EN ISO 22174:2024
(Main)Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and quantification of microorganisms - General requirements and definitions (ISO 22174:2024)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and quantification of microorganisms - General requirements and definitions (ISO 22174:2024)
This document specifies the general requirements for the in vitro amplification of nucleic acid sequences (DNA or RNA).
This document is applicable to the testing for microorganisms and viruses from the food chain using the polymerase chain reaction (PCR). This document, or parts of it, is applicable to other fields of PCR diagnostics based on a case-by-case evaluation.
The minimum requirements laid down in this document are intended to ensure that comparable and reproducible results are obtained in different laboratories.
This document has been established for microorganisms from the food chain and is applicable to:
— products intended for human consumption;
— products for feeding animals;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis von Mikroorganismen - Allgemeine Anforderungen und Definitionen (ISO 22174:2024)
Dieses Dokument legt die allgemeinen Anforderungen an die in-vitro-Amplifikation von Nukleinsäuresequenzen (DNA oder RNA) fest.
Dieses Dokument ist anwendbar für die Untersuchung auf Mikroorganismen und Viren aus der Lebensmittelkette unter Anwendung der Polymerase-Kettenreaktion (en: polymerase chain reaction, PCR). Dieses Dokument, oder Teile davon, sind auf der Grundlage einer Einzelfallbeurteilung auch für andere Bereiche der PCR-Diagnostik anwendbar.
Die in diesem Dokument festgelegten Mindestanforderungen dienen dazu, vergleichbare und reproduzierbare Ergebnisse in verschiedenen Laboren sicherzustellen.
Dieses Dokument wurde für Mikroorganismen aus der Lebensmittelkette erstellt und ist anwendbar für
- Produkte, die für den menschlichen Verzehr vorgesehen sind,
- Produkte, die Futtermittel sind,
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln,
- Proben aus dem Bereich der Primärproduktion.
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR) pour la recherche et la quantification de micro-organismes - Exigences générales et définitions (ISO 22174:2024)
Le présent document spécifie les exigences générales relatives à l’amplification in vitro des séquences d’acide nucléique (ADN ou ARN).
Le présent document s’applique aux essais pour la détection de micro‑organismes et de virus issus de la chaîne alimentaire en faisant appel à la réaction de polymérisation en chaîne (PCR). Le présent document, ou certaines de ses parties, s’applique(nt) à d’autres domaines de diagnostic par PCR sur la base d’une évaluation au cas par cas.
Les exigences minimales déclarées dans le présent document sont destinées à garantir l’obtention de résultats comparables et reproductibles dans des laboratoires différents.
Le présent document a été conçu pour les micro‑organismes issus de la chaîne alimentaire et s’applique aux:
— produits destinés à la consommation humaine;
— produits destinés à l’alimentation animale;
— échantillons environnementaux prélevés dans des zones de production et de manipulation de produits alimentaires et d’aliments pour animaux;
— échantillons de production primaire.
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za ugotavljanje prisotnosti in kvantifikacijo mikroorganizmov - Splošne zahteve in definicije (ISO 22174:2024)
Ta mednarodni standard določa splošne zahteve za in vitro povečevanje zaporedij nukleinske kisline (DNA ali RNA). Uporablja se za preskušanje prisotnosti mikroorganizmov, ki se prenašajo s hrano, v živilih in izolatih iz živil s polimerazno verižno reakcijo (PCR).
Minimalne zahteve iz tega mednarodnega standarda zagotavljajo primerljive in ponovljive rezultate v različnih laboratorijih.
