ISO/TS 20224-9:2022

Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 9: Goose DNA detection method

This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of goose-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 goose material derived from domestic breeds of swan goose (Anser cygnoides domesticus) and domestic goose (Anser anser domesticus). Cross detection of Anser brachyrhynchus, Anser indicus, Branta canadensis, Cygnus atratus, Cygnus buccinator, Cygnus cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and Stictonetta naevosa of Anseriformes is expected. The method can be applied to distinguish domestic breeds of swan goose (Anser cygnoides domesticus) and domestic goose (Anser anser domesticus) from domestic chicken, duck and turkey which are the most common adulterants of foie gras.[1] It is also able to differentiate the domestic goose from other high-end domestic poultry meats (quail, pigeon, pheasant). The target sequence is a partial fragment of the Anser cygnoides isolate SCWG-2014 breed Sichuan white goose unplaced genomic scaffold, GooseV1.0 scaffold320 (i.e. GenBank accession number NW_025927981.1)[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 9: Méthode de détection de l'ADN d'oie

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: 18-Mar-2024
• Completion Date: 05-Oct-2022

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 20224-9
First edition
2022-10
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 9:
Goose 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 9: Méthode de détection de l'ADN d'oie
Reference number
ISO/TS 20224-9:2022(E)
© ISO 2022
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ISO/TS 20224-9:2022(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
© ISO 2022 – All rights reserved
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ISO/TS 20224-9:2022(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 ..................................................................................................................................................................................... 5

8.1 General ........................................................................................................................................................................................................... 5

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 .............................................................................................................................................................................................................................19

iii
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ISO/TS 20224-9:2022(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
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ISO/TS 20224-9:2022(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 to detect domestic commercial breeds of swan goose (Anser cygnoides

domesticus) and domestic goose (Anser anser domesticus) and interpretation of results. Cross detection

of Anser brachyrhynchus, Anser indicus, Branta canadensis, Cygnus atratus, Cygnus buccinator, Cygnus

cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and Stictonetta naevosa of Anseriformes

is expected. The method can be applied to distinguish domestic goose from domestic chicken, duck

[1]

and turkey which are most common adulterants of foie gras. The method is also able to differentiate

domestic goose from other high-end domestic poultry meats (quail, pigeon, pheasant).

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/TS 20224-9:2022(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-time
PCR —
Part 9:
Goose DNA detection method
1 Scope

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

qualitative detection of goose-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 goose material derived from domestic breeds of swan goose (Anser cygnoides domesticus) and

domestic goose (Anser anser domesticus). Cross detection of Anser brachyrhynchus, Anser indicus, Branta

canadensis, Cygnus atratus, Cygnus buccinator, Cygnus cygnus, Cygnus olor, Nettapus auritus, Oxyura

jamaicensis and Stictonetta naevosa of Anseriformes is expected.

The method can be applied to distinguish domestic breeds of swan goose (Anser cygnoides domesticus)

and domestic goose (Anser anser domesticus) from domestic chicken, duck and turkey which are the

[1]

most common adulterants of foie gras. It is also able to differentiate the domestic goose from other

high-end domestic poultry meats (quail, pigeon, pheasant).

The target sequence is a partial fragment of the Anser cygnoides isolate SCWG-2014 breed Sichuan

white goose unplaced genomic scaffold, GooseV1.0 scaffold320 (i.e. GenBank accession number

[2]

NW_025927981.1) , 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 % 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.

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ISO/TS 20224-9:2022(E)

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

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Scientific basis

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 goose species-specific DNA sequence of the single-copy Anser cygnoides isolate

SCWG-2014 breed Sichuan white goose unplaced genomic scaffold, GooseV1.0 scaffold320 (i.e.

GenBank accession number NW_025927981.1) in a real-time PCR.

NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several

hundred to several thousand, while the specific target sequence in the goose genome and myostatin gene in

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

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 concentration, which is ready to use.
Commercial real-time PCR master mix may be used.
5.2.2 Oligonucleotides.

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

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ISO/TS 20224-9:2022(E)
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR

Specific sequence in Anser cygnoides isolate SCWG-2014 breed Sichuan white goose unplaced genomic scaffold

(GenBank accession number NW_025927981.1)
Goose-121bp-F 5′-ACGAGGATAGGTTGTGACAGC-3′ 400 nmol/l
Goose-121bp-R 5′-GAATCTCTGTGTCGTCTTCTCTATATG-3′ 400 nmol/l
5′-[FAM]-ACTCTGTTCAGCCTTGCGAAGACCTTATGC-[TAMRA]
Goose-121bp-P 200 nmol/l
-3′

PCR product = 113884 - ACGAGGATAG GTTGTGACAG CTGACTCTGT TCAGCCTTGC GAAGACCTTA TGCTGTCTAC

AATTACCTAA TTGGAGGATA TAGAATTATA GAATCATATA GAGAAGACGA CACAGAGATT C - 114004 - NW_025927981.1.

