ISO/TS 20224-1:2020

TECHNICAL ISO/TS
SPECIFICATION 20224-1
First edition
2020-07
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 1:
Bovine DNA detection method
Analyse de biomarqueurs moléculaires — Détection de matériaux
d'origine animale dans les denrées alimentaires et les aliments pour
animaux par PCR en temps réel —
Partie 1: Méthode de détection de l'ADN bovin
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

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
5.1 General . 2
5.2 PCR reagents . 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 . 4
8.1 General . 4
8.2 Identification . 5
9 Validation status and performance criteria . 5
9.1 General . 5
9.2 Robustness . 5
9.3 Reproducibility . 6
9.4 Sensitivity . 6
9.5 Specificity . 9
10 Test report .11
Annex A (informative) BlastN 2.9.0 results for query of GenBank refseq_genomes (6 databases) .12
Bibliography .16
Foreword
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electrotechnical standardization.
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different types of ISO documents should be noted. This document was drafted in accordance with the
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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 20224 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
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iv © ISO 2020 – All rights reserved

Introduction
Fraudulent adulteration of meat in food and feed threatens both public safety and commerce.
Adulteration can affect those adhering to ethnological dietary rules, economic development and social
stability. This document provides a real-time polymerase chain reaction (real-time PCR) analytical
method for the identification of meat animal species from nucleic acid present in the ingredients of food
and feed.
Animal-derived biological materials in food and feed 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 and interpretation of results. This document provides
guidance for PCR amplification and interpretation of results, specific to bovine DNA detection.
The ISO 20224 series consists of technical specifications that describe specific applications. New
species DNA detection methods established in the future will be added as independent parts.
TECHNICAL SPECIFICATION ISO/TS 20224-1:2020(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-
time PCR —
Part 1:
Bovine DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the
qualitative detection of bovine-specific DNA derived from food and feed. It requires the extraction of an
adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection
of bovine material derived from cattle including taurine (Bos taurus) and zebu (Bos indicus). The assay
also detects the species bison (Bison bison) and yak (Bos mutus).
The target sequence is a partial fragment of the bovine nuclear beta actin gene in chromosome 25 (i.e.
[1][2][3]
GenBank accession number NC_037352.1) , which is present as a single copy per haploid genome.
The provided PCR assay for this target has an absolute limit of detection of five copies per reaction,
with ≥ 95 % replicability at this concentration (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 — Terms and definitions
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 terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
4 Scientific basis
DNA is extracted from the test portion by applying a suitable method (see ISO 21571:2005, A.1). The
DNA analysis consists of two parts:
— verification of the quality and amplifiability of the extracted DNA using a PCR assay specific for
eukaryotes (e.g. 18S rRNA gene) or mammals and poultry (e.g. myostatin gene);
— detection of the bovine species-specific DNA sequence of the single-copy beta actin gene in
chromosome 25 (e.g. GenBank accession number NC_037352.1) in a real-time PCR.
NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several
hundred to several thousand, while the beta actin gene in the bovine genome and myostatin gene in mammals
and poultry genome are single copy. The copy number of the beta actin gene in the bovine genome was confirmed
by bioinformatics analysis at the whole genome scale (see Annex A) and digital PCR for absolute quantification.
5 Reagents and materials
5.1 General
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. The use of aerosol protected pipette tips (protection against
cross-contamination) is recommended.
5.2 PCR reagents
5.2.1 PCR master mix.
PCR master mix contains thermostable DNA polymerase, pH buffer, KCl, MgCl , uracil-DNA glycosylase
(UDG) and the four dNTPS (dATP, dGTP, dUTP, dCTP) as a dilutable concentrate, which is ready to use.
5.2.2 Oligonucleotides.
The quality of the oligonucleotides shall be sufficient for their use as primers and probes. See Table 1.
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR
a
Bovine beta actin gene as the target sequence (GenBank accession number NC_037352.1)
Bovine-62bp-F 5′-GGCCTCGGAGTGTGTATTCAG-3′ 400 nmol/l
Bovine-62bp-R 5′-GCCCCAGAATGAGGTTCACTT-3′ 400 nmol/l
b
Bovine-62bp-P 5′-[FAM]-AGGTGCACAGTACGTTC-[NFQ-MGB] -3′ 200 nmol/l
a
PCR product = 38 800 878 - GGCCTCGGAG TGTGTATTCA GTAGGTGCAC AGTACGTTCT GAAGTGAACC TCATTCTGGG
GC-38 800 939 - NC_037352.1.
b
FAM: 6-carboxyfluorescein, NFQ-MGB: non-fluorescent quencher minor groove binder.
Bovine-62bp-F is base pairs 38 800 878-38 800 898, Bovine-62bp-R is base pairs 38 800 919-
38 800 939 and Bovine-62bp-P is 38 800 900-38 800 916 of NC_037352.1 bovine nuclear beta actin
gene in chromosome 25. Equivalent reporter dyes and/or quencher dyes can be used if they yield the
same or better results.
2 © ISO 2020 – All rights reserved

6 Apparatus
Requirements concerning apparatus and materials shall follow ISO 20813. In addition to the usual
laboratory equipment, the following equipment is required.
6.1 Real-time thermocycler instrument.
A device that amplifies DNA in vitro and performs the temperature-time cycles is needed for PCR.
Additionally, 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 described in 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 described in
ISO 21571:2005, Annex A, are recommended.
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 20 µl into each reaction vial;
b) add 5 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
Total reaction volume 25 µl
Sample DNA (20 ng/µl to 200 ng/µl) or controls 5 µl
a
2 × PCR master mix 12,5 µl
Primer Bovine-62bp-F, c = 10 μmol/l and Bovine-62bp-R, c = 10 μmol/l 1,0 µl for each
Probe Bovine-62bp-P, c = 10 μmol/l 0,5 µl
Water to 25 µl
a
In the collaborative trial, a ready-to-use optimized 2 × PCR master mix containing all of the components, excluding
the template and primers, was used. The 2 × PCR master mix contains thermostable 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. If necessary, the amounts of the reagents and the
temperature-time programme can be adapted.
7.3.2 PCR controls
7.3.2.1 General
PCR controls shall be as described in 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, myostatin gene for mammals and poultry)
PCR assay using sample DNAs shall be performed to test nucleic acid amplifiability and provide control
to exclude false-negative results.
7.3.3 Real-time PCR thermocycler plate set-up
Transfer
...