SIST EN ISO 20836:2022
(Main)Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of microorganisms - Thermal performance testing of thermal cyclers (ISO 20836:2021)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of microorganisms - Thermal performance testing of thermal cyclers (ISO 20836:2021)
This document specifies requirements for the installation, maintenance, temperature calibration and temperature performance testing of standard thermal cyclers and real-time thermal cyclers. It is applicable to the detection of microorganisms as well as any other applications in the food chain using polymerase chain reaction (PCR)-based methods.
This document has been established for food testing, but is also applicable to other domains using thermal cyclers (e.g. environmental, human health, animal health, forensic testing).
Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Leistungsprüfung für PCR-Geräte (ISO 20836:2021)
Dieses Dokument legt Anforderungen an die Installation, Instandhaltung, Temperaturkalibrierung und Temperaturleistungsprüfung von Standard-Thermocyclern und Real-Time-Thermocyclern fest. Es ist anwendbar für den Nachweis von Mikroorganismen sowie alle anderen Anwendungen in der Lebensmittelkette mithilfe von Verfahren, die auf einer Polymerase-Kettenreaktion (PCR) beruhen.
Dieses Dokument wurde für Lebensmittelprüfungen erstellt, ist aber auch in anderen Bereichen anwendbar, in denen Thermocycler benutzt werden (z. B. Umwelt, menschliche Gesundheit, Tiergesundheit, forensische Tests).
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR) pour la recherche de micro-organismes - Essais de performance thermique des thermocycleurs (ISO 20836:2021)
Le présent document spécifie les exigences relatives à l’installation, à la maintenance, à l’étalonnage de la température et aux essais de performance thermique des thermocycleurs classiques et des thermocycleurs en temps réel. Il s’applique à la recherche de micro-organismes, ainsi qu’à toute autre application de la chaîne alimentaire utilisant des méthodes fondées sur la réaction de polymérisation en chaîne (PCR).
Le présent document a pour vocation de s’appliquer aux essais sur les aliments, mais est aussi applicable à d’autres domaines utilisant des thermocycleurs (par exemple, des essais relatifs à l’environnement, à la santé humaine, à la santé animale, à la médecine légale).
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za ugotavljanje prisotnosti mikroorganizmov - Preskus toplotnega delovanja cikličnih termostatov (ISO 20836:2021)
Ta dokument določa zahteve za namestitev, vzdrževanje, umerjanje temperature in preskušanje toplotnega delovanja standardnih cikličnih termostatov in cikličnih termostatov v realnem času. Uporablja se za ugotavljanje prisotnosti mikroorganizmov in druge namene v prehranjevalni verigi z uporabo
metod, ki temeljijo na verižni reakciji s polimerazo (PCR).
Ta dokument je bil pripravljen za preskušanje hrane, vendar je uporaben tudi za druga področja, ki uporabljajo ciklične termostate (npr. okolje, zdravje ljudi, zdravje živali, forenzično preskušanje).
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2022
Nadomešča:
SIST-TS CEN ISO/TS 20836:2005
Mikrobiologija v prehranski verigi - Polimerazna verižna reakcija (PCR) za
ugotavljanje prisotnosti mikroorganizmov - Preskus toplotnega delovanja cikličnih
termostatov (ISO 20836:2021)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of
microorganisms - Thermal performance testing of thermal cyclers (ISO 20836:2021)
Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR)
zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Leistungsprüfung
für PCR-Geräte (ISO 20836:2021)
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)
pour la recherche de micro-organismes - Essais de performance thermique des
thermocycleurs (ISO 20836:2021)
Ta slovenski standard je istoveten z: EN ISO 20836:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20836
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes CEN ISO/TS 20836:2005
English Version
Microbiology of the food chain - Polymerase chain reaction
(PCR) for the detection of microorganisms - Thermal
performance testing of thermal cyclers (ISO 20836:2021)
Microbiologie de la chaîne alimentaire - Réaction de Mikrobiologie von Lebensmitteln und Futtermitteln -
polymérisation en chaîne (PCR) pour la recherche de Polymerase-Kettenreaktion (PCR) zum Nachweis von
micro-organismes - Essais de performance thermique pathogenen Mikroorganismen in Lebensmitteln -
des thermocycleurs (ISO 20836:2021) Leistungsprüfung für PCR-Geräte (ISO 20836:2021)
This European Standard was approved by CEN on 19 November 2021.
