ISO/DTS 24910-1

• Abstract:

  This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of chickpea-specific DNA derived from food. PCR amplification reactions in which sufficient amounts of DNA are extracted from the relevant substrate can be used to detect the genetic material of chickpeas. The target sequence is a partial fragment of the chickpea chloroplast import apparatus 2-like gene in chromosome Ca7 (GenBank accession number CP039337.1). The provided PCR assay for this target has an absolute detection limit of 5 copies per reaction, with ≥ 95% reproducibility at this concentration (LOD95%).

• Status: Not 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 - Horizontal methods for molecular biomarker analysis
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 25-Sep-2026
• Completion Date: 25-Sep-2026

Overview

ISO/DTS 24910-1:2026, developed by ISO/TC 34/SC 16, specifies a standardized real-time polymerase chain reaction (PCR) method for the qualitative detection of chickpea-specific DNA in food and feed products. This international technical specification provides laboratories and food safety professionals with clear procedures for extracting, amplifying, and identifying chickpea-derived genetic material, supporting regulatory compliance, allergen management, and food authenticity verification. The method targets a partial fragment of the chickpea chloroplast import apparatus 2-like gene on chromosome Ca7, enabling sensitive and specific detection down to five copies per PCR reaction, with high reproducibility (LOD95%).

Key Topics

• Qualitative Chickpea DNA Detection
  The standard outlines sample preparation, DNA extraction, and real-time PCR setup for specifically identifying chickpea DNA, including guidance on controls and data interpretation.
• Food Safety and Allergen Control
  By enabling accurate identification of chickpea DNA, the method assists in detecting allergenic ingredients and undeclared or adulterated plant-based components in foods.
• Specificity and Sensitivity
  The protocol demonstrates high specificity for Cicer arietinum and closely related species, with no cross-reactivity to a wide range of other plant or animal ingredients. The detection limit of five copies per reaction ensures reliable performance even at low DNA concentrations.
• Validation and Reliability
  The method underwent internal validation for robustness, repeatability, specificity, and sensitivity, showing consistent results across different food matrices and real-time PCR platforms.

Applications

This standardized PCR method for chickpea DNA detection offers value in:

• Food Allergen Testing
  Detect trace chickpea-derived materials, assisting manufacturers and control labs in allergen management and regulatory labeling compliance.
• Food Authenticity Verification
  Confirm the presence or absence of chickpea ingredients, helping to prevent food fraud and ensure that products are labeled truthfully.
• Quality Control in Food Manufacturing
  Integrate as part of routine quality assurance programs for raw materials and finished products containing pulses and legumes.
• Import/Export and Regulatory Testing
  National authorities or customs agencies can use the method in the verification of imports, exports, or market surveillance for adherence to food safety regulations.
• Research and Product Development
  Support the development of novel chickpea-containing foods, dietary supplements, or functional ingredients, with molecular assurance of ingredient identity.

Related Standards

The following international standards provide supporting methods and vocabulary referenced within ISO/DTS 24910-1:

• ISO 16577 - Molecular biomarker analysis vocabulary for analytical methods in agriculture and food production.
• ISO 20688-1:2020 - Biotechnology - Requirements for production and quality control of synthesized oligonucleotides.
• ISO 20813 - General requirements and definitions for nucleic acid-based methods for detection of animal species in foods.
• ISO 21571 - Methods for the extraction of nucleic acids for analysis of genetically modified organisms and derived products.
• ISO 24276 - General requirements and definitions for the detection of genetically modified organisms and derived products.

Practical Value

Implementing ISO/DTS 24910-1:2026 in laboratory testing protocols enhances the reliability and comparability of chickpea DNA detection methods worldwide. This harmonization is crucial for addressing food allergen risks, ensuring product authenticity, and maintaining consumer trust in the global food supply. The clear, repeatable real-time PCR workflow supports regulatory authorities, industry laboratories, and quality assurance professionals in meeting rigorous food safety and labeling requirements.

Keywords: molecular biomarker analysis, chickpea DNA detection, real-time PCR, food authenticity, food allergen testing, ISO food standards, Cicer arietinum, plant-derived material, food safety, laboratory method.

