SIST EN ISO 11781:2025

SLOVENSKI STANDARD
01-junij-2025
Nadomešča:
SIST-TS CEN/TS 17329-1:2021
Analiza molekularnih biomarkerjev - Zahteve in navodila za validacijo kvalitativnih
metod s polimerazno verižno reakcijo (PCR) v realnem času v posameznem
laboratoriju (ISO 11781:2025)
Molecular biomarker analysis - Requirements and guidance for single-laboratory
validation of qualitative real-time polymerase chain reaction (PCR) methods (ISO
11781:2025)
Untersuchung auf molekulare Biomarker - Anforderungen und Leitfaden für die
Einzellaborvalidierung qualitativer Real-time-PCR-Verfahren (ISO 11781:2025)
Analyse de biomarqueurs moléculaires - Exigences et recommandations pour la
validation intralaboratoire des méthodes de PCR qualitative en temps réel (ISO
11781:2025)
Ta slovenski standard je istoveten z: EN ISO 11781:2025
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 11781
EUROPEAN STANDARD
NORME EUROPÉENNE
April 2025
EUROPÄISCHE NORM
ICS 67.050 Supersedes CEN/TS 17329-1:2021
English Version
Molecular biomarker analysis - Requirements and
guidance for single-laboratory validation of qualitative
real-time polymerase chain reaction (PCR) methods (ISO
11781:2025)
Analyse de biomarqueurs moléculaires - Exigences et Untersuchung auf molekulare Biomarker -
recommandations pour la validation intralaboratoire Anforderungen und Leitfaden für die
des méthodes de PCR qualitative en temps réel (ISO Einzellaborvalidierung qualitativer Real-time-PCR-
11781:2025) Verfahren (ISO 11781:2025)
This European Standard was approved by CEN on 4 April 2025.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2025 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11781:2025 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 11781:2025) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by October 2025, and conflicting national standards shall
be withdrawn at the latest by October 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN/TS 17329-1:2021.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 11781:2025 has been approved by CEN as EN ISO 11781:2025 without any modification.

International
Standard
ISO 11781
First edition
Molecular biomarker analysis —
2025-04
Requirements and guidance for
single-laboratory validation of
qualitative real-time polymerase
chain reaction (PCR) methods
Analyse de biomarqueurs moléculaires — Exigences et
recommandations pour la validation intralaboratoire des
méthodes de PCR qualitative en temps réel
Reference number
ISO 11781:2025(en) © ISO 2025
ISO 11781:2025(en)
© ISO 2025
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Email: copyright@iso.org
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Published in Switzerland
ii
ISO 11781:2025(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
5 Single-laboratory validation of the performance characteristics . 2
5.1 General .2
5.2 Limit of detection .2
5.3 Determining copies of DNA target sequences in DNA test materials .3
5.4 Evaluation of data for the limit of detection .3
5.5 PCR efficiency and variability of the measured copy number around the limit of
detection .4
5.6 Specificity .4
5.6.1 General .4
5.6.2 Bioinformatic (in silico) test for specificity .4
5.6.3 Practical test for specificity .4
5.6.4 Robustness . .5
6 Validation report . 6
Annex A (informative) Estimation of the number of copies of the DNA target sequence . 7
Annex B (informative) Determination of limit of detection, precision and PCR efficiency . 9
Annex C (informative) Generalized linear mixed model with log-log link .13
Annex D (informative) Robustness testing . 19
Bibliography .21

iii
ISO 11781:2025(en)
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,
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
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Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis, in collaboration with the European Committee for
Standardization (CEN) Technical Committee CEN/TC 275, Food analysis - Horizontal methods, in accordance
with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO 11781:2025(en)
Introduction
Qualitative real-time polymerase chain reaction (PCR) methods currently find broad application for the
detection of specific DNA sequences in food (e.g. for the detection and identification of genetically modified
organisms and the products derived thereof, for food authentication and speciation and for other purposes). It
is important that a newly developed food analytical method is fit for purpose and meets certain performance
characteristics and quality criteria as demonstrated by a particular set of validation experiments.
The data determined by the single laboratory validation are the basis for the decision to apply a method
in-house. Furthermore, it helps to decide whether the method in question should be fully validated in
the framework of a collaborative study. The statistical model described has been practically applied, e.g.
[1][2] [3]
some parts of the ISO/TS 21569 series and ISO/TS 20224 series . Other models can be applicable, see
[4]
ISO/TS 16393 .
The aim of this document is to provide a protocol for single-laboratory validation of qualitative real-time
PCR methods that are applied for food analysis. Procedures for DNA extraction from the food matrix are not
included in this document. The procedure described is a recommendation which is underpinned by practical
experience in several laboratories. Alternate approaches may be applied if they can be shown to meet the
performance criteria set in this document.