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, Turkey 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20836:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20836:2021) 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 June 2022, and conflicting national standards shall be
withdrawn at the latest by June 2022.
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 CEN ISO/TS 20836:2005.
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, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 20836:2021 has been approved by CEN as EN ISO 20836:2021 without any modification.
INTERNATIONAL ISO
STANDARD 20836
First edition
2021-11
Microbiology of the food chain —
Polymerase chain reaction (PCR)
for the detection of microorganisms
— Thermal performance testing of
thermal cyclers
Microbiologie de la chaîne alimentaire — Réaction de polymérisation
en chaîne (PCR) pour la recherche de micro-organismes — Essais de
performance thermique des thermocycleurs
Reference number
ISO 20836:2021(E)
ISO 20836:2021(E)
© ISO 2021
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 20836:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 Polymerase chain reaction . 1
3.2 Thermal cycler . 2
3.3 Temperature characteristics . 2
3.4 Temperature measurement . 5
4 Installation of thermal cyclers . .6
5 Maintenance of thermal cyclers .6
6 Performance testing of thermal cyclers . 6
6.1 General . 6
6.2 Performance testing programme . 7
6.3 Metrological traceability . 7
6.4 Temperature performance testing method . 8
6.4.1 General . 8
6.4.2 Principle . 8
6.4.3 Equipment . 8
6.4.4 Environmental conditions . 9
6.4.5 Procedure . 9
6.4.6 Performance test results . 10
6.4.7 Performance test report . 10
6.4.8 Compliancy testing . 11
6.5 Optical performance testing method . 11
Annex A (informative) Sensor locations .13
Annex B (informative) Universal temperature protocol .18
Annex C (informative) Compliancy testing .19
Annex D (informative) Example of a thermal cycler temperature profile .22
Annex E (informative) Example of performance test and compliancy test .23
Bibliography .27
iii
ISO 20836:2021(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 first edition International Standard cancels and replaces the first edition Technical Specification
(ISO/TS 20836:2005), which has been technically revised. The main changes compared with the
previous edition are as follows:
— the Scope has been extended to include both thermal cyclers and real-time thermal cyclers;
— the physical performance testing method has been described in more detail, and the biochemical
performance testing method has been taken out;
— information for laboratories regarding ISO/IEC 17025 has been included;
— the performance testing method has been aligned with ISO/IEC 17025;
— compliancy testing has been added;
— in Annex C, two procedures to set PCR-method-based specifications have been added.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO 20836:2021(E)
Introduction
This document is part of a family of International Standards under the general title Microbiology of the
food chain — Polymerase chain reaction (PCR) for the detection of food borne pathogens:
— ISO 22174, General requirements and definitions;
— ISO 20837, Requirements for sample preparation for qualitative detection;
— ISO 20836, Thermal performance testing of thermal cyclers;
— ISO 20838, Requirements for amplifications and detection for qualitative methods.
This document describes a method for performance testing for standard thermal cyclers and real-
time thermal cyclers that allows laboratories to evaluate if the thermal cycler used is suitable for the
intended use and meets the specifications set by the laboratory.
The described method is based on a physical method that measures directly in the thermal cycler block
in block-based thermal cyclers and in tubes in heated-chamber-based thermal cyclers. The described
method provides a measurement uncertainty that is sufficiently low to allow meaningful comparison to
specifications.