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

Goose-121bp-F is base pairs 113 884 – 113 904, Goose-121bp-R is base pairs 113 978 – 114 404 and

Goose-121bp-P is 113 907 – 113 936 of NW_025927981.1, Anser cygnoides domesticus unplaced genomic

scaffold. 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 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

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ISO/TS 20224-9:2022(E)

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 Goose-121bp-F, c = 10 μmol/l and Goose-121bp-R, c = 10 μmol/l 1,0 µl for each

Probe Goose-121bp-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-over PCR contamination, and a passive internal reference based on ROX dye.

Equivalent products can be used if they yield the same or better results. If necessary, the amounts of the reagents and the

temperature-time programme can be adapted.
7.3.2 PCR controls
7.3.2.1 General
PCR controls shall be as described in ISO 24276 and ISO 20813.
7.3.2.2 Inhibition control (reference gene assay)

A reference control gene (i.e. 18S rRNA gene for eukaryotes, myostatin gene for mammals and poultry)

PCR assay using sample DNAs shall be performed to test nucleic acid amplifiability and provide control

to exclude false-negative results.
7.3.3 Real-time PCR thermocycler plate set-up

Transfer the setup reaction vials to the thermocycler. The vials should be arranged to avoid any possible

edge temperature variations associated with a particular real-time thermocycler instrument. Start the

temperature-time programme.
7.4 Temperature-time programme

The temperature-time programme as outlined in Table 3 was used in the validation study. The use of

different reaction conditions and real-time PCR cycles shall be verified. The time for initial denaturation

depends on the master mix used.
Table 3 — Temperature-time programme
Fluorescence
Step Parameter Temperature Time Cycles
measurement
1 Initial denaturation 95 °C 10 min no 1
Denaturation 95 °C 15 s no
2 Amplification 45
Annealing and
60 °C 60 s yes
elongation
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ISO/TS 20224-9:2022(E)
8 Accept/reject criteria
8.1 General

A corresponding real-time PCR-instrument-specific data analysis programme shall be used for the

identification of PCR products. The amplification results can be expressed differently, depending on

the instrument used. In the absence of detectable PCR products (e.g. negative controls), the result

shall be expressed as “undetermined”, “no amplification” or the maximum number of reaction cycles

performed. If amplification of the DNA target sequence in a sample (e.g. positive controls) occurred, a

sigmoid-shaped amplification curve shall be observed. The cycle number at the crossing point of the

amplification curve and the fluorescence threshold shall be calculated [cycle threshold (C ) or cycle

quantification (C )].

If, due to atypical fluorescence measurement data, the automatic interpretation does not provide

a meaningful result, it may be necessary to set the baseline and the threshold manually prior to

interpreting the data. In such a case, the device-specific instructions provided with the interpretation

software shall be followed.
8.2 Identification
The target sequence is considered as detected if:

— goose-specific primers Goose-121bp-F and Goose-121bp-R and the probe Goose-121bp-P, produce a

sigmoid-shaped amplification curve and a C value or C value can be calculated;
t q

— PCR control reactions with no added DNA (PCR reagent control, extraction blank control) produce

no amplification;

— the amplification controls (positive DNA target control, PCR inhibition control) produce the expected

amplification and C values (or C values).
t q

Trace detections are defined as PCRs with C values later than that defined at the target LOD . In

t 95 %

the event of a trace detection or contradictory positive/negative results from different extracts of the

same sample, then the sample shall be retested. At least two new extracts shall be prepared from the

homogenized laboratory sample. A minimum of 20 PCR replicates shall be conducted across the new

extracts (e.g. ten PCR repeats for two extracted DNA, seven PCR repeats for three extracted DNA).

The target sequence shall be considered as “detected” if ≥ 95 % of the new extract PCR results show

a positive detection. The target sequence shall be considered as “not detected” if < 95 % of the new

extract PCR results show a positive detection.
9 Validation status and performance criteria
9.1 General
Validation followed a two-part process:
a) in-house validation;
b) collaborative trial validation.
9.2 Robustness

The robustness of the method was confirmed for the collaborative trial by changing the reaction

conditions for the following factors:

a) real-time PCR instruments (e.g. ABI 7500, BioRad CFX96, ABI 7900 HT Fast, Eppendorf Realplex 4 );

1) These are examples of a suitable products available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of these products.

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ISO/TS 20224-9:2022(E)

b) reaction volume: 19 µl or 21 µl PCR reagent mixture plus 5 µl sample DNA (20 ng/µl to 200 ng/µl);

c) annealing temperature: 59 °C and 61 °C;
d) primer or probe concentration: both reduced by 30 %.