v
International Standard ISO 11781:2025(en)
Molecular biomarker analysis — Requirements and guidance
for single-laboratory validation of qualitative real-time
polymerase chain reaction (PCR) methods
1 Scope
This document specifies minimum requirements and minimum performance criteria for conducting a single-
laboratory validation study for qualitative (binary) real-time polymerase chain reaction (PCR) methods
applied to the detection of specific DNA sequences present in foods.
The document is applicable to any single-laboratory validation of a qualitative real-time PCR method used
for the detection of specific DNA sequences in food and food products (e.g. for the detection of genetically
modified foodstuffs and for species determination, including species known to produce allergenic proteins).
The document does not apply to single laboratory validation of qualitative microbiological real-time PCR
methods.
The document does not apply to the evaluation of applicability and practicability with respect to the specific
scope of the PCR method.
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 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction
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
probability of detection
POD
probability of a positive analytical outcome of a qualitative method for a given matrix at a given concentration
in a single laboratory
Note 1 to entry: For a qualitative real-time PCR method, it describes the probability that, for a given number of DNA
copies of the target sequence, PCR amplification will take place.
[SOURCE: ISO 16577:2022, 3.9.12, modified — Note 1 to entry replaced Notes 1 and 2 to entry.]

ISO 11781:2025(en)
3.2
polymerase chain reaction efficiency
PCR efficiency
measured amplification rate for a DNA copy of the target sequence per PCR cycle in relation to the
theoretically achievable value of 1
Note 1 to entry: The PCR efficiency is calculated from the slope of a standard curve resulting from the decadic
semi-logarithmic plot of quantification cycle (Cq) values over the DNA concentration. The slope from the calculated
regression line can be used. The PCR efficiency can either be expressed as absolute number or as percentage.
3.3
limit of detection
LOD
95 %
mean number of DNA copies of the target sequence yielding a probability of detection (3.1) of 0,95
3.4
specificity
property of a method to respond exclusively to the characteristic or analyte under investigation
[SOURCE: ISO 24276:2006, 3.1.4]
4 Principle
Specific primers and also probes, depending on the detection system applied, are designed for specific
amplification and detection of a DNA target sequence by a qualitative real-time PCR method. In the next step
for single laboratory validation, the method’s performance characteristics shall be assessed to show that the
[5][6]
method complies with the quality criteria stipulated in relevant documents.
For a qualitative real-time PCR method, the main focus of the validation shall be the limit of detection
(LOD ) (at which the probability of detection (POD) is ≥ 95 %), the specificity for the DNA target sequence
95 %
and the robustness to small but deliberate variations in the method parameters.
On the basis of single laboratory validation data, fulfilment of the minimum required performance criteria
for a qualitative real-time PCR method can be verified and should comprise the basis for applicability of
the method by a single laboratory. A further decision whether to conduct a validation of the method in the
framework of a collaborative study can then be taken.
Determination of the reproducibility (interlaboratory transferability) and how the method performs in
different laboratories, in particular the false-positive/false-negative rate obtained with negative/positive
test samples, and the POD across laboratories, can be evaluated by a collaborative study, if the design is
[6]
appropriate.
5 Single-laboratory validation of the performance characteristics
5.1 General
Guidance for compiling the information required for a complete and detailed description of all components
that should be provided with the protocol of qualitative PCR methods (e.g. oligonucleotide sequences,
amplicon length, instrument or chemistry specifications, PCR conditions, analytical controls) is described in
[8]
other relevant documents, see References [5] and [6] and ISO 21569 .
DNA extraction shall be in accordance with the requirements specified in ISO 21571.
5.2 Limit of detection
The LOD is expressed as the number of copies of the target sequence and shall be determined by means
95 %
of a dilution series of the target DNA, where, in addition to the target DNA, each dilution contains a uniform
concentration of non-target DNA (background DNA).

