ISO/TS 20224-8:2022

Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 8: Turkey DNA detection method

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of turkey-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 turkey material derived from domestic turkey (Meleagris gallopavo) and wild turkey (Meleagris ocellata). The target sequence is a partial fragment of the Meleagris gallopavo chromosome Z DNA sequence (i.e. GenBank accession number NC_015041.2), 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 % confidence at this concentration (LOD95 %).

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 8: Méthode de détection de l'ADN de dinde

General Information

• Status: Published
• Publication Date: 04-Oct-2022
• 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: 6060 - International Standard published
• Start Date: 05-Oct-2022
• Due Date: 17-Mar-2024
• Completion Date: 05-Oct-2022

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 20224-8
First edition
2022-10
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 8:
Turkey 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 8: Méthode de détection de l'ADN de dinde
Reference number
ISO/TS 20224-8:2022(E)
© ISO 2022

---------------------- Page: 1 ----------------------
ISO/TS 20224-8:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
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
ii
  © ISO 2022 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/TS 20224-8:2022(E)
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 +2.12.0 results for query of GenBank RefSeq Genome (refseq_
genomes) and Whole-genome Shotgun Contigs (wgs) .12
Bibliography .14
iii
© ISO 2022 – All rights reserved

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ISO/TS 20224-8:2022(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 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 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.
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
complete listing of these bodies can be found at www.iso.org/members.html.
iv
  © ISO 2022 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/TS 20224-8:2022(E)
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 the genus Meleagris which
encompasses both wild and domestic breeds of turkey.
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.
v
© ISO 2022 – All rights reserved

---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 20224-8:2022(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-time
PCR —
Part 8:
Turkey DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the
qualitative detection of turkey-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 turkey material derived from domestic turkey (Meleagris gallopavo) and wild turkey (Meleagris
ocellata).
The target sequence is a partial fragment of the Meleagris gallopavo chromosome Z DNA sequence (i.e.
[1]
GenBank accession number NC_015041.2) , 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 % confidence 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 — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
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/
1
© ISO 2022 – All rights reserved

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ISO/TS 20224-8:2022(E)
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 (i.e. 18S rRNA gene) or mammals and poultry (i.e. myostatin gene);
— detection of the turkey species-specific DNA sequence of the single-copy turkey chromosome Z DNA
sequence (i.e. GenBank accession number NC_015041.2) 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 specific target sequence in the turkey genome and myostatin gene in
mammals and poultry genome are single copy. The copy number of the specific target sequence in the turkey
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 reagents 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.
In general, real-time PCR master mix contains thermostable DNA polymerase, dNTPs, MgCl , KCl and
2
buffer as a dilutable concentrated mixture, that is ready to use.
NOTE Commercial real-time PCR master mix can be used.
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
Specific sequence in Meleagris gallopavo chromosome Z DNA sequence (i.e. GenBank accession number
a
NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
b
Turkey-118bp-P 5′-[FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l
a
PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT
GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.
b
FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.
Turkey-118bp-F is base pairs 57 643 369 – 57 643 573, Turkey-118bp-R is base pairs 57 643 464 – 57 643
486 and Turkey-118bp-P is 57 643 434 – 57 643 456 of NC_015041.2, Meleagris gallopavo chromosome
Z DNA sequence. Equivalent reporter dyes and/or quencher dyes can be used if they yield the same or
better results.
2
  © ISO 2022 – All rights reserved

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ISO/TS 20224-8:2022(E)
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 as 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.
3
© ISO 2022 – All rights reserved

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ISO/TS 20224-8:2022(E)
Table 2 — Reaction setup for the ampl
...