Ta mednarodni standard je bil pripravljen za mikroorganizme, ki se prenašajo s hrano, v živilskih in krmnih matricah ali izolatih iz njih ter se uporablja za:
– proizvode, namenjene za prehrano ljudi;
– proizvode, namenjene za živalsko krmo;
– okoljske vzorce na področju proizvodnje hrane in krme; ter
– vzorce iz faze primarne proizvodnje.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-november-2024
Nadomešča:
SIST EN ISO 22174:2005
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za
ugotavljanje prisotnosti in kvantifikacijo mikroorganizmov - Splošne zahteve in
definicije (ISO 22174:2024)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and
quantification of microorganisms - General requirements and definitions (ISO
22174:2024)
Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis
von Mikroorganismen - Allgemeine Anforderungen und Definitionen (ISO 22174:2024)
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)
pour la recherche et la quantification de micro-organismes - Exigences générales et
définitions (ISO 22174:2024)
Ta slovenski standard je istoveten z: EN ISO 22174:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 22174
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2024
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 22174:2005
English Version
Microbiology of the food chain - Polymerase chain reaction
(PCR) for the detection and quantification of
microorganisms - General requirements and definitions
(ISO 22174:2024)
Microbiologie de la chaîne alimentaire - Réaction de Mikrobiologie der Lebensmittelkette - Polymerase-
polymérisation en chaîne (PCR) pour la recherche et la Kettenreaktion (PCR) zum Nachweis von
quantification de micro-organismes - Exigences Mikroorganismen - Allgemeine Anforderungen und
générales et définitions (ISO 22174:2024) Definitionen (ISO 22174:2024)
This European Standard was approved by CEN on 21 July 2024.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22174:2024 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 22174:2024) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food chain” the
secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by March 2025, and conflicting national standards shall
be withdrawn at the latest by March 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 22174:2005.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 22174:2024 has been approved by CEN as EN ISO 22174:2024 without any modification.
International
Standard
ISO 22174
Second edition
Microbiology of the food chain —
2024-08
Polymerase chain reaction (PCR)
for the detection and quantification
of microorganisms — General
requirements and definitions
Microbiologie de la chaîne alimentaire — Réaction de
polymérisation en chaîne (PCR) pour la recherche et la
quantification de micro-organismes — Exigences générales et
définitions
Reference number
ISO 22174:2024(en) © ISO 2024
ISO 22174:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 22174:2024(en)
Contents Page
Foreword .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 General terms .2
3.2 Terms related to the extraction and purification of DNA/RNA .3
3.3 Terms related to reverse transcription of RNA to DNA .4
3.4 Terms related to DNA amplification by PCR/RT-PCR .4
3.5 Terms related to controls .5
3.6 Terms related to qPCR .6
3.7 Terms related to dPCR .7
4 Principle . 8
4.1 General .8
4.2 Laboratory sample .9
4.3 Sampling, transport and storage.9
4.4 Preparation of test sample .9
5 Microbial enrichment and virus concentration . 9
5.1 Microbial enrichment .9
5.2 Virus concentration .9
6 Nucleic acid preparation . 10
6.1 General .10
6.2 Prevention of amplification of DNA from dead cells .10
6.3 Nucleic acid extraction, release and purification .10
6.4 Nucleic acid quality and quantity .10
7 PCR amplification . .11
8 Detection and confirmation of amplicons .11
9 General environmental laboratory requirements .12
9.1 General . 12
9.2 Laboratory setup . 12
9.2.1 General . 12
9.2.2 Control of flows . 13
9.2.3 Cleaning of laboratory .14
9.2.4 Environmental monitoring for nucleic acid contamination .14
10 Reagents and consumables . 14
11 Equipment . 14
12 Procedure .15
12.1 Enrichment and sample treatment . 15
12.2 Amplification .16
12.2.1 General .16
12.2.2 Control reaction .16
12.2.3 Detection of amplicon .18
12.2.4 Data analysis .18
12.3 Evaluation .19
12.3.1 Qualitative evaluation .19
12.3.2 Quantitative evaluation . 20
12.4 Test report .21
13 Performance characteristics of PCR-based methods .21
14 Validation and verification of PCR-based methods.21
14.1 General .21
iii
ISO 22174:2024(en)
14.2 Validation .21
14.3 Verification . . 22
Annex A (informative) Fluorescence signals and amplification curve .23
Bibliography .26
iv
ISO 22174:2024(en)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 TC 34, Food products, Subcommittee SC 9, Microbiology,
in collaboration with the European Committee for Standardization (CEN) Technical Committee CEN/TC 463,
Microbiology of the food chain, in accordance with the Agreement on technical cooperation between ISO and
CEN (Vienna Agreement).