For each factor tested, the PCRs were analysed in triplicate, each with 20 copies of the target sequence

and with 100 copies of the non-target sequence as negative controls. Method performance remained

satisfactory for both samples and negative controls under the changed conditions for each changed

factor.
9.3 Reproducibility

The reproducibility of the method was verified in a collaborative trial with 12 participants, organized

by the Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Customs in

[3] [4]

accordance with the IUPAC protocol and the BVL guidelines . Participants received 12 DNA samples

for the evaluation of false-positive and false-negative rates. All samples were labelled with randomized

coding numbers and consisted of six replicate samples. The 12 DNA samples were:
— six vials of goose DNA solution, 10 copies/µl;
— six vials of bovine DNA solution, 20 copies/µl.

The copy numbers were determined using the real-time PCR of this method and serial dilutions of

plasmid DNA containing the target sequence. The concentration of the plasmid-DNA (copies/µl) was

measured by digital PCR.

Participants received a PCR master mix and the oligonucleotides (primers and probes) from the

collaborative test organizer to conduct the PCR experiments.

Goose and bovine genomic DNA were extracted from goose meat and bovine meat, respectively, and

then adjusted with 0,2 × TE buffer to a nominal concentration of 10 copies/µl for goose DNA and

20 copies/µl for bovine DNA, respectively.

The collaborative trial was designed to determine false-positive and false-negative rates. Each DNA

sample was tested by the participants in a single PCR test with 5 µl of the respective DNA solution,

using the procedure and the conditions given in Tables 2 and 3. The results of the collaborative trial are

listed in Table 4.
Table 4 — Results of the collaborative trial
Number of laboratories 12
Number of laboratories submitting results 12
Number of samples per laboratory 12
Number of accepted results 144
Number of accepted samples containing goose material 72
Number of accepted samples not containing goose material 72
False-positive results 0
False-positive results (in %) 0
False-negative results 1
False-negative results (in %) 1,4
9.4 Sensitivity

The absolute limit of detection (LOD ) for the method is five DNA copies. The collaborative trial of

95 %

the goose detection method was carried out at the same time as collaborative trials for the duck, turkey

and pigeon detection methods. Goose, duck, turkey and pigeon target DNA sequences were synthesized

and cloned into the pUC57 vector (2 710 bp in length, GenBank/EMBL accession number Y14837). This

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ISO/TS 20224-9:2022(E)

constructed plasmid pUC57-gdtp (3 167 bp in length) was sequenced to ensure that only one copy

of the goose, duck, turkey and pigeon target DNA sequence was inserted (see Figure 1). No deletion

or insertion mutations were found in the inserted sequences (see Figure 2). The target sequences of

corresponding PCR methods are indicated.
Key
1 nt 1-121 = goose amplicon (121 bp)
2 nt nt 122~226 = duck amplicon (105 bp)
3 nt 227~344 = turkey amplicon (118 bp)
4 nt 345~457 = pigeon amplicon (113 bp)
5 M13 reverse promoter
6 ColE1 origin of replication
7 β-lactamase gene (ampicillin resistance gene)
8 ampicillin resistance gene promoter
9 M13 forward promoter
Figure 1 — Map of the multi-target DNA plasmid
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ISO/TS 20224-9:2022(E)
Key
single bold underline nt 1~121 = goose amplicon (121 bp)
double underline nt 122~226 = duck amplicon (105 bp)
dashed underline nt 227~344 = turkey amplicon (118 bp)
dotted underline nt 345~457 = pigeon amplicon (113 bp)

Figure 2 — Complete sequence of nucleotides (nt) and annotation of the insertion in plasmid

pUC57

Each participant in the collaborative trial received a solution containing plasmid pUC57 DNA adjusted

to 1 000 copies/µl of the target sequence (see Figures 1 and 2) in 20 ng/µl sonicated salmon sperm

DNA. The concentration was measured before distribution by digital PCR (QX100 Droplet Digital PCR

System ). Serial dilutions were produced by the 12 laboratories in the range of 0,02 copies/µ

...

© ISO 2022 – All rights reserved
ISO/PRF TS 20224-9:2022(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 9: Goose 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 9: Méthode de

détection de l'ADN d'oie
---------------------- Page: 1 ----------------------
ISO/PRF TS 20224-9:2022(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.
© ISO 2022 – All rights reserved iii
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ISO/PRF TS 20224-9:2022(E)
Contents

Foreword ................................................................................................................................................................ v

Introduction ......................................................................................................................................................... vi

1 Scope .......................................................................................................................................................... 1

2 Normative references .......................................................................................................................... 1

3 Terms and definitions ......................................................................................................................... 2

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 ................................................................................................................................................. 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 .............................................................................................................................................13

Annex A (informative) BlastN +2.12.0 results for query of GenBank RefSeq Genome

(refseq_genomes) and Whole-genome Shotgun Contigs (wgs) ...........................................14

Bibliography ........................................................................................................................................................22

iv © ISO 2022 – All rights reserved
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ISO/PRF TS 20224-9:2022(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 v
---------------------- Page: 4 ----------------------
ISO/PRF TS 20224-9:2022(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 to detect domestic commercial breeds of swan goose (Anser cygnoides

domesticus) and domestic goose (Anser anser domesticus) and interpretation of results. Cross detection

of Anser brachyrhynchus, Anser indicus, Branta canadensis, Cygnus atratus, Cygnus buccinator, Cygnus

cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and Stictonetta naevosa of Anseriformes is

expected. The method can be applied to distinguish domestic goose from domestic chicken, duck and

[1]

turkey which are most common adulterants of foie gras. The method is also able to differentiate

domestic goose from other high-end domestic poultry meats (quail, pigeon, pheasant).

