ISO/FDIS 22753

Molecular biomarker analysis -- Method for the statistical evaluation of analytical results obtained in testing sub-sampled groups of genetically modified seeds and grains -- General requirements and definitions

Analyse moléculaire de biomarqueurs— Méthode pour l'évaluation statistique des résultats d'analyse obtenus lors des essais de sous-échantillons multiples de semences et de graines génétiquement modifiées -- Exigences générales et définitions

General Information

• Status: Published
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16/WG 9 - Subsampling of seeds and grains
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 31-May-2021
• Completion Date: 31-May-2021

Standards Content (sample)

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22753
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Method for the statistical evaluation of
Voting begins on:
2021­05­31 analytical results obtained in testing
sub-sampled groups of genetically
Voting terminates on:
2021­07­26
modified seeds and grains — General
requirements and definitions
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/FDIS 22753:2021(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 2021
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ISO/FDIS 22753:2021(E)
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© ISO 2021

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ISO/FDIS 22753:2021(E)
Contents Page

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

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

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

4.1 General ........................................................................................................................................................................................................... 4

4.2 Preparation of seed/grain groups .......................................................................................................................................... 5

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain groups ....... 5

4.4 Statistical evaluation .......................................................................................................................................................................... 5

5 Reagents ........................................................................................................................................................................................................................ 6

6 Apparatus and equipment .......................................................................................................................................................................... 6

7 Design of testing plan ...................................................................................................................................................................................... 6

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

7.2 Single­stage testing plan ................................................................................................................................................................. 6

7.3 Double­stage testing plan .............................................................................................................................................................. 7

8 Selection of qualitative methods ......................................................................................................................................................... 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Performance criteria .......................................................................................................................................................................... 8

9 Interpretation .......................................................................................................................................................................................................... 8

10 Expression of results .....................................................................................................................................................................................10

10.1 Classification of a seed/grain lot into “accept” or “reject” category ......................................................10

10.2 Estimation of the level of molecular biomarker in the seed/grain lot .................................................10

11 Test report ................................................................................................................................................................................................................10

Annex A (informative) Terms and definitions comparison table ........................................................................................12

Annex B (informative) Implementation of the method to evaluate GMO content in seeds/

grains example.....................................................................................................................................................................................................14

Annex C (informative) Estimation of the limit of detection for a testing plan to detect GM

seeds/grains in seed lots ..........................................................................................................................................................................21

Annex D (informative) Experimental determination of maximum group size ......................................................25

Bibliography .............................................................................................................................................................................................................................26

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ISO/FDIS 22753:2021(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 non­governmental, 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.

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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ISO/FDIS 22753:2021(E)
Introduction

Seed and grain testing is used throughout the world to commercially define the purity of seed and grain

lots.

Commercial requirements for labelling agricultural products with genetically modified organism (GMO)

content at a specified threshold level both as a seed/grain contaminant and a food ingredient have

become common to satisfy regulations and consumer demands. Conformance with these specifications

is evaluated at various points of the supply chain, often starting with the harvested grain.

Quantitative real-time polymerase chain reaction (PCR) can be used to determine the GMO content by

analysis of the ratio of GMO DNA copy numbers to plant-species specific DNA copy numbers followed by

a conversion to genetically modified (GM) mass fraction.

Multiple events stacked in a crop, such as those generated by crossing two or more single events,

are widely used in agricultural production. A stacked event seed or grain containing GMO DNA

corresponding to two or more GM events commingled in lot cannot be differentiated by quantitative

PCR alone from multiple seeds within the lot each containing a single GM event. Consequently, if the

actual measured GMO arises only from GM stacked event seeds, GM content measured by quantitative

real­time PCR of a single sample will lead to an overestimation of the actual number of GM seeds or

grains present.

The group testing strategy described in this document provides a reliable alternative to estimate the

GM content on the basis of the fact that whole seeds/grains are the sample material.

The process described in this document can provide a method to accurately estimate the percentages

of GM seeds/grains in a lot irrespective of the presence of stacked event seeds/grains. GM content is

determined for representative subsampled groups of seed/grain from a lot and statistically analysed.

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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 22753:2021(E)
Molecular biomarker analysis — Method for the statistical
evaluation of analytical results obtained in testing sub-
sampled groups of genetically modified seeds and grains —
General requirements and definitions
1 Scope

This document describes general requirements, procedures and performance criteria for evaluating

the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes

qualitative analysis of sub-sampled groups followed by statistical evaluation of the results.