This second edition cancels and replaces ISO 22174:2005, ISO 20837:2006, ISO 20838:2006 and
ISO 22119:2011, which have been technically revised.
The main changes are as follows:
— inclusion of requirements for the implementation of digital PCR;
— inclusion of requirements for laboratory flows monitoring including environmental monitoring for PCR;
— extension of 12.2.2 control reaction with descriptions of the different controls;
— change of 12.3 to include quantitative evaluation;
— inclusion of Clause 14 on validation and verification.
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.
v
International Standard ISO 22174:2024(en)
Microbiology of the food chain — Polymerase chain reaction
(PCR) for the detection and quantification of microorganisms
— General requirements and definitions
1 Scope
This document specifies the general requirements for the in vitro amplification of nucleic acid sequences
(DNA or RNA).
This document is applicable to the testing for microorganisms and viruses from the food chain using the
polymerase chain reaction (PCR). This document, or parts of it, is applicable to other fields of PCR diagnostics
based on a case-by-case evaluation.
The minimum requirements laid down in this document are intended to ensure that comparable and
reproducible results are obtained in different laboratories.
This document has been established for microorganisms from the food chain and is applicable to:
— products intended for human consumption;
— products for feeding animals;
— environmental samples in the area of food and feed production and handling;
— samples from the primary production stage.
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 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological examinations
ISO 20836, Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms
— Thermal performance testing of thermal cyclers
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
ISO 22174:2024(en)
3.1 General terms
3.1.1
laboratory sample
sample as prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 7002:1986, A.19]
3.1.2
test sample
sample prepared from the laboratory sample (3.1.1) according to the procedure specified in the method of
test and from which test portions (3.1.3) are taken
3.1.3
test portion
measured (volume or mass) representative sample taken from the laboratory sample (3.1.1)
[SOURCE: ISO 6887-1:2017, 3.5, modified — “for use in the preparation of the initial suspension” and Note 1
to entry deleted.]
3.1.4
reference material
material, sufficiently homogeneous and stable with respect to one or more specified properties, which has
been established to be fit for its intended use in a measurement process
Note 1 to entry: Reference material producers fulfilling the requirements of ISO 17034 are considered to be competent.
[SOURCE: ISO Guide 30:2015, 2.1.1, modified — Notes to entry deleted and a new Note 1 to entry added.]
3.1.5
matrix
all the components of the sample
[SOURCE: ISO 16140-1:2016, 2.38, modified — “(product)” deleted in the term.]
3.1.6
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form
3.1.7
deoxyribonuclease
DNase
enzyme which degrades deoxyribonucleic acid (DNA) (3.1.6)
3.1.8
amplicon
deoxyribonucleic acid (DNA) (3.1.6) amplified by polymerase chain reaction (PCR) (3.1.17)
3.1.9
ribonucleic acid
RNA
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
3.1.10
ribonuclease
RNase
enzyme which degrades ribonucleic acid (RNA) (3.1.9)
3.1.11
nucleic acid
polymer of deoxyribonucleotides or ribonucleotides
ISO 22174:2024(en)
3.1.12
target nucleic acid sequence
nucleic acid sequence selected for amplification
3.1.13
endogenous sequence
nucleic acid sequence naturally present in the tested matrix (3.1.5)
3.1.14
exogenous sequence
nucleic acid sequence naturally absent in the tested matrix (3.1.5)
3.1.15
detection of polymerase chain reaction product
detection of amplicon
process which signals the presence of an amplicon (3.1.8)