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.

vi © ISO 2022 – All rights reserved
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TECHNICAL SPECIFICATION ISO/PRF TS 20224-9:2022(E)
Molecular biomarker analysis — Detection of animal-derived
materials in foodstuffs and feedstuffs by real-time PCR —
Part 9: Goose DNA detection method
1 Scope

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

detection of goose-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 goose

material derived from domestic breeds of swan goose (Anser cygnoides domesticus) and domestic goose

(Anser anser domesticus). Cross detection of Anser brachyrhynchus, Anser indicus, Branta canadensis,

Cygnus atratus, Cygnus buccinator, Cygnus cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and

Stictonetta naevosa of Anseriformes is expected.

The method can be applied to distinguish domestic breeds of swan goose (Anser cygnoides domesticus)

and domestic goose (Anser anser domesticus) from domestic chicken, duck and turkey which are the most

[1]

common adulterants of foie gras. It is also able to differentiate the domestic goose from other high-end

domestic poultry meats (quail, pigeon, pheasant).

The target sequence is a partial fragment of the Anser cygnoides domesticus unplaced genomic scaffold

[2]

(i.e. GenBank accession number NW_013185870.1) , 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 % 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
© ISO 2022 – All rights reserved 1
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ISO/PRF TS 20224-9:2022(E)
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
— 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 goose species-specific DNA sequence of the single-copy Anser cygnoides domesticus

unplaced genomic scaffold (i.e. GenBank accession number NW_013185870.1) in a real-time PCR.

NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several

hundred to several thousand, while the specific target sequence in the goose genome and myostatin gene in

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

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 concentration, which is ready to use.
NOTE: Commercial real-time PCR master mix may 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
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ISO/PRF TS 20224-9:2022(E)
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR

Specific sequence in Anser cygnoides domesticus unplaced genomic scaffold as the target sequence (GenBank

accession number: NW_013185870.1)
Goose-121bp-F 5′-ACGAGGATAGGTTGTGACAGC-3′ 400 nmol/l
Goose-121bp-R 5′-GAATCTCTGTGTCGTCTTCTCTATATG-3′ 400 nmol/l
Goose-121bp-P 5′- [FAM]-ACTCTGTTCAGCCTTGCGAAGACCTTATGC-[TAMRA] -3′ 200 nmol/l

PCR product = 869408 - acgaggatag gttgtgacag ctgactctgt tcagccttgc gaagacctta tgctgtctac aattacctaa ttggaggata tagaattata

gaatcatata gagaagacga cacagagatt c - 869528 - NW_013185870.1.
FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.

Goose-121bp-F is base pairs 869 408 – 869 428, Goose-121bp-R is base pairs 869 502 – 869 528 and

Goose-121bp-P is 869 431 – 869 460 of NW_013185870.1, Anser cygnoides domesticus unplaced genomic

scaffold. 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 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.
© ISO 2022 – All rights reserved 3
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ISO/PRF TS 20224-9:2022(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 Goose-121bp-F, c = 10 μmol/l and Goose-121bp-R, c = 10 μmol/l 1,0 µl for each

Probe Goose-121bp-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-over PCR contamination, and a passive internal reference based on ROX dye. Equivalent products can be

used if they yield the same or better results. If necessary, the amounts of the reagents and the temperature-time programme can

be adapted.
7.3.2 PCR controls
7.3.2.1 General
PCR controls shall be as described in ISO 24276 and ISO 20813.
7.3.2.2 Inhibition control (reference gene assay)

A reference control gene (i.e. 18S rRNA gene for eukaryotes, myostatin gene for mammals and poultry)

PCR assay using sample DNAs shall be performed to test nucleic acid amplifiability and provide control

to exclude false-negative results.
7.3.3 Real-time PCR thermocycler plate set-up

Transfer the setup reaction vials to the thermocycler. The vials should be arranged to avoid any possible

edge temperature variations associated with a particular real-time thermocycler instrument. Start the

temperature-time programme.
4 © ISO 2022 – All rights reserved
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ISO/PRF TS 20224-9:2022(E)
7.4 Temperature-time programme

The temperature-time programme as outlined in Table 3 was used in the validation study. The use of

different reaction conditions and real-time PCR cycles shall be verified. The time for initial denaturation

depends on the master mix used.
Table 3 — Temperature-time programme
Fluorescence
Step Parameter Temperature Time Cycles
measurement
1 Initial denaturation 95 °C 10 min no 1
Denaturation 95 °C 15 s no
2 Amplification 45
Annealing and
60 °C 60 s yes
elongation
8 Accept/reject criteria
8.1 General

A corresponding real-time PCR-instrument-specific data analysis programme shall be used for the

identification of PCR products. The amplification results can be expressed differently, depending on the

instrument used. In the absence of detectable PCR products (e.g. negative controls), the result shall be

expressed as “undetermined”, “no amplification” or the maximum number of reaction cycles performed.