This document is applicable to group testing strategy estimating the GM content on a percentage seed/

grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where

seed/grain lots are carrying stacked events.
This document is not applicable to processed products.

NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and

[20].
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 — Terms and definitions

ISO 21572, Foodstuffs — Molecular biomarker analysis — Immunochemical methods for the detection and

quantification of proteins

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 and the following apply.

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 https:// www .electropedia .org/
3.1
absolute PCR limit of detection
absolute polymerase chain reaction limit of detection
absolute PCR LOD

lowest nominal (average) number of target copies in the template volume distributed to individual PCRs

that would allow for an acceptable probability of detecting the target
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ISO/FDIS 22753:2021(E)
3.2
AQL
acceptable quality limit

level of impurity that is acceptable to the producer and that production practices can support

3.3
consumer risk
consumer (beta) risk
probability of accepting a lot at the lower quality limit (3.10)
3.4
deviant seed/grain

considered non-conforming based on the presence or absence of a specific trait or characteristic

Note 1 to entry: For the purpose of this document, a deviant seed is considered to possess a GM characteristic

that is not expected or is unintended based on the expected or known GM characteristics of the seed/grain.

3.5
false negative rate
FNR

probability that a known positive (seed/grain group) test sample (3.20) has been classified as negative

by the method

Note 1 to entry: The false negative rate is the number of misclassified known positives divided by the total

number of positive test samples (3.20).

[SOURCE: ISO 16577:2016, 3.63, modified — the abbreviation has been added, “positive test sample”

has been changed to “positive (seed/grain group) test sample”, and the formula has been deleted.]

3.6
false positive rate
FPR

probability that a known negative (seed/grain group) test sample (3.20) has been classified as positive

by the method

Note 1 to entry: The false positive rate is the number of misclassified known negatives divided by the total

number of negative test samples (3.20).

[SOURCE: ISO 16577:2016, 3.65, modified — the abbreviation has been added, “negative test sample”

has been changed to “negative (seed/grain group) test sample”, and the formula has been deleted.]

3.7
group size
number of seeds/grains comprising a group
3.8
group testing

statistical evaluation of analyte contents based on qualitative analysis results (i.e. positive or negative)

from each seed/grain group in the test sample (3.20)
3.9
laboratory sample
sample or subsample(s) received by the laboratory

Note 1 to entry: The seed/grain sample received is expected to represent the seed/grain lot (3.18).

[SOURCE: ISO 16577:2016, 3.89, modified — Note 1 to entry has been added.]
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ISO/FDIS 22753:2021(E)
3.10
LQL
lower quality limit
highest impurity that is acceptable to the consumer
Note 1 to entry: This can be equivalent to the threshold (3.22).
3.11
mass fraction

ratio of GM seeds/grains relative to the total seeds/grains corresponding to mass ratio

3.12
number of deviant seed/grain groups

number of seed/grain groups (3.17) including one or more deviant seeds/grains (3.4)

3.13
operating characteristic curve
OC curve

graph plotting the percentage of deviant seeds/grains and the probability of acceptance respectively

on the horizontal and the vertical axes and used in quality control to determine the probability of

accepting seed/grain lots (3.18) in a testing plan (3.21)
3.14
producer risk
producer (alpha) risk
probability of rejecting a lot at the AQL (3.2)
3.15
representative sample

sampling units (samples or groups) that have been extracted from a lot with the process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
3.16
reject/accept criterion

maximum number of deviant seed/grain groups (3.12) that can be detected in the test sample (3.20) of an

acceptable seed/grain lot (3.18)
3.17
seed/grain group
group

determined number of seeds/grains prepared from a seed/grain test sample (3.20) by representative

sampling
3.18
seed/grain lot
lot
population for which sampling is intended to estimate the measured parameter
3.19
stacked event

accumulation of two or more transformation events as a result of traditional breeding and/or successive

transformation steps)

Note 1 to entry: In the context of this document a stacked event refers to a stack in which the two or more events

are not genetically linked.
[SOURCE: ISO 16577:2016, 3.197, modified — Note 1 to entry has been added.]
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ISO/FDIS 22753:2021(E)
3.20
test sample

sample prepared for testing or analysis, the whole quantity or part of it being used for testing or

analysis at one time
Note 1 to entry: The test sample is prepared from the laboratory sample (3.9).

Note 2 to entry: The test sample is expected to represent the laboratory sample (3.9).

[SOURCE: ISO 16577:2016, 3.210, modified — Note 1 to entry and Note 2 to entry have been added.]