Furthermore, the method does meet the criteria of a metrological traceable calibration method in case
it is used by ISO/IEC 17025-compliant laboratories.
v
INTERNATIONAL STANDARD ISO 20836:2021(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of microorganisms —
Thermal performance testing of thermal cyclers
1 Scope
This document specifies requirements for the installation, maintenance, temperature calibration
and temperature performance testing of standard thermal cyclers and real-time thermal cyclers. It is
applicable to the detection of microorganisms as well as any other applications in the food chain using
polymerase chain reaction (PCR)-based methods.
This document has been established for food testing, but is also applicable to other domains using
thermal cyclers (e.g. environmental, human health, animal health, forensic testing).
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/IEC Guide 98-3:2008, Uncertainty of measurement — Part 3: Guide to the expression of uncertainty in
me a s ur ement (GUM: 1995)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Polymerase chain reaction
3.1.1
polymerase chain reaction
PCR
enzymatic procedure that allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005, 3.4.1]
3.1.2
PCR method
test method based on the PCR (3.1.1) technique
Note 1 to entry: Examples include, but are not limited to, PCR, quantitative real-time PCR (qPCR), reverse
transcription PCR (RT PCR) and reverse transcription quantitative real-time PCR (RT qPCR).
ISO 20836:2021(E)
3.2 Thermal cycler
3.2.1
thermal cycler
automatic device that performs defined heating and cooling cycles necessary for PCR (3.1.1) or real-
time PCR
Note 1 to entry: The thermal cycler can be a block-based or (individual) reaction-chamber-based thermal cycler.
[SOURCE: ISO 22174:2005, 3.4.20, modified — “or real-time PCR” and Note 1 to entry have been added.]
3.2.2
reaction block
heated and cooled metal block in which PCR reaction vials, containing the PCR reaction mix, can be
inserted
Note 1 to entry: The block can be heated and cooled by a number of technologies, among which Peltier heating
and cooling is the most abundantly used.
3.2.3
reaction chamber
heated and cooled chamber in which PCR reaction vials, containing the PCR reaction mix, can be
inserted directly or in a rotor
Note 1 to entry: The chamber can be heated and cooled by a number of technologies, among which air heating
and cooling is the most abundantly used.
3.2.4
heated lid
heated cover of a thermal cycler (3.2.1), which is applied in block-based thermal cyclers onto reaction
tubes to prevent condensate of reaction mix to collect inside the cap of the reaction tube or onto the
seal, and evaporation from the reaction tube, and which applies pressure onto the tubes to ensure
proper thermal contact
3.2.5
PCR temperature protocol
heating and cooling cycles required for PCR (3.1.1), typically consisting of denaturation, annealing and
extension temperature steps, which are typically repeated 30 to 45 times
Note 1 to entry: In certain PCR methods (3.1.2), a two-step temperature protocol is used in which annealing and
extension are combined to one step.
3.3 Temperature characteristics
3.3.1
thermal cycler temperature profile
graph of the course of the temperature by performing measurements at defined intervals
Note 1 to entry: See Annex D for an example graph of a thermal cycler temperature profile.
3.3.2
Tt
i
()
temperature in °C of sensor i at time t in s
3.3.3
set temperature
T
set
target temperature programmed to be reached in °C
ISO 20836:2021(E)
3.3.4
average temperature
T
avg
N
T ()t
i
T ()t =
avg ∑
N
i=1
where
Tt
() is average temperature in °C at time t;
avg
i is sensor i of N;
N is total number of sensors.