If amplification of the DNA target sequence in a sample (e.g. positive controls) occurred, a sigmoid-shaped

amplification curve shall be observed. The cycle number at the crossing point of the amplification curve

and the fluorescence threshold shall be calculated [cycle threshold (C ) or cycle quantification (C )].

t q

If, due to atypical fluorescence measurement data, the automatic interpretation does not provide a

meaningful result, it may be necessary to set the baseline and the threshold manually prior to interpreting

the data. In such a case, the device-specific instructions provided with the interpretation software shall

be followed.
8.2 Identification
The target sequence is considered as detected if:

— goose-specific primers Goose-121bp-F and Goose-121bp-R and the probe Goose-121bp-P, produce a

sigmoid-shaped amplification curve and a C value or C value can be calculated;
t q

— PCR control reactions with no added DNA (PCR reagent control, extraction blank control) produce

no amplification;

— the amplification controls (positive DNA target control, PCR inhibition control) produce the expected

amplification and C values (or C values).
t q

Trace detections are defined as PCRs with C values later than that defined at the target LOD . In the

t 95 %

event of a trace detection or contradictory positive/negative results from different extracts of the same

sample, then the sample shall be retested. At least two new extracts shall be prepared from the

homogenized laboratory sample. A minimum of 20 PCR replicates shall be conducted across the new

extracts (e.g. ten PCR repeats for two extracted DNA, seven PCR repeats for three extracted DNA). The

target sequence shall be considered as “detected” if ≥ 95 % of the new extract PCR results show a positive

© ISO 2022 – All rights reserved 5
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ISO/PRF TS 20224-9:2022(E)

detection. The target sequence shall be considered as “not detected” if < 95 % of the new extract PCR

results show a positive detection.
9 Validation status and performance criteria
9.1 General
Validation followed a two-part process:
a) in-house validation;
b) collaborative trial validation.
9.2 Robustness

The robustness of the method was confirmed for the collaborative trial by changing the reaction

conditions for the following factors:

a) real-time PCR instruments (e.g. ABI 7500, BioRad CFX96, ABI 7900 HT Fast, Eppendorf Realplex 4 );

b) reaction volume: 19 µl or 21 µl PCR reagent mixture plus 5 µl sample DNA (20 ng/µl to 200 ng/µl);

c) annealing temperature: 59 °C and 61 °C;
d) primer or probe concentration: both reduced by 30 %.

For each factor tested, the PCRs were analysed in triplicate, each with 20 copies of the target sequence

and with 100 copies of the non-target sequence as negative controls. Method performance remained

satisfactory for both samples and negative controls under the changed conditions for each changed factor.

9.3 Reproducibility

The reproducibility of the method was verified in a collaborative trial with 12 participants, organized by

the Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Customs in

[3] [4]

accordance with the IUPAC protocol and the BVL guidelines . Participants received 12 DNA samples

for the evaluation of false-positive and false-negative rates. All samples were labelled with randomized

coding numbers and consisted of six replicate samples. The 12 DNA samples were:
— six vials of goose DNA solution, 10 copies/µl;
— six vials of bovine DNA solution, 20 copies/µl.

The copy numbers were determined using the real-time PCR of this method and serial dilutions of

plasmid DNA containing the target sequence. The concentration of the plasmid-DNA (copies/µl) was

measured by digital PCR.

Participants received a PCR master mix and the oligonucleotides (primers and probes) from the

collaborative test organizer to conduct the PCR experiments.

Goose and bovine genomic DNA were extracted from goose meat and bovine meat, respectively, and then

adjusted with 0,2 × TE buffer to a nominal concentration of 10 copies/µl for goose DNA and 20 copies/µl

for bovine DNA, respectively.

These are examples of a suitable products available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of these products.