3.21
testing plan

plan specifying group testing (3.8) conditions including group size (3.7), the number of seed/grain groups

(3.17) and the number of deviant seed/grain groups (3.12) in test sample (3.20) resulting in rejection of

seed/grain lot (3.18)
3.22
threshold
maximum acceptable content of GMO presence in a lot
Note 1 to entry: This can be a prescribed value.

Note 2 to entry: Thresholds can be expressed in mass fraction (3.11) with the proviso that an uncertainty factor

is involved in the conversion to a seed/grain percentage threshold.
4 Principle
4.1 General

In this method, the test sample is divided into a predetermined number of groups. Each group consists

of a determined number of seed/grain and is tested qualitatively for the presence or absence of a GM

target. A statistical evaluation is performed on the number of GM positive groups relative to the total

number of seed/grain groups to determine the GM content in mass fraction.

A statistical calculation determines the optimal testing conditions, namely, the number of seeds/grains

per group (group size), the number of seed/grain groups, and the maximum number of GMO positive

seed/grain groups for seed/grain lot acceptance. Alternatively, a statistical calculation provides an

estimate of the percentage by number of the GM seeds/grains in a lot, according to a given testing plan.

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ISO/FDIS 22753:2021(E)
4.2 Preparation of seed/grain groups
Key
1 bulk seed/grain lot
2 laboratory sample
3 test sample
4 seed/grain groups
5 deviant seed/grain
NOTE Each group is represented as an array on the right.

Figure 1 — Sampling illustration of the obtention of seed/grain groups from a bulk seed/grain

lot

The process of forming seed/grain groups from a series of sampling steps starting with the bulk seed/

grain lot is shown in Figure 1, (1).

Although the procedures for obtaining a laboratory sample from a seed/grain lot is not the subject

of this document, a laboratory sample (2) from a seed/grain lot shall be obtained appropriately. The

procedures can be designed according to the References [3], [6], [10], [11], [12], [15], [19] and [23].

The laboratory sample shall be thoroughly mixed and divided/reduced to create the test sample (3).

Likewise, the test sample shall be thoroughly mixed (i.e. homogeneous) and divided into seed/grain

groups (each group represented as an array in Figure 1, (4)) following simple random sampling

principles. The seed/grain groups can vary in size from one single seed/grain up to the complete test

sample (i.e. a single bulk). In most cases, multiple seed/grain groups are created from the test sample.

A determined number of seeds/grains can either be obtained by weighing or a volumetric measurement,

where an approximation of number is made based on a determined conversion factor (e.g. thousand

seeds/grains weight). For the case that weight is used to obtain the seed/grain groups, the operator

shall have an estimate of the variability introduced by using weight rather than seed/grain count.

The group testing procedure described in Clause 7 is carried out on the collective qualitative (positive

(e) or negative) results for each seed/grain group.

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain

groups
[21]

In general, GMO detection methods are categorized into two classes . The first class of assays targets

a nucleic acid sequence for detecting GMO presence. The second class includes methods for detecting a

specified protein that confers a specific transgenic trait. Detection methods from either or both classes

should be selected considering fitness-for-purpose. Guidance on the selection of qualitative methods is

[4]
provided in Clause 8. Further details can be found in ISO 21569 and ISO 21572.
4.4 Statistical evaluation

Sampling and measurement uncertainty shall be considered. Sampling uncertainty can be adequately

[18][2]

considered using the binomial distribution . The FPR and the FNR of the qualitative assay should

[2]
be considered . The LOD of the applied detection method should be considered.
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ISO/FDIS 22753:2021(E)

The group testing described here can be used to set reject/accept criteria based on a given threshold by

GMO content, as well as to estimate the GMO content and associated upper and lower confidence limits.

5 Reagents
All reagents used in the analysis should be those specified in the method.
Otherwise, all reagents should be of molecular biology grade.

These reagents shall be stored and used as recommended by the supplier or according to the laboratory

quality assurance specifications. It can also be appropriate to aliquot the reaction solutions required

for the analytical method in order to avoid subjecting them to repeated freeze–thaw cycles, or to reduce

the chances of cross contamination or both. Further details shall refer to ISO 24276 and ISO 21572.

6 Apparatus and equipment

The laboratory should use properly maintained equipment suitable for the methods employed.

Further details shall refer to ISO 24276 and ISO 21572.
7 Design of testing plan
7.1 General

The number of seeds/grains tested, the reject/accept criteria, the sample preparation steps and the

method used for testing shall be determined depending on the analytical purpose.