average of measured values of all active temperature sensors in °C at a specific time in s
3.3.5
temperature deviation
T
dev
Tt()=TT()t −
devavg set
average temperature (3.3.4) minus set temperature (3.3.3) in °C at a specific time in s
3.3.6
minimum temperature
T
min
Tt()=min()TT()tt. ()
min iN
minimum value of all active temperature sensors in °C at a specific time in s
3.3.7
maximum temperature
T
max
TTtt=max .T t
() ()() ()
max iN
maximum value of all active temperature sensors in °C at a specific time in s
3.3.8
temperature uniformity
T
uniformity
Tt()=TT()tt− ()
uniformity maxmin
homogeneity of the temperature distribution within the reaction block (3.2.2) or chamber, defined as
maximum temperature (3.3.7) minus minimum temperature (3.3.6) in °C at a specific time in s
3.3.9
temperature transition
T
transition
phase of fast temperature change from one set temperature to another set temperature
3.3.10
ramp rate
heat or cool rate of thermal cycler (3.2.1) in °C/s
3.3.11
average ramp rate
V
t
N
TT−
ii,%90 ,%10
V ==
t
∑
tt−
ii,%90 ,%10
i=1
where
ISO 20836:2021(E)
V is ramp rate in °C/s;
t
i is sensor i of N;
N is total number of sensors;
T
is T at 10 % temperature of the ramp slope in °C;
i,%10
i
T
is T at 90 % temperature of the ramp slope in °C;
i,%90
i
t is time in s.
heat or cool rate of thermal cycler (3.2.1) calculated between 10 % and 90 % time of the heating or
cooling slope
Note 1 to entry: The heat rate is a positive ramp rate (3.3.10). The cool rate is a negative ramp rate.
3.3.12
maximum ramp rate
V
t max
maximum heat or cool rate during heating or cooling slope in °C/s
3.3.13
maximum temperature overshoot
T
i,ovs,max
tholds=15
Tt=TT−=tholds30
() ()
ii,ovs,max ,max tholds=0 i
maximum temperature (3.3.7) value in °C of all active temperature sensors during temperature
overshoot above the average temperature (3.3.4) of the reaction block (3.2.2) or chamber temperature at
hold when heating up
Note 1 to entry: The maximum temperature overshoot is calculated between start and end of the overshoot and
is expressed relative to the temperature at 30 s hold time (3.3.18).
Note 2 to entry: The overshoot occurs typically between 0 s and 15 s hold time. See Annex D for an example
thermal cycler temperature profile (3.3.1).
3.3.14
minimum temperature undershoot
T
i,uns,min
tholds=15
Tt=TT−=tholds30
() ()
ii,uns,min ,min tholds=0 i
minimum temperature (3.3.6) value in °C of all active temperature sensors during temperature
undershoot below the average temperature (3.3.4) of reaction block (3.2.2) or chamber temperature at
hold when cooling down
Note 1 to entry: The maximum temperature undershoot is calculated between start and end of the undershoot
and is expressed relative to the temperature at 30 s hold time (3.3.18). An undershoot is an overshoot in negative
direction.
Note 2 to entry: The undershoot occurs typically between 0 s and 15 s hold time. See Annex D for an example
thermal cycler temperature profile (3.3.1).
3.3.15
average temperature overshoot
T
ovs,avg
N
T
i,ovs,max
T =
ovs,avg ∑
N
i=1
average value of maximum temperature overshoots (3.3.13) of all active block temperature sensors in °C
ISO 20836:2021(E)
3.3.16
average temperature undershoot
T
uns,avg
N
T
i,uns,min
T =
uns,avg ∑
N
i=1
average value of minimum temperature undershoots (3.3.14) of all active block temperature sensors in °C
3.3.17
overshoot duration
time elapsed between start and end of the overshoot in s
Note 1 to entry: The start of the overshoot is defined as the moment in time where the average temperature
(3.3.4) exceeds the average hold temperature, calculated at 30 s hold, at the beginning of the overshoot. The
end of the overshoot is defined as the moment in time where the average temperature reaches the average hold
temperature at the finish of the overshoot.
3.3.18
hold time
time elapsed between start and end of a temperature hold in s
Note 1 to entry: See Annex D for an example to determine start and end of hold.