6 © ISO 2022 – All rights reserved
---------------------- Page: 11 ----------------------
ISO/PRF TS 20224-9:2022(E)

The collaborative trial was designed to determine false-positive and false-negative rates. Each DNA

sample was tested by the participants in a single PCR test with 5 µl of the respective DNA solution, using

the procedure and the conditions given in Tables 2 and 3. The results of the collaborative trial are listed

in Table 4.
Table 4 — Results of the collaborative trial
Number of laboratories 12
Number of laboratories submitting results 12
Number of samples per laboratory 12
Number of accepted results 144
Number of accepted samples containing goose material 72
Number of accepted samples not containing goose material 72
False-positive results 0
False-positive results (in %) 0
False-negative results 1
False-negative results (in %) 1.,4
9.4 Sensitivity

The absolute limit of detection (LOD ) for the method is five DNA copies. The collaborative trial of the

95 %

goose detection method was carried out at the same time as collaborative trials for the duck, turkey, and

pigeon detection methods. Target DNA sequences for separate identification of detection of goose, duck,

turkey and pigeon were synthesized and cloned into the pUC57 vector (2 710 bp in length,

GenBank/EMBL accession number Y14837). This constructed plasmid pUC57-gdtp (3 167 bp in length)

was sequenced to ensure that only one copy of the goose, duck, turkey and pigeon target DNA sequence

was inserted (see Figure 1). No deletion or insertion mutations were found in the inserted sequences (see

Figure 2). The target sequences of corresponding PCR methods are indicated.
© ISO 2022 – All rights reserved 7
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ISO/PRF TS 20224-9:2022(E)
Key
1 nt 1-121 = goose amplicon (121 bp)
2 nt nt 122~226 = duck amplicon (105 bp)
3 nt 227~344 = turkey amplicon (118 bp)
4 nt 345~457 = pigeon amplicon (113 bp)
5 M13 reverse promoter
6 ColE1 origin of replication
7 β-lactamase gene (ampicillin resistance gene)
8 ampicillin resistance gene promoter
9 M13 forward promoter
Figure 1 — Map of the multi-target DNA plasmid
8 © ISO 2022 – All rights reserved
---------------------- Page: 13 ----------------------
ISO/PRF TS 20224-9:2022(E)
Key
single bold underline nt 1~121 = goose amplicon (121 bp)
double underline nt 122~226 = duck amplicon (105 bp)
dashed underline nt 227~344 = turkey amplicon (118 bp)
dotted underline nt 345~457 = pigeon amplicon (113 bp)

Figure 2 — Complete sequence of nucleotides (nt) and annotation of the insertion in plasmid

pUC57

Each participant in the collaborative trial received a solution containing plasmid pUC57 DNA adjusted to

1 000 copies/µl of the target sequence (see Figures 1 and 2) in 20 ng/µl sonicated salmon sperm DNA.

The concentration was measured before distribution by digital PCR (QX100 Droplet Digital PCR System ).

Serial dilutions were produced by the 12 laboratories in the range of 0,02 copies/µl to 4 copies/µl using

0,2 × TE buffer containing 20 ng/µl sonicated salmon sperm DNA. Each participant measured six

replicates per concentration level. A positive result was achieved for five copies per PCR in 72 out of 72

tests (see Table 5).

Probability of detection (POD) describes the probability that PCR amplification will take place at a given

number of copies of the target sequences (see ISO/TS 16393). Qualitative data generated across all

laboratories and dilution levels (see Table 5) was used to determine the POD = 0,95 of the detection

method (see Table 6) as described in Reference [3]. Standard deviation was determined to be 0,29 and

the LOD was 3,1 copies; both parameters well below the required maximum of 1 and 20 copies,

95 %
[5]
respectively.

This is a product supplied by Bio-Rad GmbH. This information is given for the convenience of users of this

document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used

if they can be shown to lead to the sam
...

FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 20224-9
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 9:
Goose 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 9: Méthode de détection de l'ADN d'oie
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 20224-9:2022(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 20224-9:2022(E)
FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 20224-9
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Detection of animal-derived materials
Voting begins on:
in foodstuffs and feedstuffs by real-
time PCR —
Voting terminates on:
Part 9:
Goose 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 9: Méthode de détection de l'ADN d'oie
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or ISO’s member body in the country of the requester.
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTS 20224-9:2022(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 20224-9:2022(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 ..................................................................................................................................................................................... 5

8.1 General ........................................................................................................................................................................................................... 5

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 .............................................................................................................................................................................................................................19

iii
© ISO 2022 – All rights reserved
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ISO/DTS 20224-9:2022(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
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ISO/DTS 20224-9:2022(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 to detect domestic commercial breeds of swan goose (Anser cygnoides

domesticus) and domestic goose (Anser anser domesticus) and interpretation of results. Cross detection

of Anser brachyrhynchus, Anser indicus, Branta canadensis, Cygnus atratus, Cygnus buccinator, Cygnus

cygnus, Cygnus olor, Nettapus auritus, Oxyura jamaicensis and Stictonetta naevosa of Anseriformes

is expected. The method can be applied to distinguish domestic goose from domestic chicken, duck

[1]

and turkey which are most common adulterants of foie gras. The method is also able to differentiate

domestic goose from other high-end domestic poultry meats (quail, pigeon, pheasant).