ISO 11781:2025(en)
A minimum of six target DNA concentration levels with 12 replicates per level are required.
The lowest dilution level, i.e. the lowest number of copies for which all 12 replicates are positive, is considered
to be an approximate value for the LOD (see Clause B.2).
95 %
The LOD of qualitative real-time PCR methods should not exceed 20 copies of the target sequence.
95 %
NOTE 1 This document is applicable to the validation of new methods. However, for method verification, 10
replicates can be sufficient.
NOTE 2 If the LOD equals 20 copies of the target sequence, the amplification probability (λ) of the entire PCR is
95 %
[7]
approximately 15 % based on the parameter of the Poisson distribution (λ·LOD = 2,996).
95 %
Annex A provides additional detailed information regarding copy number estimation of target DNA.
Practical guidance for determining the LOD experimentally is given in Annex B.
95 %
Annex C provides the basics of the specific statistical model adapted for PCR methods.
5.3 Determining copies of DNA target sequences in DNA test materials
A determination of copies of DNA target sequences is required for the validation.
The number of copies of the target sequence for a specified mass of nucleic acid (DNA) can be calculated on
basis of haploid genome equivalents using the measured DNA concentration (see ISO 21571:2005, Annex B)
[9][10][11]
and the genome mass. The use of digital PCR equipment (e.g. droplet digital PCR) is an alternative
approach that allows an accurate determination of the number of copies of a DNA target sequence or the
[12]
concentration of a DNA solution.
The quality and the concentration (very high or very low) of the background DNA used for the dilution can
influence the validation experiment. It is therefore highly recommended to use DNA tested for the absence
of PCR inhibitors (e.g. commercial molecular biology grade DNA preparations) and a concentration that is
[13]
relevant for extracted DNA.
5.4 Evaluation of data for the limit of detection
The LOD , the mean POD curve and the 95 % confidence interval should be determined by means of a
95 %
statistical model.
The experimental work that also includes a Poisson component is based on the generalized linear mixed
model (GLMM) with a complementary log-log link function. This model has been found to be most effective
when the DNA copy number is low and follows a Poisson distribution.
Details for the GLMM with complementary log-log link function are given in Annex C. To perform this
calculation, the nominal copies that are added to the PCR reaction, the number of replicates performed and
the number of positive results obtained are required.
Using the results data of the dilution series, the LOD , the 95 % confidence interval and the mean POD
95 %
[14]
curve, along with the corresponding 95 % confidence range, can be calculated via a web service or by
[15]
using the R package POD (see Clause C.5 ).
Check the LOD for plausibility. A value smaller than 2,996 suggests that the number of copies of the
95 %
target sequence that were actually added to the PCR reaction did not correspond to the (nominal) numbers
[7]
of copies estimated for the DNA solutions.
If more than two results are positive at the level with 0,1 copies of the target sequence per PCR, the DNA
dilutions cannot be considered as verified and the number of copies shall be re-examined.
NOTE 1 The calculation of LOD is only valid if false-positive results are negligible, i.e. if the specificity testing
95 %
was successful and PCR carry-over contamination can be excluded.
NOTE 2 The level that will be the result of a tenfold dilution of 1 nominal copy is designated “level with 0,1 copies
per PCR” for the sake of better readability throughout this document.