3.4 Temperature measurement
3.4.1
temperature measurement system
temperature measurement and data logging instrument
3.4.2
sampling frequency
number of samples per second taken from a time-continuous signal to make a time-discrete signal
3.4.3
response time
time required for the temperature measurement system (3.4.1), when subjected to a change in
temperature, to react to this change
3.4.4
measurement uncertainty
parameter associated with the result of a measurement that characterizes the dispersion of the values
that could reasonably be attributed to the quantity intended to be measured
[SOURCE: ISO/IEC Guide 98-3:2008, B.2.18, modified — “quantity intended to be measured” and
replaced “measurand” and the notes to entry have been deleted.]
3.4.5
performance test
test procedure that determines the performance of a thermal cycler (3.2.1)
3.4.6
calibration
operation that, under specified conditions, in a first step, establishes a relation between the quantity
values with measurement uncertainties (3.4.4) provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in a second step, uses this information to
establish a relation for obtaining a measurement result from an indication
Note 1 to entry: Calibration should not be confused with adjustment of a measuring system, nor with auto-check,
self-verification test, verification, normalization, installation qualification (IQ), operational qualification (OQ) or
performance qualification (PQ).
ISO 20836:2021(E)
[SOURCE: ISO/IEC Guide 99:2007, 2.39, modified — Note 1 to entry has replaced the original Notes 1, 2
and 3 to entry.]
4 Installation of thermal cyclers
The manufacturer’s instructions shall be followed.
The following should be taken into consideration.
a) Thermal cyclers should be installed and operated at suitable environmental conditions that do not
invalidate the results or adversely affect the required quality of any test.
b) The environmental conditions that should at minimum be taken into account are room temperature
and relative humidity.
See the manual of the thermal cycler for recommended room conditions.
Thermal cyclers shall be located in such a way that free circulation of air is permanently allowed.
See ISO 22174 for guidelines for contamination prevention and separation of incompatible laboratory
activities.
5 Maintenance of thermal cyclers
The laboratory shall establish a maintenance programme, where appropriate, and keep records to
ensure proper functioning and prevent deterioration of the thermal cyclers.
6 Performance testing of thermal cyclers
6.1 General
If the performance testing method of this document is used as a metrological traceable temperature
calibration, as a conformity test or as a reference method, the performance test shall be carried out
with a minimum number of sensors that represent at least 12,5 % of wells for reaction blocks or
chambers < 96 wells or 12 wells for reaction blocks or chambers > 96 wells (see 6.4.5.1) and metrological
traceability (see 6.3) shall be provided up to the level of the thermal cycler. If the performance testing
method is used for other purposes, such as supplier’s quality control or supplier’s after sales service,
the number of sensors may be reduced to a minimum number of sensors that represent at least 8 % of
wells for reaction blocks or chambers < 96 wells or 8 wells for reaction blocks or chambers > 96 wells
and metrological traceability shall be provided up to the level of the temperature measurement system.
In case of individual reaction chambers, each of the individual reaction chambers shall be tested.
The decision chart in Figure 1 can be
...
SLOVENSKI STANDARD
01-februar-2022
Nadomešča:
SIST-TS CEN ISO/TS 20836:2005
Mikrobiologija živil in krme - Polimerazna verižna reakcija (PCR) za ugotavljanje
prisotnosti mikroorganizmov - Preskus toplotnega delovanja cikličnih termostatov
(ISO 20836:2021)
Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of
microorganisms - Thermal performance testing of thermal cyclers (ISO 20836:2021)
Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR)
zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Leistungsprüfung
für PCR-Geräte (ISO 20836:2021)
Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)
pour la recherche de micro-organismes - Essais de performance thermique des
thermocycleurs (ISO 20836:2021)
Ta slovenski standard je istoveten z: EN ISO 20836:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20836
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2021
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes CEN ISO/TS 20836:2005
English Version
Microbiology of the food chain - Polymerase chain reaction
(PCR) for the detection of microorganisms - Thermal
performance testing of thermal cyclers (ISO 20836:2021)
Microbiologie de la chaîne alimentaire - Réaction de Mikrobiologie von Lebensmitteln und Futtermitteln -
polymérisation en chaîne (PCR) pour la recherche de Polymerase-Kettenreaktion (PCR) zum Nachweis von
micro-organismes - Essais de performance thermique pathogenen Mikroorganismen in Lebensmitteln -
des thermocycleurs (ISO 20836:2021) Leistungsprüfung für PCR-Geräte (ISO 20836:2021)
This European Standard was approved by CEN on 19 November 2021.