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 20224-9:2022(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-time
PCR —
Part 9:
Goose DNA detection method
1 Scope

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

qualitative detection of goose-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 goose material derived from domestic breeds of swan goose (Anser cygnoides domesticus) and

domestic goose (Anser anser domesticus). Cross detection of Anser brachyrhynchus, Anser indicus, Branta

canadensis, Cygnus atratus, Cygnus buccinator, Cygnus cygnus, Cygnus olor, Nettapus auritus, Oxyura

jamaicensis and Stictonetta naevosa of Anseriformes is expected.

The method can be applied to distinguish domestic breeds of swan goose (Anser cygnoides domesticus)

and domestic goose (Anser anser domesticus) from domestic chicken, duck and turkey which are the

[1]

most common adulterants of foie gras. It is also able to differentiate the domestic goose from other

high-end domestic poultry meats (quail, pigeon, pheasant).

The target sequence is a partial fragment of the Anser cygnoides domesticus unplaced genomic scaffold

[2]

(i.e. GenBank accession number NW_013185870.1) , 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 % 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.

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ISO/DTS 20224-9:2022(E)

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

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Scientific basis

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 goose species-specific DNA sequence of the single-copy Anser cygnoides domesticus

unplaced genomic scaffold (i.e. GenBank accession number NW_013185870.1) in a real-time PCR.

NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several

hundred to several thousand, while the specific target sequence in the goose genome and myostatin gene in

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

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 concentration, which is ready to use.
Commercial real-time PCR master mix may be used.
5.2.2 Oligonucleotides.

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

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ISO/DTS 20224-9:2022(E)
Table 1 — Oligonucleotides
Final concentration
Name DNA sequence of the oligonucleotide
in PCR

Specific sequence in Anser cygnoides domesticus unplaced genomic scaffold as the target sequence (GenBank

accession number NW_013185870.1)
Goose-121bp-F 5′-ACGAGGATAGGTTGTGACAGC-3′ 400 nmol/l
Goose-121bp-R 5′-GAATCTCTGTGTCGTCTTCTCTATATG-3′ 400 nmol/l
5′-[FAM]-ACTCTGTTCAGCCTTGCGAAGACCTTATGC-[TAMRA]
Goose-121bp-P 200 nmol/l
-3′

PCR product = 869408 - acgaggatag gttgtgacag ctgactctgt tcagccttgc gaagacctta tgctgtctac aattacctaa ttggaggata

tagaattata gaatcatata gagaagacga cacagagatt c - 869528 - NW_013185870.1.
FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine.

Goose-121bp-F is base pairs 869 408 – 869 428, Goose-121bp-R is base pairs 869 502 – 869 528 and

Goose-121bp-P is 869 431 – 869 460 of NW_013185870.1, Anser cygnoides domesticus unplaced genomic

scaffold. 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 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

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ISO/DTS 20224-9:2022(E)

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 Goose-121bp-F, c = 10 μmol/l and Goose-121bp-R, c = 10 μmol/l 1,0 µl for each

Probe Goose-121bp-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-over PCR contamination, and a passive internal reference based on ROX dye.

Equivalent products can be used if they yield the same or better results. If necessary, the amounts of the reagents and the

temperature-time programme can be adapted.
7.3.2 PCR controls
7.3.2.1 General
PCR controls shall be as described in ISO 24276 and ISO 20813.
7.3.2.2 Inhibition control (reference gene assay)

A reference control gene (i.e. 18S rRNA gene for eukaryotes, myostatin gene for mammals and poultry)

PCR assay using sample DNAs shall be performed to test nucleic acid amplifiability and provide control

to exclude false-negative results.
7.3.3 Real-time PCR thermocycler plate set-up

Transfer the setup reaction vials to the thermocycler. The vials should be arranged to avoid any possible

edge temperature variations associated with a particular real-time thermocycler instrument. Start the

temperature-time programme.
7.4 Temperature-time programme

The temperature-time programme as outlined in Table 3 was used in the validation study. The use of

different reaction conditions and real-time PCR cycles shall be verified. The time for initial denaturation

depends on the master mix used.
Table 3 — Temperature-time programme
Fluorescence
Step Parameter Temperature Time Cycles
measurement
1 Initial denaturation 95 °C 10 min no 1
Denaturation 95 °C 15 s no
2 Amplification 45
Annealing and
60 °C 60 s yes
elongation
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ISO/DTS 20224-9:2022(E)
8 Accept/reject criteria
8.1 General

A corresponding real-time PCR-instrument-specific data analysis programme shall be used for the

identification of PCR products. The amplification results can be expressed differently, depending on

the instrument used. In the absence of detectable PCR products (e.g. negative controls), the result

shall be expressed as “undetermined”, “no amplification” or the maximum number of reaction cycles

performed. If amplification of the DNA target sequence in a sample (e.g. positive controls) occurred, a

sigmoid-shaped amplification curve shall be observed. The cycle number at the crossing point of the

amplification curve and the fluorescence threshold shall be calculated [cycle threshold (C ) or cycle

quantification (C )].

