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
Current Stage
6060 - International Standard published
Start Date
05-Oct-2022
Due Date
17-Mar-2024
Completion Date
05-Oct-2022
Ref Project

Buy Standard

Technical specification
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 Released:5. 10. 2022
English language
14 pages
sale 15% off
Preview
sale 15% off
Preview
Draft
REDLINE ISO/DTS 20224-8 - Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 8: Turkey DNA detection method Released:5. 07. 2022
English language
14 pages
sale 15% off
Preview
sale 15% off
Preview
Draft
ISO/DTS 20224-8 - Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 8: Turkey DNA detection method Released:5. 07. 2022
English language
14 pages
sale 15% off
Preview
sale 15% off
Preview

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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
© 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
---------------------- Page: 3 ----------------------
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.
© 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.

© 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/
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
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

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

NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
Turkey-118bp-P 5′-[FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l

PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT

GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.

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.
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
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.
© ISO 2022 – All rights reserved
---------------------- Page: 8 ----------------------
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: copyright@iso.org
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

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

NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
Turkey-118bp-P 5′- [FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l

PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT

GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.

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
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

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
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

IMPORTANT — Please use this updated version dated 2022-07-06, and

the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below

discard any previous version of this DTS. Ballot dates have been
or ISO’s member body in the country of the requester.
modified to align with ISO/DTS 20224-9.
RECIPIENTS OF THIS DRAFT ARE INVITED TO
ISO copyright office
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
CP 401 • Ch. de Blandonnet 8
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
CH-1214 Vernier, Geneva
DOCUMENTATION.
Phone: +41 22 749 01 11
IN ADDITION TO THEIR EVALUATION AS
Reference number
Email: copyright@iso.org
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTS 20224-8:2022(E)
Website: www.iso.org
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
Published in Switzerland
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
© ISO 2022 – All rights reserved
NATIONAL REGULATIONS. © ISO 2022
---------------------- Page: 2 ----------------------
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

iii
© ISO 2022 – All rights reserved
---------------------- Page: 3 ----------------------
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.
© ISO 2022 – All rights reserved
---------------------- Page: 4 ----------------------
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.

© ISO 2022 – All rights reserved
---------------------- Page: 5 ----------------------
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/
© ISO 2022 – All rights reserved
---------------------- Page: 6 ----------------------
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

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

NC_015041.2)
Turkey-118bp-F 5′-TGAACAAATCCACTTCCCTTTAACC-3′ 400 nmol/l
Turkey-118bp-R 5′-TCATTTCTGCTGCACAAAGTGAG-3′ 400 nmol/l
Turkey-118bp-P 5′-[FAM]-TGATGACGAGCCGCAGCCACACC-[TAMRA] -3′ 200 nmol/l

PCR product = 57 643 369 - TGAACAAATC CACTTCCCTT TAACCTCAGG AACATCCAGC ATATTGGTAA ACAGAGGGAT

GTGGGGGTGT GGCTGCGGCT CGTCATCACC TGCAGCTCAC TTTGTGCAGC AGAAATGA - 57 643 486 - NC_015041.2.

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.
© ISO 2022 – All rights reserved
---------------------- Page: 7 ----------------------
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
...

Questions, Comments and Discussion

Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.