM cotton events. GM cotton levels below the detection

limit of the method or unknown GM cotton events that do not contain any of the elements or the construct

tested cannot be determined by this detection method. When one or more screening methods indicate

that GM elements are present, the sample should be considered as derived from GM cotton.

Further investigation for the identification of GM-cotton events present in the sample is not part of this

document as such, but some guidance is provided in Annex A as to how further identification of the

related cotton events can be achieved.
0.3 Structure

The structure of this document is illustrated in Figure 1. Clause 4 describes the principle of the

screenings protocol. Clause 5 describes sample preparation for different types of material. Clause 6

describes the DNA isolation method that allows for successful DNA isolation from the respective

cotton-related products. Clause 7 describes the DNA quality control for the different cotton species.

Clause 8 describes the screening of GM-related DNA sequences in a cotton sample. Clause 9 describes

recommendations on the test report (outcome). Annex A gives an overview of known GMO cotton

events. Annex B gives an overview of detection methods applied by RIKILT . Annex C provides

1) https: //www .wur .nl/en/Research -Results/Research -Institutes/rikilt .htm
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more information on the inhouse validation as carried out by RIKILT. Annex D provides a list of the

contributos to the International Workshop.
Figure 1 — Structure of this document
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International Workshop Agreement IWA 32:2019(E)
Screening of genetically modified organisms (GMOs) in
cotton and textiles

WARNING — The method described in this document implies the use of reagents that pose a

hazard to health. This document does not claim to address all associated safety problems. It

is the responsibility of the user of this document to take appropriate measures for health and

safety protection.
1 Scope

This document provides requirements and recommendations to laboratories that perform genetically

modified organism (GMO) analyses in cottonseed, leaf, cotton fibre and cotton fibre-derived materials.

The following are within the scope of this document:

a) identifying the materials to be assessed, based on the probability of obtaining good quality, fit for

purpose DNA from the materials in subsequent steps in the cotton cloth production process;

b) specifying a method for efficient DNA isolation from cotton and cotton-derived materials described

under point a);

c) specifying the cotton-specific method(s) to be used as control for amplifiable DNA;

d) specifying the screening procedure that provides optimal chances to detect GMOs as a result of the

performance of the lowest number of genetically modified (GM) element screening assays.

NOTE 1 The protocol allows for the screening of all currently known GM cotton events and is set up in a way

that optimizes the probability of also detecting unknown GM cotton events that possibly contain similar DNA

sequences. Further information is given in CEN/TS 16707.
Sampling is outside of the scope of this document.

NOTE 2 A recommended sampling method is given in ISO 6497. General guidance for the sampling of bulk

materials or for cotton-based products is available in standards such as ASTM D1441-12 and CEN/TS 15568.

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 21570:2005, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Quantitative 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
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
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IWA 32:2019(E)

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
cottonseed
seed from cotton plants
3.2
cotton leaf
leaves from the cotton plant
3.3
seed cotton
raw cotton that contains both the seed and the fibre before it has been ginned
3.4
cotton lint
raw fibre that has gone through the ginning process
3.5
greige yarn

unprocessed long continuous length of interlocked cotton lint that results from the cleaning and

subsequent spinning of the cotton lint
3.6
greige fabric

unprocessed textiles formed by weaving, knitting or crocheting the yarn and non-wovens

3.7
processed yarn
yarn that has undergone processing, to develop its full textile potential
3.8
processed fabric
fabric that has undergone processing, to develop its full textile potential
4 Principle

This document describes a method for the screening of GMO in cotton and textiles. The screening is

based on realtime PCR methods which depends on obtaining good quality amplifiable DNA. Good quality

DNA samples (those fit for purpose) are defined as those where the amplification of an endogenous

cotton gene (positive control) is observed. The amplification and detection of endogenous cotton is

achieved through isolation methods that result in good quality DNA, applied to cotton and textiles,

while the targeted amplification of six genetic elements can allow for the detection of GM-cotton in

these samples.

NOTE Experimental results have shown that good quality DNA can be isolated from the production stages

of cottonseed up to greige yarn and greige fabric, while it showed not to be possible to isolate amplifiable DNA

in processed yarn and processed fabric. Processed yarn and processed fabric are therefore excluded from this

protocol. See Clause C.3 for the assessment of isolation of good quality DNA at different cotton production stages

by RIKILT.
5 Sample preparation
Homogenize the sample using suitable methods and avoiding excessive heating.
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Sample preparation is dependent on sample type. Prepare samples by using either one of the following

techniques: 'teasing', 'cutting', 'crushing' or 'shredding'.

Prepare at least two replicates per sample. Include appropriate controls, as specified in ISO 21571 on

DNA extraction.

The recommended sample preparation for different types of material is as follows.

— Cottonseed: Crush the seeds thoroughly with a suitable method. Use 100 mg in the DNA isolation

procedure.

— Cotton leaf: Crush the leaves thoroughly with a suitable method. Use 100 mg in the DNA isolation

procedure.

— Seed cotton: Seperate the seeds from the fibres, crush the seeds thoroughly with a suitable method.

Use 100 mg in the DNA isolation procedure.

— Cotton lint: The fibre material can be teased thoroughly applying suitable method. Use 100 mg in

the DNA isolation procedure.

— Yarn: Cut the yarn with a suitable method into small parts of a maximum of approximately 0,5 cm

length. Use 100 mg in the DNA isolation procedure.