If, due to atypical fluorescence measurement data, the automatic interpretation does not provide

a meaningful result, it may be necessary to set the baseline and the threshold manually prior to

interpreting the data. In such a case, the device-specific instructions provided with the interpretation

software shall be followed.
8.2 Identification
The target sequence is considered as detected if:

— goose-specific primers Goose-121bp-F and Goose-121bp-R and the probe Goose-121bp-P, produce a

sigmoid-shaped amplification curve and a C value or C value can be calculated;
t q

— PCR control reactions with no added DNA (PCR reagent control, extraction blank control) produce

no amplification;

— the amplification controls (positive DNA target control, PCR inhibition control) produce the expected

amplification and C values (or C values).
t q

Trace detections are defined as PCRs with C values later than that defined at the target LOD . In

t 95 %

the event of a trace detection or contradictory positive/negative results from different extracts of the

same sample, then the sample shall be retested. At least two new extracts shall be prepared from the

homogenized laboratory sample. A minimum of 20 PCR replicates shall be conducted across the new

extracts (e.g. ten PCR repeats for two extracted DNA, seven PCR repeats for three extracted DNA).

The target sequence shall be considered as “detected” if ≥ 95 % of the new extract PCR results show

a positive detection. The target sequence shall be considered as “not detected” if < 95 % of the new

extract PCR results show a positive detection.
9 Validation status and performance criteria
9.1 General
Validation followed a two-part process:
a) in-house validation;
b) collaborative trial validation.
9.2 Robustness

The robustness of the method was confirmed for the collaborative trial by changing the reaction

conditions for the following factors:

a) real-time PCR instruments (e.g. ABI 7500, BioRad CFX96, ABI 7900 HT Fast, Eppendorf Realplex 4 );

1) These are examples of a suitable products available commercially. This information is given for the convenience

of users of this document and does not constitute an endorsement by ISO of these products.

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ISO/DTS 20224-9:2022(E)

b) reaction volume: 19 µl or 21 µl PCR reagent mixture plus 5 µl sample DNA (20 ng/µl to 200 ng/µl);

c) annealing temperature: 59 °C and 61 °C;
d) primer or probe concentration: both reduced by 30 %.

For each factor tested, the PCRs were analysed in triplicate, each with 20 copies of the target sequence

and with 100 copies of the non-target sequence as negative controls. Method performance remained

satisfactory for both samples and negative controls under the changed conditions for each changed

factor.
9.3 Reproducibility

The reproducibility of the method was verified in a collaborative trial with 12 participants, organized

by the Technical Center for Animal, Plant and Food Inspection and Quarantine, Shanghai Customs in

[3] [4]

accordance with the IUPAC protocol and the BVL guidelines . Participants received 12 DNA samples

for the evaluation of false-positive and false-negative rates. All samples were labelled with randomized

coding numbers and consisted of six replicate samples. The 12 DNA samples were:
— six vials of goose DNA solution, 10 copies/µl;
— six vials of bovine DNA solution, 20 copies/µl.

The copy numbers were determined using the real-time PCR of this method and serial dilutions of

plasmid DNA containing the target sequence. The concentration of the plasmid-DNA (copies/µl) was

measured by digital PCR.

Participants received a PCR master mix and the oligonucleotides (primers and probes) from the

collaborative test organizer to conduct the PCR experiments.

Goose and bovine genomic DNA were extracted from goose meat and bovine meat, respectively, and

then adjusted with 0,2 × TE buffer to a nominal concentration of 10 copies/µl for goose DNA and

20 copies/µl for bovine DNA, respectively.

The collaborative trial was designed to determine false-positive and false-negative rates. Each DNA

sample was tested by the participants in a single PCR test with 5 µl of the respective DNA solution,

using the procedure and the conditions given in Tables 2 and 3. The results of the collaborative trial are

listed in Table 4.
Table 4 — Results of the collaborative trial
Number of laboratories 12
Number of laboratories submitting results 12
Number of samples per laboratory 12
Number of accepted results 144
Number of accepted samples containing goose material 72
Number of accepted samples not containing goose material 72
False-positive results 0
False-positive results (in %) 0
False-negative results 1
False-negative results (in %) 1,4
9.4 Sensitivity

The absolute limit of detection (LOD ) for the method is five DNA copies. The collaborative trial

95 %

of the goose detection method was carried out at the same time as collaborative trials for the duck,

turkey and pigeon detection methods. Target DNA sequences for separate identification of detection of

goose, duck, turkey and pigeon were synthesized and cloned into the pUC57 vector (2 710 bp in length,

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ISO/DTS 20224-9:2022(E)
GenBank/EMBL accession number Y14837). This constructe
...