oSIST prEN ISO 22174:2023
(Main)Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and quantification of microorganisms - General requirements and definitions (ISO/DIS 22174:2022)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and quantification of microorganisms - General requirements and definitions (ISO/DIS 22174:2022)
This International Standard gives the general requirements for the in vitro amplification of nucleic
acid sequences (DNA or RNA). It is applicable to the testing of foodstuffs and isolates obtained
from foodstuffs for food-borne microorganisms using the polymerase chain reaction (PCR).
The minimum requirements laid down in this International Standard are intended to ensure that
comparable and reproducible results are obtained in different laboratories.
This International Standard has been established for food-borne microorganisms in or isolated
from food and feed matrices and is applicable to:
— products intended for human consumption,
— products intended for animal feeding,
— environmental samples in the area of food and feed production, handling, and
— samples from the primary production stage.
Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis von Mikroorganismen - Allgemeine Anforderungen und Definitionen (ISO/DIS 22174:2022)
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/DIS 22174:2022)
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za ugotavljanje prisotnosti in kvantifikacijo mikroorganizmov - Splošne zahteve in definicije (ISO/DIS 22174:2022)
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN ISO 22174:2023
01-januar-2023
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za
ugotavljanje prisotnosti mikroorganizmov - Splošne zahteve in definicije (ISO/DIS
22174:2022)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection and
quantification of microorganisms - General requirements and definitions (ISO/DIS
22174:2022)
Mikrobiologie der Lebensmittelkette - Polymerase-Kettenreaktion (PCR) zum Nachweis
von Mikroorganismen - Allgemeine Anforderungen und Definitionen (ISO/DIS
22174:2022)
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/DIS 22174:2022)
Ta slovenski standard je istoveten z: prEN ISO 22174
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 22174:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 22174:2023
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oSIST prEN ISO 22174:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 22174
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2022-10-20 2023-01-12
Microbiology of the food chain — 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 pathogènes dans les aliments — Exigences générales et définitions
ICS: 07.100.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
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THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
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BEING ACCEPTABLE FOR INDUSTRIAL,
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NATIONAL REGULATIONS.
ISO/DIS 22174:2022(E)
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
DRAFT INTERNATIONAL STANDARD
ISO/DIS 22174
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
Microbiology of the food chain — 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 pathogènes dans les aliments — Exigences générales et définitions
ICS: 07.100.30
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2022
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
NOT BE REFERRED TO AS AN INTERNATIONAL
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WHICH REFERENCE MAY BE MADE IN
Reference number
Email: copyright@iso.org
NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 22174:2022(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
ii
© ISO 2022 – All rights reserved
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2022
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 7
4.1 General . 7
4.2 Test material . 8
4.3 Sampling, transport and storage. 8
4.4 Preparation of test sample . 8
5 Microbial enrichment and virus concentration . 8
5.1 Microbial enrichment . 8
5.2 Virus concentration . 8
6 Nucleic acid preparation . 9
6.1 General . 9
6.2 DNA removal from dead cells . 9
6.3 Nucleic acid extraction, release and purification . 9
6.4 Nucleic acid quality and quantity . 9
7 PCR amplification . .10
8 Detection and confirmation of PCR products .10
9 General environmental laboratory requirements .11
9.1 General . 11
9.2 Laboratory setup . 11
9.2.1 General . 11
9.2.2 Control of flows . 12
9.2.3 Cleaning of laboratory .13
9.2.4 Environmental monitoring for PCR . 13
10 Reagents and consumables .13
11 Equipment .14
11.1 General . 14
11.2 Specific equipment for PCR . 14
11.2.1 Thermal cycler . 14
11.2.2 System for detection of PCR products, comprising . 14
11.2.3 Real-time thermal cycler . 14
11.2.4 Digital PCR thermal cycler (for dPCR) . 14
11.2.5 Pipettes (for all PCR formats) . 15
12 Procedure .15
12.1 Enrichment and sample treatment . 15
12.2 Amplification . 15
12.2.1 General .15
12.2.2 Control reaction .15
12.2.3 Detection of amplicon . 16
12.2.4 Data analysis . 16
12.3 Evaluation . 16
12.3.1 Qualitative evaluation . 16
12.3.2 Quantitative evaluation . 17
12.4 Test report . 18
13 Performance characteristics of PCR based methods .18
iii
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
14 Validation and verification of PCR based methods .18
14.1 General . 18
14.2 Validation . 19
14.3 Verification . . 19
Bibliography .20
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(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 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 the first edition (ISO 22174:2005), which has been technically
revised.
