Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 6: Horse DNA detection method

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of horse-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 horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus ♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The assay also detects the species Przewalski's horse (Equus przewalskii) and zebra (Equus burchellii). The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (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 6: Méthode de détection de l'ADN de cheval

General Information

Status

Published

Publication Date

30-Jul-2020

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/WG 8 - Meat speciation

Current Stage

6060 - International Standard published

Start Date

31-Jul-2020

Due Date

21-May-2021

Completion Date

31-Jul-2020

Ref Project

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ISO/TS 20224-6:2020 - Molecular biomarker analysis -- Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR

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TECHNICAL ISO/TS
SPECIFICATION 20224-6
First edition
2020-07
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse 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 6: Méthode de détection de l'ADN de cheval
Reference number
ISO/TS 20224-6:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO/TS 20224-6:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 20224-6:2020(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 2.9.0 results for query of GenBank refseq_genomes (452

databases) ................................................................................................................................................................................................................12

Bibliography .............................................................................................................................................................................................................................15

© ISO 2020 – All rights reserved iii
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ISO/TS 20224-6:2020(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 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/TS 20224-6:2020(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 horse DNA detection.

The ISO 20224 series consists of technical specifications that describe specific applications. New

species DNA detection methods established in the future will be added as independent parts.

© ISO 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
TECHNICAL SPECIFICATION ISO/TS 20224-6:2020(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse DNA detection method
1 Scope

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the

qualitative detection of horse-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 horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus

♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The

assay also detects the species Przewalski’s horse (Equus przewalskii) and zebra (Equus burchellii).

The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28,

[1]

EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3) , which

is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute

limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD ).

95 %
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 16577, Molecular biomarker analysis — Terms and definitions

ISO 20813, Molecular biomarker analysis — Methods of analysis for the detection and identification

of animal species in foods and food products (nucleic acid-based methods) — General requirements and

definitions

ISO 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — Nucleic acid extraction

ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
© ISO 2020 – All rights reserved 1
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ISO/TS 20224-6:2020(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 (e.g. 18S rRNA gene) or mammals and poultry (e.g. myostatin gene);

— detection of the horse species-specific DNA sequence of the single-copy Equus caballus isolate

Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence.

(GenBank accession number NC_009171.3) 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 horse genome and myostatin gene in

mammals and poultry genome are single copy. The copy number of the specific target sequence in the horse

genome was confirmed by bioinformatics analysis at the whole genome scale (see Annex A) and digital PCR for

absolute quantification.
5 Reagents and materials
5.1 General

For this document, only chemicals and water of recognized analytical grade, appropriate for molecular

biology, shall be used. Unless stated otherwise, solutions should be prepared by dissolving the

corresponding reagents in water followed by autoclave sterilization. For all operations in which gloves

are used, gloves shall be powder free. The use of aerosol protected pipette tips (protection against

cross-contamination) is recommended.
5.2 PCR reagents
5.2.1 PCR master mix.

PCR master mix contains thermostable DNA polymerase, pH buffer, KCl, MgCl , uracil-DNA glycosylase

(UDG) and the four dNTPS (dATP, dGTP, dUTP, dCTP) as a dilutable concentrate, which is ready to use.

5.2.2 Oligonucleotides.

The quality of the oligonucleotides shall be sufficient for their use as primers and probes. See Table 1.

Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR

Specific DNA sequence in Equus caballus isolate Twilight breed thoroughbred chromosome 28, EquCab3.0,

whole genome shotgun sequence (GenBank accession number NC_009171.3)
Horse-125bp-F 5′-ACTCATCAAACGCCGCTCTC-3′ 400 nmol/l
Horse-125bp-R 5′-GCTGTGAAGACCCCGTTGG-3′ 400 nmol/l
Horse-125bp-P 5′-[FAM]-CCAGGGCTCGGTGCTTCCAATCGC-[TAMRA] -3′ 200 nmol/l

PCR product = 41 821 144 - ACTCATCAAA CGCCGCTCTC GAGATCCGTG CACATCGTTC AATGGAAACT TCATTTTAAA

AAAGAGAAAA AGGCGATTGG AAGCACCGAG CCCTGGGTAG CGTGTGCCAA CGGGGTCTTC ACAGC - 41 821 268 - NC_009171.3.

FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.

Horse-125bp-F is base pairs 41 821 144-41 821 163, Horse-125bp-R is base pairs 41 821 250-41 821 268

and Horse-125bp-P is 41 821 216-41 821 239 of NC_009171.3, horse chromosome 28 DNA sequence.

Equivalent reporter dyes and/or quencher dyes can be used if they yield the same or better results.

2 © ISO 2020 – All rights reserved
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ISO/TS 20224-6:2020(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 is needed for PCR.

Additionally, the device shall be capable of exciting fluorescence molecules at specific wavelengths and

detecting sufficient emitted fluorescent light of the fluorophore used to perform TaqMan format assays.

7 Procedure
7.1 Preparation of the test portion/sample

The test sample used for DNA extraction shall be representative of the laboratory sample and

homogeneous, e.g. by grinding or homogenizing the laboratory sample to a fine mixture. Test portion/

sample preparation shall follow the general requirements and specific methods described in ISO 21571

and ISO 20813.
7.2 Preparation of DNA extracts

The extraction/purification and quantification of DNA from the test portion shall follow the

general requirements and methods provided in ISO 21571. DNA extraction methods described in

ISO 21571:2005, Annex A, are recommended.
7.3 PCR setup
7.3.1 Reaction mixes

The method is for a total volume of 25 μl per PCR. The reaction setup is given in Table 2. Reagents

shall be completely thawed at room temperature. Each reagent shall be carefully mixed and briefly

centrifuged immediately before pipetting. A PCR reagent mixture is prepared to contain all components

except for the sample DNA. The required total amount of the PCR reagent mixture prepared depends

on the number of reactions to be performed, including at least one additional reaction as a pipetting

reserve. The number of sample and control replicates
...

TECHNICAL ISO/TS
SPECIFICATION 20224-6
First edition
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse 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 6: Méthode de détection de l'ADN de cheval
PROOF/ÉPREUVE
Reference number
ISO/TS 20224-6:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO/TS 20224-6:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
ii PROOF/ÉPREUVE © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 20224-6:2020(E)
Contents Page