IWA 32:2019
(Main)Screening of genetically modified organisms (GMOs) in cotton and textiles
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
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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)
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© ISO 2019
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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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IWA 32:2019(E)
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
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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
1)
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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IWA 32:2019(E)
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
2)
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
2)
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
3)
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).
3
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
3)
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
...
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