The main changes are as follows:
— inclusion of standards ISO 20837:2006, ISO 20838:2006 and ISO 22119;
— inclusion of requirements for the implementation of digital PCR;
— inclusion of requirements for laboratory flows monitoring including environmental monitoring for
PCR;
— change of the title and inclusion of clause 12.5 to include quantification;
— inclusion of a new clause dealing with validation and verification;
— inclusion of methods from microbiology of the food chain utilizing PCR in the bibliography for
information;
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
Introduction
Because of the large variety of food and feed products, this horizontal method can not be appropriate
in every detail for certain products. In this case, different methods which are specific to these products
may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt should
be made to apply this horizontal method as far as possible.
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oSIST prEN ISO 22174:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 22174:2022(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection and quantification of
microorganisms — General requirements and definitions
1 Scope
This document establishes the general requirements for the in vitro amplification of nucleic acid
sequences (DNA or RNA). It is applicable to the testing for microorganisms and viruses from the food
chain using the polymerase chain reaction (PCR).
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.
Validation of alternative methods, single laboratory validation, interlaboratory validation for non-
proprietary or alternative confirmation methods are not covered by this document. These items in the
frame of the microbiology of the food chain are covered by the ISO 16140 series.
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 7218, Microbiology of food and animal feeding stuffs — 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. For definitions concerning
validation, see ISO 3534-1, ISO 5725-1 and ISO 16140-1.
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/
3.1
annealing
pairing of complementary single strands of nucleic acids to form a double-stranded molecule
1
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
3.2
background fluorescence
“background”
intrinsic level of fluorescence resulting from the reagents, consumables and instruments used
3.3
confirmation of PCR product
process which demonstrates that the PCR product originates from the target sequence
3.4
denaturation
process which results in the separation of the double-stranded nucleic acid into single-stranded nucleic
acid
3.5
deoxyribonucleoside triphosphate
dNTP
solution containing dATP, dCTP, dGTP, dTTP and/or dUTP
3.6
detection
recognition of the presence of the target nucleic acid
3.7
detection of PCR product
process which signals the presence of a PCR product
3.8
digital PCR
dPCR
procedure in which nucleic acid templates are randomly and independently distributed across multiple
partitions 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 PCR products, providing
a count of the number of partitions with a positive and negative signal for the target template
[SOURCE: ISO 20395:2019, 3.10, modified — Removed Note 1 to entry and Note 2 to entry]
3.9
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
3.10
DNA polymerase
thermostable enzyme which catalyses DNA 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, for example, in Taq-, Tth- and Tfl-polymerase.
3.11
DNA probe
nucleic acid molecule with a defined sequence used to detect target DNA by hybridization
3.12
target nucleic acid sequence
nucleic acid sequence selected for amplification
3.13
endogeneous sequence
nucleic acid sequence naturally present in the tested matrix
2
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
3.14
endpoint PCR
procedure using PCR amplification followed by separate detection of PCR products after the completion
of the PCR cycle
3.15
external amplification control
control DNA or RNA 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. This DNA or RNA sequence can
be endogenous (naturally present in the tested matrix) or exogeneous (naturally absent in the tested
matrix)
3.16
exogenous sequence
nucleic acid sequence naturally absent in the tested matrix
3.17
fluorescent probe
oligonucleotide or oligonucleotide analogue 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 which can be detected by the specific equipment can be used as a fluorescent probe.