FINAL DRAFT
Technical
Specification
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Detection of plant-derived materials
Voting begins on:
in food by real-time polymerase
2026-09-25
chain reaction (PCR) —
Voting terminates on:
2026-11-20
Part 1:
Chickpea DNA detection method
Analyse de biomarqueurs moléculaires — Détection de matières
d’origine végétale dans les aliments par polymérisation en chaîne
(PCR) en temps réel —
Partie 1: Méthode de détection de l’ADN de pois chiche
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
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
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.
Reference number
FINAL DRAFT
Technical
Specification
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Detection of plant-derived materials
Voting begins on:
in food by real-time polymerase
chain reaction (PCR) —
Voting terminates on:
Part 1:
Chickpea DNA detection method
Analyse de biomarqueurs moléculaires — Détection de matières
d’origine végétale dans les aliments par polymérisation en chaîne
(PCR) en temps réel —
Partie 1: Méthode de détection de l’ADN de pois chiche
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.
© ISO 2026
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Scientific basis . 2
5 Reagents and materials . 2
6 Apparatus . 3
7 Procedure . 3
7.1 Preparation of the test portion/sample .3
7.2 Preparation of DNA extracts .3
7.3 PCR setup.3
7.3.1 Reaction mixes .3
7.3.2 PCR controls .4
7.3.3 Real-time PCR thermocycler plate set-up.4
7.4 Temperature-time programme.4
8 Accept/reject criteria . 5
8.1 General .5
8.2 Identification .5
9 Validation status and performance criteria . 5
9.1 General .5
9.2 Internal validation.6
9.2.1 Robustness . .6
9.2.2 Sensitivity .6
9.2.3 Specificity .8
9.2.4 Repeatibilty .10
9.2.5 Actual sample measurement .10
9.3 Collaborative validation among laboratories .11
9.3.1 Reproducibility .11
9.3.2 Sensitivity . 12
10 Test report .13
Annex A (informative) BlastN 2.14.0 results for query of GenBank refseq-genomes (6 databases) . 14
Bibliography .16

iii
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 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
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.
A list of all parts in the ISO 24910 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
Introduction
Chickpeas are plant-based materials that contain allergens. Chickpea allergies involve reactions to proteins
such as vicilin, legumin subunits and PR-10, causing symptoms from hives to anaphylaxis. While rare,
chickpea allergies can be serious. Fraudulent adulteration of plant-derived ingredients in food can be
accidental or economically motivated. This document provides a real-time polymerase chain reaction PCR)
assay method for the identification of plant component species from nucleic acids present in food ingredients.
Plant-derived biological materials in food are detected and identified in the laboratory with the following
successive (or simultaneous) steps:
— preparation of the test portion/sample;
— nucleic acid extraction and purification;
— PCR amplification;
— interpretation of results.
This document provides guidance for the reporting of PCR amplification and chickpea DNA test results.

v
FINAL DRAFT Technical Specification ISO/DTS 24910-1:2026(en)
Molecular biomarker analysis — Detection of plant-derived
materials in food by real-time polymerase chain reaction
(PCR) —
Part 1:
Chickpea DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (PCR) method for the qualitative detection
of chickpea-specific DNA in food and feed. It includes extraction of an adequate amount of PCR-amplifiable
DNA from the relevant matrix.
This document is applicable to the detection of genetic material from chickpeas (Cicer arietinum L., Cicer
reticulatum, Cicer echinospermum, Cicer bijugum, Cicer judaicum, Cicer pinnatifidum, Cicer cuneatum, Cicer
chorassanicum, Cicer yamashitaend) and other closely related wild species.
The target sequence is a partial fragment of the chickpea chloroplast import apparatus 2-like gene in
[1]
chromosome Ca7 (GenBank accession number CP039337.1 ) which is present in a single copy per haploid
genome. The PCR assay provided for this target has an absolute limit of detection (LOD) of five copies per
reaction, with a ≥95 % confidence limit (LOD ).
95%
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 20688-1:2020, Biotechnology — Nucleic acid synthesis — Part 1: Requirements for the production and
quality control of synthesized oligonucleotides
ISO 20813, Molecular biomarker analysis — Methods of analysis for the detection and identification of animal
species in foods and food products (nucleic acid-based methods) — General requirements and definitions
ISO 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction
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 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/