In seed/grain sample classification, it can be determined whether the number of deviant seeds/grains

or seed/grain groups is above a given reject/accept criterion or not. Then, it can be decided to reject or

accept the seed/grain lot based on the test results.
A basic testing plan for group testing consists of three fundamental parameters:
a) the number of seed/grain groups;
b) the size of the seed/grain groups;

c) the maximum number of deviant seed/grain groups for seed/grain lot acceptance (reject/accept

criterion).

The risks associated with the AQL and the LQL are the producer (alpha) and consumer (beta) risks

respectively, and together with the FPR and FNR allow the design of an appropriate testing plan.

The OC curve can be used to develop a testing plan. Explanations for the estimation of the LOD for a

zero deviant testing plan, the effect of the genome size on the group size if methods targeting DNA are

applied, and the effect of the individual seed size on the sample preparation are given in Annex C.

Annex D provides guidance on the determination of the maximum group size whatever analytical

method is used in the laboratory.
[16]

NOTE Seedcalc is a statistical program (Microsoft Excel spreadsheet application) that is freely available

from the International Seed Testing Association and has procedures to facilitate the design. Seedcalc is located

on the ISTA website.
7.2 Single-stage testing plan

A single­stage testing plan consists of one testing stage. Groups are taken from the test sample and

evaluated once, and a decision is then made based on the results to accept or reject the seed/grain

test sample. In a single-stage testing plan, a specified number of individual seeds/grains or seed/

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ISO/FDIS 22753:2021(E)

grain groups shall be selected randomly from the test sample and tested. Depending on the number

of deviants detected and the maximum number of deviants specified in the plan, the seed/grain lot is

either accepted or rejected.

The probability that an individual seed/grain or seed/grain group is deviant, p , can be calculated as

given in Formula (1):
pP=−11=− 1−p (1)
where
P is the probability that there are no deviant seeds/grains in the group;
p is the true unknown impurity in the seed/grain lot;

m is the number of individual seeds/grains in a seed/grain group (if seeds/grains are tested indi­

vidually, m = 1).

Then, the probability that a lot will be accepted, P(a) is calculated as given in Formula (2):

 
ni−
P()a = pp()1− (2)
 
∑ b b
 
i=0
where
P(a) is the probability that a lot will be accepted;
n is the number of individual seeds/grains or seed/grain groups tested;
c is the maximum number of deviant seed/grain groups for acceptance.
By combining Formulae (1) and (2), P(a) is a function of p, n, m and c.

After n, m and c are determined, an OC curve can be drawn by plotting p and P(a) on the x-axis and

y-axis, respectively.
7.3 Double-stage testing plan

A double-stage testing plan is generally set up so that additional seed/grain groups are tested in the

second stage. Initial seed/grain groups are taken from the test sample and tested. Based on this test

result, three different decisions can be made:
a) accept the seed/grain lot;
b) reject the seed/grain lot; or
c) draw a second set of seed/grain groups from the test sample and retest.

The test results from the first and second stages of testing are combined and used to determine whether

the seed/grain lot is accepted or rejected (see Figure B.1). In Annex B examples for implementation of a

double­stage testing plan to evaluate GMO content in seeds/grains are provided. Subclause B.1 can also

be applied for cases where seed/grain lots are carrying stacked events.
Some additional terms are defined as follows:

— n , the number of independent seed/grain groups to be tested in the first stage;

— n , the number of independent seed/grain groups to be tested in the second stage;

— c , the maximum number of allowable deviant seed/grain groups for acceptance in the first stage;

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— c , the minimum number of deviant seed/grain groups that will result in rejection at the first stage;

— c , the maximum number of deviant seed/grain groups in the first and second stages combined

allowed for acceptance;
— d , the number of deviant seed/grain groups in the first stage;
— d , the number of deviant seed/grain groups in the second stage.
P(a) is calculated as given in Formula (3):
n n n2
       
c c −1 ci-
11ni−−ni nj−
1 i 2 i 3 j
11 2
P()a = pp()11− + pp()− × 1pp()−
      
∑ b b ∑  b b ∑ b b 
i=0 ic=+1 j=0
i i j
       
(3)
8 Selection of qualitative methods
8.1 General

An analytical method shall be chosen to meet the purpose of testing. The performance characteristics

of the method should be determined before application in seed/grain testing.

Analytical methods have been developed to detect specific genes encoding transgenic traits or specific

characteristics expressed by specific genes in seeds/grains. Nucleic-acid-based methods such as PCR

[4][5]
are available that detect specific DNA sequences encod
...