3.1.16
confirmation of polymerase chain reaction product
confirmation of amplicon
process which demonstrates that the amplicon (3.1.8) originates from the target nucleic acid sequence
(3.1.12)
3.1.17
polymerase chain reaction
PCR
enzymatic procedure that allows in vitro amplification of deoxyribonucleic acid (DNA) (3.1.6)
3.1.18
endpoint polymerase chain reaction
endpoint PCR
procedure using PCR amplification followed by separate detection of amplicons (3.1.8) after the completion
of the PCR
3.1.19
real-time polymerase chain reaction
real-time PCR
procedure which combines PCR amplification with the detection and/or quantification of specific amplicons
(3.1.8) during the amplification process
3.1.20
multiplex polymerase chain reaction
multiplex PCR
PCR (3.1.17) allowing the detection of multiple targets simultaneously within a single reaction tube, where
more primer pairs (and probes) are used within one master mix (3.4.4)
3.2 Terms related to the extraction and purification of DNA/RNA
3.2.1
nucleic acid extraction
sample treatment for the release of nucleic acids (3.1.11)
3.2.2
nucleic acid purification
method to reduce the amount of polymerase chain reaction (PCR) (3.1.17) inhibitors in the eluate
ISO 22174:2024(en)
3.3 Terms related to reverse transcription of RNA to DNA
3.3.1
reverse transcriptase
enzyme which catalyses the reverse transcription (3.3.2) of ribonucleic acid (RNA) (3.1.9) to a complementary
single-stranded deoxyribonucleic acid (cDNA)
3.3.2
reverse transcription
RT
synthesis of complementary single-stranded deoxyribonucleic acid (cDNA) from a ribonucleic acid (RNA)
template using a reverse transcriptase (3.3.1)
3.3.3
reverse transcription-polymerase chain reaction
RT-PCR
method consisting of two reactions, a reverse transcription (RT) (3.3.2) of ribonucleic acid (RNA) (3.1.9) to
single-stranded complementary deoxyribonucleic acid (cDNA), followed by a PCR (3.1.17)
Note 1 to entry: One-step RT-PCR is performed in a single tube.
Note 2 to entry: Two-step RT-PCR can either be performed sequentially in a single tube or in two different tubes.
3.4 Terms related to DNA amplification by PCR/RT-PCR
3.4.1
deoxyribonucleic acid polymerase
DNA polymerase
thermostable enzyme which catalyses DNA (3.1.6) synthesis
Note 1 to entry: DNA polymerase can also cleave a hybridized nucleic acid molecule using its 5′-3′-exonuclease activity.
It is dependent on the type of enzyme and can be present in, for example, Taq-, Tth- and Tfl-polymerase.
3.4.2
deoxyribonucleoside triphosphate
dNTP
solution containing deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP),
deoxyguanosine triphosphate (dGTP), deoxythymidine triphosphate (dTTP) and/or deoxyuridine
triphosphate (dUTP)
3.4.3
thermal cycler
automatic device that performs defined heating and cooling cycles usable for polymerase chain reaction
(PCR) (3.1.17) or real-time PCR (3.1.19) or digital PCR (3.7.1)
Note 1 to entry: The thermal cycler can be a block-based or (individual) reaction-chamber-based thermal cycler.
[SOURCE: ISO 20836:2021, 3.2.1, modified — “or digital PCR” added.]
3.4.4
master mix
mixture of reagents needed for nucleic acid amplification except for the target nucleic acid sequence (3.1.12)
[SOURCE: ISO 17822:2020, 3.27, modified — “nucleic acid sequence” replaced “DNA and the controls”.]
3.4.5
primer
oligonucleotide of defined length and sequence complementary to a segment of the target nucleic acid
sequence (3.1.12), used to signal the starting point for deoxyribonucleic acid (DNA) polymerase to extend the
new DNA strand
ISO 22174:2024(en)
3.4.6
fluorescent probe
oligonucleotide of defined sequence coupled with one or more fluorescent molecules
Note 1 to entry: Any system emitting a fluorescence signal after specific hybridization to the target nucleic acid
sequence (3.1.12) which can be detected by the specific equipment can be used as a fluorescent probe.