4 Scientific basis
[2]
DNA is extracted from the test portion by applying a suitable method (see ISO 21571:2005 , Clause A.1).
The DNA analysis consists of the following two parts:
— verification of the quality and PCR amplifiability of the extracted DNA using a real-time PCR assay
[3]
targeting an endogenous marker reference gene for eukayotes (e.g. eukaryotic 18S rRNA );
NOTE The 18S rRNA gene is a highly conserved, foundational component of eukaryotic ribosomes, acting as a
crucial, universally applicable internal control for qPCR, gene expression normalization and phylogenetic studies.
A highly conserved sequence of the 18S rRNA gene was chosen as the quality and PCR amplifiability control.
— detection of the chickpea-species-specific DNA sequences of the chloroplast import apparatus 2-like
gene in chromosome Ca7 (GenBank accession number CP039337.1) in a real-time PCR.
5 Reagents and materials
For this document, only chemicals and water of recognized analytical grade, appropriate for molecular
biology, shall be used. Unless stated otherwise, solutions should be prepared by dissolving the corresponding
reagents in water followed by autoclave sterilization. For all operations in which gloves are used, gloves shall
be powder free. Aerosol-protected pipette tips should be used (protection against cross-contamination).
5.1 PCR master mix, a pre-mixed, concentrated solution (2X) containing thermo-stable (heat resistant)
DNA polymerase, KCl, MgCl , four dNTPs (dATP, dCTP, dGTP, dTTP) and buffer excluding primers, probes
and template that is ready to use.
5.2 Oligonucleotides.
The quality of the oligonucleotides shall be sufficient for their use as primers and probes in accordance with
ISO 20688-1:2020. See Table 1.
Table 1 — Oligonucleotides
Name DNA sequence of the oligonucleotide Final concentration in
PCR
a
Chloroplast import apparatus 2-like gene as the target sequence (GenBank accession number CP039337.1)
Chickpea-90bp-F 5ʹ- GCGAACGGTGTTCCTTTATCA -3ʹ 400 nmol/l
Chickpea-90bp-R 5ʹ- GGAATTGCCAAAAAGGAAAACC -3ʹ 400 nmol/l
b
Chickpea-90bp-P 5ʹ-[FAM]-ACCAAGCATTCCGAACGCCTTCG-[BHQ] -3′ 200 nmol/l
Primers targeting the Phaseoleae environmental sample clone Elev 18S 1274 18S ribosomal RNA gene (GenBank acces-
c
sion: EF024772.1 )
18S-64bp-F 5ʹ- CCTGAGAAACGGCTACCA -3ʹ 400 nmol/l
18S-64bp-R 5ʹ- CGTGTCAGGATTGGGTAAT -3ʹ 400 nmol/l
d
18S-64bp-P 5ʹ-[FAM] - TGCGCGCCTGCTGCCTTCCT - [BHQ1] -3ʹ 400 nmol/l
a
PCR product = 2 586 220 - GCGAACGGTG TTCCTTTATC AGACCAAGCA TTCCGAACGC CTTCGTAATT CAATTTCAAC
AACAAACCGG TTTTCCTTTT TGGCAATTCC - 2 586 309 - CP039337.1.
b
FAM: 6-carboxyfluorescein, BHQ: non-fluorescent quencher minor groove binder.
c
PCR product = 394 - CCTGAGAAAC GGCTACCACA TCCAAGGAAG GCAGCAGGCG CGCAAATTAC CCAATCCTAA CACG – 457 -
EF024772.1.
d
FAM: 6-carboxyfluorescein, BHQ1: non-fluorescent quencher minor groove binder.
NOTE 1 Chickpea-90bp-F is base pairs 2 586 220 - 2 586 240, Chickpea-90bp-R is base pairs 2 586 287 - 2 586 309 and
Chickpea-90bp-P is 2 586 242 - 2 586 264 of CP039337.1 chickpea chloroplast import apparatus 2-like gene in chromosome Ca7.
Equivalent reporter dyes and/or quencher dyes can be used if they yield the same or better results.
NOTE 2 18S-F is 394-411 is base pairs of the EF024772.1 Phaseoleae environmental sample clone Elev_18S_1274 18S ribosomal
RNA gene on GenBank, 18S-R is 439-457 base pairs and 18S-P is 418-437 base pairs.