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.
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EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20836:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20836:2021) 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 June 2022, and conflicting national standards shall be
withdrawn at the latest by June 2022.
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 CEN ISO/TS 20836:2005.
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, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 20836:2021 has been approved by CEN as EN ISO 20836:2021 without any modification.
INTERNATIONAL ISO
STANDARD 20836
First edition
2021-11
Microbiology of the food chain —
Polymerase chain reaction (PCR)
for the detection of microorganisms
— Thermal performance testing of
thermal cyclers
Microbiologie de la chaîne alimentaire — Réaction de polymérisation
en chaîne (PCR) pour la recherche de micro-organismes — Essais de
performance thermique des thermocycleurs
Reference number
ISO 20836:2021(E)
ISO 20836:2021(E)
© ISO 2021
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 20836:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
3.1 Polymerase chain reaction . 1
3.2 Thermal cycler . 2
3.3 Temperature characteristics . 2
3.4 Temperature measurement . 5
4 Installation of thermal cyclers . .6
5 Maintenance of thermal cyclers .6
6 Performance testing of thermal cyclers . 6
6.1 General . 6
6.2 Performance testing programme . 7
6.3 Metrological traceability . 7
6.4 Temperature performance testing method . 8
6.4.1 General . 8
6.4.2 Principle . 8
6.4.3 Equipment . 8
6.4.4 Environmental conditions . 9
6.4.5 Procedure . 9
6.4.6 Performance test results . 10
6.4.7 Performance test report . 10
6.4.8 Compliancy testing . 11
6.5 Optical performance testing method . 11
Annex A (informative) Sensor locations .13
Annex B (informative) Universal temperature protocol .18
Annex C (informative) Compliancy testing .19
Annex D (informative) Example of a thermal cycler temperature profile .22
Annex E (informative) Example of performance test and compliancy test .23
Bibliography .27
iii
ISO 20836:2021(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 first edition International Standard cancels and replaces the first edition Technical Specification
(ISO/TS 20836:2005), which has been technically revised. The main changes compared with the
previous edition are as follows:
— the Scope has been extended to include both thermal cyclers and real-time thermal cyclers;
— the physical performance testing method has been described in more detail, and the biochemical
performance testing method has been taken out;
— information for laboratories regarding ISO/IEC 17025 has been included;
— the performance testing method has been aligned with ISO/IEC 17025;
— compliancy testing has been added;
— in Annex C, two procedures to set PCR-method-based specifications have been added.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
ISO 20836:2021(E)
Introduction
This document is part of a family of International Standards under the general title Microbiology of the
food chain — Polymerase chain reaction (PCR) for the detection of food borne pathogens:
— ISO 22174, General requirements and definitions;
— ISO 20837, Requirements for sample preparation for qualitative detection;
— ISO 20836, Thermal performance testing of thermal cyclers;
— ISO 20838, Requirements for amplifications and detection for qualitative methods.
This document describes a method for performance testing for standard thermal cyclers and real-
time thermal cyclers that allows laboratories to evaluate if the thermal cycler used is suitable for the
intended use and meets the specifications set by the laboratory.
The described method is based on a physical method that measures directly in the thermal cycler block
in block-based thermal cyclers and in tubes in heated-chamber-based thermal cyclers. The described
method provides a measurement uncertainty that is sufficiently low to allow meaningful comparison to
specifications.