3.4.7
background fluorescence
background
intrinsic level of fluorescence resulting from the reagents, consumables and instruments used
3.4.8
molecular beacon
fluorescent probe consisting of three different parts: a central part complementary to the target nucleic acid
sequence (3.1.12), plus a 5′-part and a 3′-part which are complementary, and where the reporter is attached
to one arm of the molecule, while the end of the other arm carries the quencher
3.4.9
hybridization probe
system of two fluorescent probes coupled with one fluorescent molecule each, where one molecule serves as
fluorescence resonance energy transfer (FRET) donor and the other serves as FRET acceptor
3.4.10
hydrolysis probe
fluorescent probe coupled with a fluorophore and quencher which are sterically separated by the
5′-3′-exonuclease activity of the enzyme during the amplification process
3.4.11
denaturation
process which results in the separation of the double-stranded nucleic acid into single-stranded nucleic acids
3.4.12
hybridization
specific binding of complementary nucleic acid sequences under suitable reaction conditions
3.4.13
annealing
pairing of complementary single strands of nucleic acids to form a double-stranded molecule
3.4.14
hot-start polymerase chain reaction
hot-start PCR
activation of thermostable deoxyribonucleic acid (DNA) polymerase by an initial heating step to avoid non-
specific amplification
3.5 Terms related to controls
3.5.1
negative process control
target free sample which is run through all stages of the analytical process
Note 1 to entry: The process can include sample preparation, enrichment, nucleic acid extraction and target
amplification.
3.5.2
positive process control
sample, spiked with a microorganism, treated in the same way as the test samples (3.1.2) to monitor the
entire process of the polymerase-chain-reaction-based method
ISO 22174:2024(en)
3.5.3
internal process control
control used for the quality assessment of the entire protocol, which is therefore added to the investigated
sample material to undergo the same procedure as the target microorganism
Note 1 to entry: Due to its mode of action, an internal control shall be selected which assumingly is naturally absent in
the tested matrix.
3.5.4
negative extraction control
extraction blank
control carried through all steps of the nucleic acid extraction procedure in the absence of a test sample (3.1.2)
3.5.5
internal amplification control
nucleic acid (3.1.11) added to each reaction in a defined amount or copy number which serves as an internal
control for amplification
Note 1 to entry: This nucleic acid sequence can be endogenous (naturally present in the tested matrix) or exogenous
(naturally absent in the tested matrix).
Note 2 to entry: An exogenous internal amplification control can be homologous (amplified using the same primers
as used for amplification of the target) or heterologous (amplified using different primers than those used for
amplification of the target). A homologous internal amplification control amplicon shall be distinguishable from the
microbial target amplicon (e.g. by size or by insertion of a different probe-binding sequence).
3.5.6
external amplification control
control nucleic acid added to an aliquot of the extracted nucleic acid in a defined amount or copy number
serving as a control for amplification in a separate reaction
Note 1 to entry: It is strongly recommended that the external amplification control amplicon is distinguishable from
the target amplicon (e.g. by insertion of a restriction enzyme target sequence).
3.5.7
positive polymerase chain reaction control
positive PCR control
reaction containing the target nucleic acid in a defined amount or copy number
3.5.8
negative polymerase chain reaction control
negative PCR control
no-template control
NTC
PCR control made with water (or other PCR-inert substrate such as grinding or elution buffer) free of target
nucleic acid and PCR inhibitors
3.6 Terms related to qPCR
3.6.1
quantitative polymerase chain reaction
qPCR
method allowing the quantification in a nucleic acid template of the number of a specific nucleic acid
sequence using specific oligonucleotides
ISO 22174:2024(en)
3.6.2
quantification cycle
C
q
cycle at which the fluorescence signal can be distinguished from the
background fluorescence (3.4.7) entering into the exponential phase of target amplification
Note 1 to entry: Quantification cycle is a generic term which includes cycle threshold (C ), crossing point (C ), take off
t p
point (TOP) and all other instrument specific terms referring to the fractional cycle used to detect or quantify the
target in the real-time PCR assay.