— Fabric: Cut the fabric with a suitable method in small parts of a maximum of approximately

0,5 × 0,5 cm in size. Use 100 mg in the DNA isolation procedure.
6 DNA isolation
6.1 General

In order to obtain amplifiable DNA from cottonseed, cotton and textiles as per the protocol’s scope, a

DNA isolation method has been selected that allows for successful DNA isolation from the respective

cotton-related products. This method allows for rapid purification of genomic DNA suitable for PCR

with a limited number of protocol steps. The protocol works well for cotton-derived materials that can

contain relatively high levels of PCR inhibitors.

NOTE 1 The DNA isolation procedure described in this document is the QIAamp® Fast DNA Stool Mini Kit. The

rest of this protocol refers to the QIAamp® Fast DNA Stool Mini Kit .

NOTE 2 As an alternative strategy to the DNA isolation method described below, the cotton-adjusted CTAB-

protocol (e.g. CRLVL-14/05XP: JRC 2006) or any other suitable DNA isolation method can be applied, provided

that this method has been proven by means of in-house validation against the QIAamp® Fast DNA Stool Mini

Kit to perform equally well or better compared to the QIAamp® Fast DNA Stool Mini Kit. For seed, certified

reference materials are used for validation.
6.2 Principle

The DNA isolation procedure is based on an inhibition buffer, a lysis buffer and a DNA-binding spin

column. DNA binds specifically to the silica-gel membrane in the spin column, while contaminants pass

through. No phenol-chloroform extraction is required. PCR inhibitors are separated from DNA by the

inhibition buffer.

QIAamp® Fast DNA Stool Mini Kit is an example of a suitable product available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of this product.

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6.3 Chemicals, reagents and equipment

Use only reagents of recognized analytical grade. Appropriate facilities should be used in order to avoid

contamination during the steps of preparation and measurement (e.g. uses of laminar flow benches or

comparable clean facilities) .

Unless otherwise stated, only reagents that conform to the specifications of ISO 24276 were used.

6.3.1 Reagents

6.3.1.1 Inhibition buffer: contains lithium chloride (>=1 – 10 % w/w) and sodium dodecyl sulfate

(>=1 - <10 % w/w) (e.g. Inhibitex Buffer Qiagen Cat No./ID: 51604), as provided by the manufacturer.

6.3.1.2 Lysis buffer: lysis buffer contains guanidine hydrochloride (>=30 - <50 % w/w) and maleic

acid (>=0.1 - <1 % w/w), as provided by the manufacturer.

6.3.1.3 Wash Buffer 1; ethanol solution to denature proteins contains guanidine hydrochloride (>=50

- <70 % w/w) ), as provided by the manufacturer.

6.3.1.4 Wash Buffer 2: Tris-based ethanol solution to remove salts, contains sodium azide), as

provided by the manufacturer.
6.3.1.5 Ethanol 96 % to 100 %.

6.3.1.6 Elution Buffer: contains 10 mM Tris-HCl pH8.3, 0.1 mM EDTA, 0.04 % NaN (sodium azide).

6.3.1.7 Proteinase K (>=1 - <10 % w/w).
6.3.1.8 Molecular biology grade water or water of equivalent purity.

6.3.1.9 DNA degrading solution (e.g. 1 % bleach) household bleach (hypochloric acid).

6.3.2 Apparatus and equipment
6.3.2.1 Silica-based mini spin columns, as provided by the manufacturer.
6.3.2.2 Disposable spatulas.
6.3.2.3 Sterile filter pipette tips protecting against aerosols.
6.3.2.4 Microcentrifuge tubes of 1,5 ml and 2,0 ml.
6.3.2.5 Disposable gloves (powder-free).
6.3.2.6 Analytical scale and top weigher.
6.3.2.7 Waterbath and/or thermoshaker (e.g. 24 ml × 2,0 ml).
6.3.2.8 Centrifuge for microcentrifuge tubes (at least 20 000 x g).

Reference to a given product is given for for the convenience of users of this document and does not constitute an endorsement by ISO of

the product named. Equivalent products may be used if they can be shown to lead to the same results.

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IWA 32:2019(E)
6.3.2.9 Suitable prepared homogenization equipment.
6.3.2.10 Autoclave, 121 °C, 20 minutes.
6.3.2.11 Pipettes (1-10 μl, 2-20 μl, 20-200 μl, 200-1 000 μl).
6.3.2.12 Vortex.
6.3.2.13 Refrigerator.
6.3.2.14 Freezer.
6.3.2.15 Clean lab coat.
6.4 Procedure
6.4.1 General
The DNA extraction procedure comprises the following steps:

— lysis of, and separation of, impurities from samples in guanidine hydrochloride-containing buffer;

— purification of DNA on mini spin columns.
6.4.2 Protocol

All centrifugation steps should be carried out at room temperature (15 °C to 25 °C).

Perform the DNA isolation according to the protocol of the chosen isolation method or see the

manufacturer’s instructions.

— (1) Weigh 100 mg (+/− 10 mg) homogenized sample, as prepared in Clause 5, in a 2 ml

microcentrifuge tube.

— (2) Add 1 ml inhibition buffer to each sample. Vortex continuously for 1 min or until the sample is

thoroughly mixed.
— (3) Centrifuge sample at 20 000 x g for 1 min to pellet particles.
— (4) Pipette 25 μl proteinase K into a new 2 ml microcentrifuge tube.

— (5) Pipette 600 μl supernatant from step (3) into the 2 ml microcentrifuge tube containing

proteinase K.
— (6) Add 600 μl lysis buffer and vorte
...