3.18
fluorescence resonance energy transfer
FRET
distance-dependent energy transfer from a donor molecule to an acceptor molecule resulting in
enhanced fluorescence of the acceptor molecule after excitation with electromagnetic radiation of a
defined wavelength
3.19
hot-start PCR
activation of thermostable DNA polymerase by an initial heating step to avoid non-specific amplification
3.20
hybridization
specific binding of complementary nucleic acid sequences under suitable reaction conditions
3.21
hybridization probe
system of two fluorescent probes coupled with one fluorescent molecule each, where one molecule
serves as FRET donor and the other serves as FRET acceptor
3.22
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
3.23
internal amplification control
DNA added to each reaction in a defined amount or copy number which serves as an internal control
for amplification. This DNA sequence can be endogeneous (naturally present in the tested matrix) or
exogeneous (naturally absent in the tested matrix)
3.24
laboratory sample
Sample prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 7002:1986, A.19]
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
3.25
mastermix
mixture of reagents needed for PCR nucleic acid amplification with the exception ofexcept for the target
DNAtarget nucleic acid sequencenucleic acid from the test sample
[SOURCE: ISO 17822-2:2020:2016, 3.27, modified — “DNA and the controls” has been removed and
replaced by target nucleic acid sequencenucleic acid from the test sample.]
3.26
matrix
all the components of the sample
[SOURCE: ISO 16140-1:2016, 2.38, modified — In the term, “(product)” has been removed.]
3.27
molecular beacon
fluorescent probe consisting of three different parts: a central part complementary to the target nucleic
acid sequence, plus a 5′-part and a 3′-part which are complementary; the reporter is attached to one
arm of the molecule, while the end of the other carries a quencher
3.28
multiplex PCR
PCR allowing the detection of multiple targets simultaneously within a single reaction
3.29
negative extraction control
extraction blank
control carried through all steps of the DNA or RNA extraction procedure in the absence of a test sample
3.30
negative PCR control
PCR control made with water (or other PCR-inert substrate such as grinding or elution buffer) free of
target nucleic acid and PCR inhibitors
3.31
negative process control
target free sample of the matrix which is run through all stages of the analytical process
Note 1 to entry: The process can include sample preparation, enrichment, DNA or RNA extraction and target
amplification.
3.32
nucleic acid
polymer of deoxyribonucleotides or ribonucleotides
3.33
nucleic acid extraction
sample treatment for the release of target nucleic acid
3.34
nucleic acid purification
method to reduce the amount of inhibitors.
3.35
one-step RT-PCR
method combining reverse transcription (RT) of RNA to cDNA and PCR in a single reaction tube
3.36
passive reference
fluorescent molecules present in the reaction mix used to normalize the signal
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
3.37
PCR product
amplicon
DNA amplified by PCR
3.38
PCR quality DNA
DNA template of sufficient length, purity and quantity for PCR
3.39
polymerase chain reaction
PCR
enzymatic procedure that allows in vitro amplification of DNA
3.40
positive PCR control
reaction containing the target DNA or RNA in a defined amount or copy number
3.41
positive process control
sample, spiked with a microorganism or virus, which should be treated in the same way as the test
samples to monitor the reaction
3.42
primer
oligonucleotide of defined length and sequence complementary to a segment of the target nucleic acid
sequence
3.43
primer extension
enzymatic reaction which leads to the synthesis of a new DNA strand by the addition of single
deoxyribonucleotides to the 3'-end of the primer sequence
3.44
quencher
fluorescent molecule serving as an energy acceptor and thus quenching the fluorescence signal of the
reporter (donor)
3.45
real-time PCR
procedure which combines PCR amplification with the detection and/or quantification of specific PCR
products during the amplification process
3.46
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 — The original Notes to entry have been deleted and a
new Note 1 to entry was added]
3.47
reporter
fluorescent molecule used to detect the hybridization of specific probes by excitation with
electromagnetic radiation of an appropriate wavelength
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oSIST prEN ISO 22174:2023
ISO/DIS 22174:2022(E)
3.48
reverse transcription
RT
synthesis of DNA from an RNA template using a reverse transcriptase enzyme
3.49
reverse transcriptase
enzyme which catalyses the reverse transcription of RNA to DNA
3.50
ribonuclease
RNase
enzyme which de
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