Note 2 to entry: The quantification cycle is based either on a threshold applied to all samples or on a regression
analysis of the signal, for each sample.
[SOURCE: ISO 20395:2019, 3.8, modified — The definition has been revised. “real-time PCR” replaced “qPCR”
in the domain and Note 1 to entry.]
3.6.3
relative quantification by real-time polymerase chain reaction
relative quantification by real-time PCR
procedure involving PCR amplification to determine the levels of changes of a target nucleic acid relative to
the levels of a reference nucleic acid of known concentration, measured within the same or in separate PCR
reactions
3.6.4
absolute quantification by real-time polymerase chain reaction
absolute quantification by real-time PCR
procedure involving PCR amplification to determine the concentration of a target nucleic acid in a sample by
comparison with a standard curve, derived from standards containing a defined amount of target
3.6.5
passive reference
fluorescent molecules present in the reaction mix used to normalize
the signal
3.7 Terms related to dPCR
3.7.1
digital polymerase chain reaction
digital PCR
dPCR
procedure in which nucleic acid templates are randomly and independently distributed across multiple
partitions (3.7.2) of nominally equivalent volume, such that some partitions contain template and others do
not, followed by PCR amplification of target sequences and detection of specific amplicons (3.1.8), providing
a count of the number of partitions with a positive and negative signal for the target template
Note 1 to entry: dPCR can also provide the qualitative results “detected” or “not detected”.
Note 2 to entry: In certain instances, whole cells or organisms can be partitioned and lysis is performed in the
individual partitions to allow amplification of target templates.
[SOURCE: ISO 20395:2019, 3.10, modified — “are randomly and independently” added before “distributed”.
Original Notes 1 and 2 to entry deleted and new Notes to entry added.]
3.7.2
partition
droplets or chambers of nominally equivalent volume into which digital polymerase chain reaction (dPCR)
(3.7.1) mix of reagents and template is randomly distributed and then amplified by PCR
[SOURCE: ISO 20395:2019, 3.22]
ISO 22174:2024(en)
3.7.3
negative cluster
set of negative results from partitions that contained the reaction mix, without the target sequence,
representing the negative partitions
3.7.4
positive cluster
set of positive results from partitions that contained the reaction mix, with amplification of the target
sequence, representing the positive partitions
3.7.5
separability
distance between the negative cluster (3.7.3) and the positive cluster (3.7.4) in the analysis diagram (1D Dot
Plot) generated after reading the partition signals
Note 1 to entry: Separability can vary depending on the quality of the sample and the amount of target. Therefore, it is
important to pay attention to the separability of these two clusters when a weak positive sample is detected.
3.7.6
analysis threshold
limit of separation between the negative cluster (3.7.3) and the positive
cluster (3.7.4)
Note 1 to entry: The threshold limit can be set automatically by the analysis software or manually by the operator
3.7.7
limit of blank
LoB
highest number of partitions appearing positive, with more than 95 %
probability, when testing samples in the absence of the target nucleic acid sequence of the pathogen, which
determines the target sequence specific “false positive” limit
Note 1 to entry: The LoB should be determined, as a minimum, from replicates of the negative amplification control
(amplification LoB) (e.g. water, elution buffer) and negative samples containing the matrix (full method LoB). The
number of negative control replicates and negative samples should be justified by the user laboratory and should be
consistent with the validation tests performed by the developer.
3.7.8
passive reference
fluorescent molecule in the reaction medium allowing to count the
partitions (3.7.2) containing the reaction mixture
3.7.9
absolute quantification by digital polymerase chain reaction
absolute quantification by dPCR
procedure involving PCR amplification and target copy quantification which does not require a standard
curve to determine the concentration of a target nucleic acid in a sample
4 Principle
4.1 General
The procedure comprises the following consecutive steps:
a) preliminary microbial enrichment of the food-borne microorganism or concentration of the virus from
the laboratory sample, if required (see Clause 5);
b) nucleic acid extraction and purification, if required (see Clause 6);
ISO 22174:2024(en)
c) amplification of the target nucleic acid sequence by PCR or RT-PCR using specific primers with the
addition of fluorescent probes or DNA double strand binding dyes in the case of real-time and digital
format (see Clause 7);
d) detection and confirmation of the specific amplicons on electrophoresis gel (endpoint PCR) or by
monitoring the fluorescence signal with an optical detection system (real-time or digital format) (see
Clause 8);
e) data analysis;
f) evaluation.
