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

ISO/DTS 24910-1 is an ISO Technical Specification for a real-time PCR method to qualitatively detect chickpea-specific DNA in food and feed. It specifies the target sequence, sample preparation, PCR setup, result interpretation, validation, and reporting for laboratories checking whether chickpea-derived material is present. The method targets a partial fragment of the chickpea chloroplast import apparatus 2-like gene on chromosome Ca7 and reports an absolute detection limit of five copies per reaction with LOD95%.

What does ISO/DTS 24910-1 specify?

ISO/DTS 24910-1 specifies a laboratory method for detecting chickpea DNA by real-time polymerase chain reaction (PCR). It is built for extracted, PCR-amplifiable DNA from food and feed matrices, and it is aimed at qualitative detection rather than quantification.

The document is organized into Clauses 1 to 10, plus one informative annex and a bibliography. The flow is practical: scope, references, terms, scientific basis, reagents and materials, apparatus, procedure, accept/reject criteria, validation, and the test report. That structure helps a laboratory move from sample preparation through to a documented result.

Annex	What it covers
Annex A	BLASTN results for the 90-base query against GenBank refseq-genomes, used to support specificity claims

What are the key requirements of ISO/DTS 24910-1?

Sample handling and DNA extraction

Clause 7 requires a representative, homogeneous test portion and DNA extraction that yields enough PCR-amplifiable DNA. In practice, the sample needs to be prepared so that the DNA extract reflects the laboratory sample, because a poor or uneven subsample can give a misleading negative result.

Clause 4 ties the method to a two-step logic: first verify extract quality and amplifiability with an endogenous marker, then test for chickpea-specific DNA. That matters because a negative chickpea signal is only meaningful if the DNA itself can still amplify.

Target, reagents, and instrument capability

Clause 5 specifies analytical-grade reagents, powder-free gloves, and aerosol-protected tips, and it defines the primer and probe set for the chickpea target. The method depends on contamination control and on oligonucleotides that match the stated target sequence.

Clause 6 requires a real-time thermocycler instrument that can excite and detect fluorescence for a TaqMan-format assay. A basic PCR machine is not enough, because the result depends on fluorescence monitoring during cycling.

PCR setup and controls

Clause 7 sets out a fixed reaction setup and requires the sample DNA, a positive DNA target control, an extraction blank control, and a reagent control. It also uses an endogenous reference gene assay, such as 18S rRNA, as an inhibition or amplifiability control.

In practice, these controls tell the operator whether a result is real or whether the run is compromised by contamination, inhibition, or failed extraction. If the reference gene does not amplify as expected, a negative chickpea result cannot be treated as reliable.

Cycling conditions and verification of changes

Clause 7 includes a defined temperature-time programme that was used in validation. The document also says that if a laboratory changes reagents, reaction conditions, or instrument settings, those changes need to be verified.

That is important for routine work because laboratories often use different thermocyclers or commercial master mixes. The method can still be used, but the laboratory has to show that the modified setup performs comparably.

How to call a result

Clause 8 says a positive result needs a sigmoid amplification curve and a calculable Ct or Cq value. The no-template controls must show no amplification, while the positive target control and internal reference control must behave as expected.

Trace detections and contradictory results trigger re-testing from new extracts. The practical meaning is that a borderline or inconsistent result is not accepted at face value; the sample is re-extracted and re-amplified until the detection outcome is clear.

Validation and performance

Clause 9 reports internal validation for robustness, sensitivity, specificity, repeatability, and actual sample measurement, followed by collaborative laboratory validation. The stated performance includes an absolute detection limit of five copies per reaction and specificity checks against chickpea relatives, other plants, fungi, and animal materials.

For users, this section shows how the method behaved during validation and what type of sample evidence supports its use. It is especially relevant for laboratories deciding whether the method is fit for routine screening.

Test report

Clause 10 requires a report that identifies the sample, the standard used, the method, the result with its calculation clause, any deviations, unusual observations, and the test date. That gives buyers, quality managers, and regulators a traceable record of how the result was obtained.

What terms does ISO/DTS 24910-1 define?

ISO/DTS 24910-1 relies on the vocabulary in ISO 16577 and uses several core PCR terms in a specific way.

• Real-time PCR - PCR in which fluorescence is measured during amplification so the target DNA can be detected as the reaction runs.
• Ct / Cq - the cycle at which the fluorescence signal crosses the threshold; it is the main number used to express a positive amplification.
• LOD95% - the detection level at which the assay detects the target with at least 95% reproducibility.
• Endogenous reference gene - a natural sample gene, such as eukaryotic 18S rRNA, used to show that the extract can amplify.
• Extraction blank control - a control taken through extraction without sample DNA, used to check for contamination introduced during preparation.
• PCR reagent control - a no-template control used to check that reagents and setup do not produce false amplification.
• Sigmoid-shaped amplification curve - the characteristic real-time PCR curve that indicates successful target amplification.

Who uses ISO/DTS 24910-1?

ISO/DTS 24910-1 is used by laboratories that test food and feed for chickpea presence. Typical users include molecular biology and analytical laboratories, quality assurance teams, and product authentication staff.

It is relevant where chickpea ingredients need to be confirmed, screened, or investigated in the context of allergen management, ingredient verification, and fraud or adulteration checks. It also supports buyers and specifiers who need a defined DNA-based method for checking claims about plant-derived ingredients.

Which standards are used with ISO/DTS 24910-1?

Standard	What it contributes
ISO 16577	Vocabulary for molecular biomarker analytical methods in agriculture and food production
ISO 20688-1:2020	Quality requirements for synthesized oligonucleotides used as primers and probes
ISO 20813	General requirements and definitions for nucleic-acid-based methods for animal species in foods and food products
ISO 21571	Nucleic acid extraction for food and related samples
ISO 24276	General requirements and definitions for GMO-related analytical methods and PCR controls

These references provide the shared rules for extraction, oligonucleotide quality, terminology, and PCR method requirements that ISO/DTS 24910-1 depends on.

What does the ISO/DTS 24910-1 document contain?

The document contains the target definition, reagent list, apparatus requirements, PCR setup, acceptance criteria, validation data, and reporting instructions. It also includes tables for the oligonucleotides, reaction setup, cycling programme, specificity outcomes, sample measurements, and collaborative validation summaries.

Figures show the validation plasmid map and the inserted target sequence used in sensitivity work. Annex A provides BLASTN search results that support the specificity of the 90-base chickpea target sequence.

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
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
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INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
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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
...