6 Apparatus
Requirements concerning apparatus and materials shall follow ISO 20813 and ISO 24276 . In addition to the
usual laboratory equipment, the following equipment shall be used.
6.1 Real-time thermocycler instrument, which amplifies DNA in vitro and performs the temperature-
time cycles for PCR. In addition, the device shall be capable of exciting fluorescence molecules at specific
wavelengths and detecting sufficient emitted fluorescent light of the fluorophore used to perform TaqMan
format assays.
7 Procedure
7.1 Preparation of the test portion/sample
The test sample used for DNA extraction shall be representative of the laboratory sample and homogeneous
(e.g. by grinding or homogenizing the laboratory sample to a fine mixture). Test portion/sample preparation
shall follow the general requirements and specific methods in accordance with ISO 21571 and ISO 20813.
7.2 Preparation of DNA extracts
The extraction/purification and quantification of DNA from the test portion shall follow the general
requirements and methods provided in ISO 21571. DNA extraction methods in accordance with
[2]
ISO 21571:2005 , Annex A, should be used.
7.3 PCR setup
7.3.1 Reaction mixes
The method is for a total volume of 25 μl per PCR. The reaction setup is given in Table 2. Reagents shall
be completely thawed at room temperature. Each reagent shall be carefully mixed and briefly centrifuged
immediately before pipetting. A PCR reagent mixture is prepared to contain all components except for the
sample DNA. The required total amount of the PCR reagent mixture prepared depends on the number of
reactions to be performed, including at least one additional reaction as a pipetting reserve. The number of
sample and control replicates shall follow ISO 20813. Set up the PCR tests as follows:
a) mix the PCR reagent mixture, centrifuge briefly and pipette 23 μl into each reaction vial;
b) add 2 μl of each sample DNA (20 ng/μl to 200 ng/μl) or positive DNA target control or extraction blank
control or water to the respective reaction vials;
c) mix and centrifuge briefly.

Table 2 — Reaction setup for the amplification
Parameter Value
µl
Total reaction volumes 25
Sample DNA (20 ng/μl to 200 ng/μl) or controls 2
a
2X PCR master mix 12,5
b
Primer chickpea - 90 bp - F, c=10 μmol/l 1,0
b
Primer chickpea - 90 bp - R, c=10 μmol/l 1,0
b
Probe chickpea - 90 bp - P, c=10 μmol/l 1,0
ddH 0 to 25
a
In the collaborative trial, a ready-to-use optimized 2X PCR master mix containing all of the components, excluding the
template and primers, was used. The 2X PCR reaction mixture contains a mixture of thermostable (heat-resistant) DNA
polymerase, a blend of dNTPs with dUTP and uracil-UDG to minimize carry-over PCR contamination and a passive internal
reference based on ROX dye. Equivalent products can be used if they yield the same or better results. The total reaction volume
of the real-time fluorescence PCR is 25 μl. Reagent quantity and the temperature-time programme can be adjusted according to
different manufacturers’ reagents and real-time fluorescence PCR instruments.
b
The primer and probe concentrations for 18S ribosomal RNA were consistent with those used for chickpea.
7.3.2 PCR controls
7.3.2.1 General
PCR controls shall be in accordance with ISO 24276 and ISO 20813.
7.3.2.2 Inhibition control (reference gene assay)
A reference control gene (e.g. 18S rRNA gene for eukaryotes) PCR assay using sample DNAs shall be
performed to test nucleic acid amplifiabili
...