ISO 11781:2025(en)
5.5 PCR efficiency and variability of the measured copy number around the limit of
detection
For an optional determination of the copy number variability around the LOD , assign the copy numbers
95 %
to the respective Cq values on the basis of an additional calibration series of target DNA (for preparation of a
calibration series, see Clause B.2).
The variability of the measured number of copies around LOD should be assessed by comparing the
95 %
repeatability standard deviations with the theoretical values of the Poisson model. An adjusted standard
deviation of > 30 % indicates that the LOD that can be achieved in routine analysis can be subject to high
95 %
variability (see Clause B.5).
The experimental data collected will also permit determination of the PCR efficiency (see Clause B.6),
the slope of the amplification curve and the coefficient of determination (R ). A PCR without inhibitory
influences will amplify as an exponential doubling with a slope (Cq/number of initial copies) of −3,321 9.
An acceptable PCR efficiency should not deviate by more than ±10 % from the theoretical value of 100 %
(corresponding to a slope in the interval −3,1 to −3,6). Where the PCR efficiency is found to be greater
than the theoretical maximum, errors due to reaction setup and PCR inhibition should be considered. The
coefficient of determination should be at least 0,98 (or 98 %).
5.6 Specificity
5.6.1 General
In silico analysis and experimental results from testing the method with genomic sequence databases
and material containing the target sequence shall be provided. If available, testing should be inclusive and
exclusive with relevant and representative data and materials according to the scope of the method.
5.6.2 Bioinformatic (in silico) test for specificity
Bioinformatic specificity tests shall be carried out, examining the oligonucleotide and the amplicon sequences
[16]
with available bioinformatics tools (e.g. primer-dimer formation with primer 3). Homology to other
®1)[17]
sequences shall be tested by searches in nucleic acid sequence databases (e.g. BLAST in GenBank ).
The in silico analysis should not reveal any sequence similarities between the target and the sequences that
can be present in the sample capable of influencing the analytical result. The oligonucleotide sequence(s)
should be adapted accordingly, if appropriate. Additional guidance on in silico analysis can be found in
Reference [18].
5.6.3 Practical test for specificity
Perform tests for unexpected cross-reactions with non-target DNA. Check the PCR detection system for
cross-reactivity with DNA from organisms that have similar (homologous) genetic elements, genes or
genetic constructs. Also check for species that are often present in food as ingredients (e.g. corn, soya, rape
seed, rice, potato, wheat, cattle, chicken, pig, sheep, turkey, horse).
If non-target DNA is tested and a negative result is expected, at least 2 500 copies should be added to
the PCR reaction. If no reference material with sufficiently high concentrations of the non-target DNA is
available, lower concentrations can be used and the number of copies added should be indicated. Verify the
amplifiability of the non-target DNA by means of an independent test.
Perform tests with target DNA. Add target DNA for which a positive result is expected in copy numbers in
the range of the limit of quantification (LOQ) (empirically the copy number for LOD can be multiplied by
95 %
a factor of 3, i.e. in general 20 to 60 copies per PCR). Add non-target DNA at a concentration of 100 ng/25 µl
to 200 ng/25 µl of PCR mix to the target DNA in order to simulate conditions that are relevant in practice
and could influence the outcome. ®
1) BLAST in GenBank 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.

ISO 11781:2025(en)
It is sufficient to carry out each of the PCR tests for inclusivity (using target DNA) and exclusivity (using non-
target DNA) in duplicate determination.
PCR results for in silico and the experimental analyses should meet or exceed the requirements and criteria
of the method.
If there is cross-reactivity that is considered acceptable, it shall be indicated and taken into account in the
scope of the method.
5.6.4 Robustness
The robustness of a qualitative real-time PCR method shall be evaluated. The evaluation of robustness shall
include small but deliberate variations, e.g. in the following method parameters:
— different types of real-time PCR equipment, if available;
— PCR reagent kits;
— annealing temperature applied in the thermal cycling program;
— master mix volume;
— primer and probe concentrations.
A practicable example of factors and modifications in the procedure conditions controlling all relevant
aspects of qualitative real-time PCR is given in Table 1. Other factors or modifications may be applied if the
gain of sufficient informative value is indicated by the addition.
[19]
Testing should be carried out in a multifactorial experimental design. The PCR reactions with the
different combinations of factors should be done with target DNA at a concentration around the number of
copies corresponding to the LOD multiplied by a factor of 3 (corresponding to approximately 20 to 60
95 %
copies per PCR). Dilute the target DNA in non-target DNA (background DNA), e.g. 20 ng/µl. For each factor-
level combination, PCR tests should at least be performed in triplicate. An example of the procedure is given
in Table D.1.
The method shall yield positive results for all combinations despite the modified conditions.
In the case of negative results, the PCR test for the corresponding combinations shall be repeated. In the
case of repeated negative results, the method is not sufficiently robust and shall be optimized.
Considerable deviations between Cq values can be an indication that the robustness of the method is
insufficient.
Tables 1 and 2 describe statistically proven examples for robustness testing. Other options can be applied
when they produce comparable statistically proven outcomes. If the single laboratory is the basis for a
collaborative study, the tests of several variables is strongly recommended, see Annex D.
Table 1 — Example of a robustness test of factors and modifications in the procedure
...