4.2 Laboratory sample
Any material from the food chain is suitable as a laboratory sample, provided it has been established by the
use of a relevant control (see Table 1) that the nucleic acid solution prepared from the test portion does not
completely inhibit the PCR.
4.3 Sampling, transport and storage
Sampling is not part of the method specified in this document. Follow the specific International Standard
dealing with the product concerned. Further details of sampling certain products are given in specific
standards that are listed in ISO 7218.
If there is no specific International Standard dealing with the sampling of the product concerned, it is
recommended that the parties concerned come to an agreement on this subject.
It is important that the laboratory receives a sample that is representative. Samples transported to the
laboratory are kept under conditions which will minimize any alteration in the number of microorganisms
present. The sample should not have been damaged or changed during transport or storage.
The recommended temperatures for samples during transport and storage are described in ISO 7218.
4.4 Preparation of test sample
Prepare the test sample from the laboratory sample in accordance with the specific International Standard
dealing with the product concerned. Follow the procedures specified in the ISO 6887 series. The preparation
of the test sample for the detection of viruses can be different and is specified in, for example, the ISO 15216
series for some products. If there is no specific International Standard available, it is recommended that the
parties concerned come to an agreement on this subject.
5 Microbial enrichment and virus concentration
5.1 Microbial enrichment
Microbial enrichment may start directly from the test portion. This can be done by stimulating growth of
the target microorganism by culturing the test portion in selective or non-selective liquid nutrient media.
Filtration and/or concentration such as immunomagnetic separation can be also used. Specific antimicrobial
compounds can be used to inhibit the growth of non-target microorganisms.
5.2 Virus concentration
The foodstuffs and food surfaces are often highly complex matrices and the target viruses can be present
at low concentrations. It is therefore necessary to carry out matrix-specific virus extraction and/or
concentration in order to provide a substrate for subsequent common parts of the process. The method
depends upon the matrix, some of which are specified in the ISO 15216 series.
ISO 22174:2024(en)
6 Nucleic acid preparation
6.1 General
The purpose of the nucleic acid preparation step is to obtain a nucleic acid solution from the laboratory
sample that does not significantly inhibit the PCR. The microbial cells in the laboratory sample or enriched/
concentrated culture (see Clause 5) are lysed to release their nucleic acid (DNA and/or RNA). If required, a
concentration stage (e.g. centrifugation or filtration) is performed prior to lysis and/or a purification step
is performed following lysis. The nucleic acid solution obtained from the extraction step should contain a
sufficient amount of target nucleic acid of suitable quality.
The resulting nucleic acid solution should contain as few as possible substances with detectable PCR
inhibition or fluorescence interference.
NOTE 1 Fluorescence is often derived from coloured reaction vessels and some ingredients of enrichment broths.
Depending on the sample received it can be necessary to pre-treat or dilute the test portion, i.e. some
products can contain compounds that inhibit PCR. If PCR inhibition is identified (by the use of a control
reaction, see 12.3), the enriched test portion or the lysate can be diluted prior to the PCR step.
NOTE 2 The method of extraction can have a great influence on nucleic acids degradation and/or yield and/or
amplification efficiency. The most suitable extraction method strongly depends on the characteristics of the test
portion.
6.2 Prevention of amplification of DNA from dead cells
Some test portions can contain high amounts of DNA originating from dead cells of the target organism (e.g.
highly processed samples, environmental samples collected after disinfection or phage treated samples).
These dead cells, if present in high enough concentrations, can lead to PCR positive re
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