© ISO 2022 – All rights reserved
ISO/DTS PRF TS 20224-8:20212022(E)
Date: 2022-06-21
ISO TC 03434/SC 16/WG 8
Secretariat: ANSI
Molecular biomarker analysis — Detection of animal-derived
materials in foodstuffs and feedstuffs by real-time PCR —
Part 8: Turkey 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 8: Méthode de
détection de l'ADN de dinde

---------------------- Page: 1 ----------------------
ISO/PRF TS 20224-8:2022(E)
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of
this publication may be reproduced or utilized otherwise in any form or by any means, electronic or
mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO at the address below or ISO’s member body in the
country of the requester.
ISO Copyright Office
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: [e-mail]
Website: www.iso.org
Published in Switzerland.
ii © ISO 2022 – All rights reserved

---------------------- Page: 2 ----------------------
ISO/PRF TS 20224-8:2022(E)
Contents
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
5.2.1 PCR master mix. . 2
5.2.2 Oligonucleotides. . 2
6 Apparatus . 3
6.1 Real-time thermocycler instrument. . 3
7 Procedure . 3
7.1 Preparation of the test portion/sample . 3
7.2 Preparation of DNA extracts . 3
7.3 PCR setup . 4
7.3.1 Reaction mixes . 4
7.3.2 PCR controls . 4
7.3.3 Real-time PCR thermocycler plate set-up . 4
7.4 Temperature-time programme . 5
8 Accept/reject criteria . 5
8.1 General . 5
8.2 Identification . 5
9 Validation status and performance criteria . 6
9.1 General . 6
9.2 Robustness . 6
9.3 Reproducibility . 6
9.4 Sensitivity . 7
9.5 Specificity . 10
10 Test report . 12
Annex A (informative) BlastN +2.12.0 results for query of GenBank RefSeq Genome
(refseq_genomes) and Whole-genome Shotgun Contigs (wgs) . 13
Bibliography . 15
iii © ISO 2022 – All rights reserved

---------------------- Page: 3 ----------------------
ISO/PRF TS 20224-8:2022(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 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 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.
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
complete listing of these bodies can be found at www.iso.org/members.html.
iv © ISO 2022 – All rights reserved

---------------------- Page: 4 ----------------------
ISO/PRF TS 20224-8:2022(E)
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 the genus Meleagris which
encompasses both wild and domestic breeds of turkey.
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.
v © ISO 2022 – All rights reserved

---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/PRF TS 20224-8:2022(E)

Molecular biomarker analysis — Detection of animal-derived
materials in foodstuffs and feedstuffs by real-time PCR —
Part 8: Turkey DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative
detection of turkey-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 turkey
material derived from domestic turkey (Meleagris gallopavo) and wild turkey (Meleagris ocellata).
The target sequence is a partial fragment of the Meleagris gallopavo chromosome Z DNA sequence (i.e.
[1]
GenBank accession number NC_015041.2) , 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 % confidence 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 16393, Molecular biomarker analysis — Determination of the performance characteristics of
qualitative measurement methods and validation of methods
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
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 terminologicalterminology databases for use in standardization at the following
addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
© ISO 2022 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO/PRF TS 20224-8:2022(E)
— IEC Electropedia: available at https://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 (i.e. 18S rRNA gene) or mammals and poultry (i.e. myostatin gene);
— detection of the turkey species-specific DNA sequence of the single-copy turkey chromosome Z DNA
sequence (i.e. GenBank accession number NC_015041.2) 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 specific target sequence in the turkey genome and myostatin gene in
mammals and poultry genome are single copy. The copy number of the specific target sequence in the turkey
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 reagents 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.
In general, real-time PCR master mix contains thermostable DNA polymerase, dNTPs, MgCl , KCl, and
2
buffer as a dilutable concentrated mixture, that is ready to use.
NOTE Commercial real-time PCR master mix can be used.
5.2.2 Oligonucleotides.
The quality of the oligonucleotides shall be sufficient for their use as primers and probes. See Table 1.

2 © ISO 2022 – All rights reserved

---------------------- Page: 7 ----------------------
ISO/PRF TS 20224-8:2022(E)
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR
Specific sequence in Meleagris gallopavo chromosome Z DNA sequence (i.e. GenBank accession number
a
NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
b
Turkey-118bp-P 5′- [FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l
a
  PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT
GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.
b
  FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.

Turkey-118bp-F is base pairs 57 643 369 – 57 643 573, Turkey-118bp-R is base pairs 57 643 464 – 57
643 486 and Turkey-118bp-P is 57 643 434 – 57 643 456 of NC_015041.2, Meleagris gallopavo
chromosome Z DNA sequence. Equivalent reporter dyes and/or quencher dyes can be used if they yield
the same or better results.
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 as 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.
© ISO 2022 – All rights reserved 3

---------------------- Page: 8 ----------------------
ISO/PRF TS 20224-8:2022(E)
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 Turkey-118bp-F, c = 10 μmol/l and Turkey-118bp-R, c = 10 μmol/l 1,0 µl for each
Probe Turkey-118bp-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-ove
...

FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 20224-8
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Detection of animal-derived materials
Voting begins on:
2022-07-20 in foodstuffs and feedstuffs by real-
time PCR —
Voting terminates on:
2022-09-14
Part 8:
Turkey 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 8: Méthode de détection de l'ADN de dinde
IMPORTANT — Please use this updated version dated 2022-07-06, and
discard any previous version of this DTS. Ballot dates have been
modified to align with ISO/DTS 20224-9.
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/DTS 20224-8:2022(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 2022

---------------------- Page: 1 ----------------------
ISO/DTS 20224-8:2022(E)
FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 20224-8
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Detection of animal-derived materials
Voting begins on:
2022-07-20 in foodstuffs and feedstuffs by real-
time PCR —
Voting terminates on:
2022-09-14
Part 8:
Turkey 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 8: Méthode de détection de l'ADN de dinde
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NATIONAL REGULATIONS. © ISO 2022

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ISO/DTS 20224-8:2022(E)
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 +2.12.0 results for query of GenBank RefSeq Genome (refseq_
genomes) and Whole-genome Shotgun Contigs (wgs) .12
Bibliography .14
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ISO/DTS 20224-8:2022(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 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 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.
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
complete listing of these bodies can be found at www.iso.org/members.html.
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ISO/DTS 20224-8:2022(E)
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 the genus Meleagris which
encompasses both wild and domestic breeds of turkey.
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.
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TECHNICAL SPECIFICATION ISO/DTS 20224-8:2022(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-time
PCR —
Part 8:
Turkey DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the
qualitative detection of turkey-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 turkey material derived from domestic turkey (Meleagris gallopavo) and wild turkey (Meleagris
ocellata).
The target sequence is a partial fragment of the Meleagris gallopavo chromosome Z DNA sequence (i.e.
[1]
GenBank accession number NC_015041.2) , 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 % confidence 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 — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
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/
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ISO/DTS 20224-8:2022(E)
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 (i.e. 18S rRNA gene) or mammals and poultry (i.e. myostatin gene);
— detection of the turkey species-specific DNA sequence of the single-copy turkey chromosome Z DNA
sequence (i.e. GenBank accession number NC_015041.2) 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 specific target sequence in the turkey genome and myostatin gene in
mammals and poultry genome are single copy. The copy number of the specific target sequence in the turkey
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 reagents 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.
In general, real-time PCR master mix contains thermostable DNA polymerase, dNTPs, MgCl , KCl and
2
buffer as a dilutable concentrated mixture, that is ready to use.
NOTE Commercial real-time PCR master mix can be used.
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
Specific sequence in Meleagris gallopavo chromosome Z DNA sequence (i.e. GenBank accession number
a
NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
b
Turkey-118bp-P 5′-[FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l
a
PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT
GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.
b
FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.
Turkey-118bp-F is base pairs 57 643 369 – 57 643 573, Turkey-118bp-R is base pairs 57 643 464 – 57 643
486 and Turkey-118bp-P is 57 643 434 – 57 643 456 of NC_015041.2, Meleagris gallopavo chromosome
Z DNA sequence. Equivalent reporter dyes and/or quencher dyes can be used if they yield the same or
better results.
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ISO/DTS 20224-8:2022(E)
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 as 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
centr
...