Furthermore, the method does meet the criteria of a metrological traceable calibration method in case
it is used by ISO/IEC 17025-compliant laboratories.
v
INTERNATIONAL STANDARD ISO 20836:2021(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of microorganisms —
Thermal performance testing of thermal cyclers
1 Scope
This document specifies requirements for the installation, maintenance, temperature calibration
and temperature performance testing of standard thermal cyclers and real-time thermal cyclers. It is
applicable to the detection of microorganisms as well as any other applications in the food chain using
polymerase chain reaction (PCR)-based methods.
This document has been established for food testing, but is also applicable to other domains using
thermal cyclers (e.g. environmental, human health, animal health, forensic testing).
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/IEC Guide 98-3:2008, Uncertainty of measurement — Part 3: Guide to the expression of uncertainty in
me a s ur ement (GUM: 1995)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1 Polymerase chain reaction
3.1.1
polymerase chain reaction
PCR
enzymatic procedure that allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005, 3.4.1]
3.1.2
PCR method
test method based on the PCR (3.1.1) technique
Note 1 to entry: Examples include, but are not limited to, PCR, quantitative real-time PCR (qPCR), reverse
transcription PCR (RT PCR) and reverse transcription quantitative real-time PCR (RT qPCR).
ISO 20836:2021(E)
3.2 Thermal cycler
3.2.1
thermal cycler
automatic device that performs defined heating and cooling cycles necessary for PCR (3.1.1) or real-
time PCR
Note 1 to entry: The thermal cycler can be a block-based or (individual) reaction-chamber-based thermal cycler.
[SOURCE: ISO 22174:2005, 3.4.20, modified — “or real-time PCR” and Note 1 to entry have been added.]
3.2.2
reaction block
heated and cooled metal block in which PCR reaction vials, containing the PCR reaction mix, can be
inserted
Note 1 to entry: The block can be heated and cooled by a number of technologies, among which Peltier heating
and cooling is the most abundantly used.
3.2.3
reaction chamber
heated and cooled chamber in which PCR reaction vials, containing the PCR reaction mix, can be
inserted directly or in a rotor
Note 1 to entry: The chamber can be heated and cooled by a number of technologies, among which air heating
and cooling is the most abundantly used.
3.2.4
heated lid
heated cover of a thermal cycler (3.2.1), which is applied in block-based thermal cyclers onto reaction
tubes to prevent condensate of reaction mix to collect inside the cap of the reaction tube or onto the
seal, and evaporation from the reaction tube, and which applies pressure onto the tubes to ensure
proper thermal contact
3.2.5
PCR temperature protocol
heating and cooling cycles required for PCR (3.1.1), typically consisting of denaturation, annealing and
extension temperature steps, which are typically repeated 30 to 45 times
Note 1 to entry: In certain PCR methods (3.1.2), a two-step temperature protocol is used in which annealing and
extension are combined to one step.
3.3 Temperature characteristics
3.3.1
thermal cycler temperature profile
graph of the course of the temperature by performing measurements at defined intervals
Note 1 to entry: See Annex D for an example graph of a thermal cycler temperature profile.
3.3.2
Tt
i
()
temperature in °C of sensor i at time t in s
3.3.3
set temperature
T
set
target temperature programmed to be reached in °C
ISO 20836:2021(E)
3.3.4
average temperature
T
avg
N
T ()t
i
T ()t =
avg ∑
N
i=1
where
Tt
() is average temperature in °C at time t;
avg
i is sensor i of N;
N is total number of sensors.
average of measured values of all active temperature sensors in °C at a specific time in s
3.3.5
temperature deviation
T
dev
Tt()=TT()t −
devavg set
average temperature (3.3.4) minus set temperature (3.3.3) in °C at a specific time in s
3.3.6
minimum temperature
T
min
Tt()=min()TT()tt. ()
min iN
minimum value of all active temperature sensors in °C at a specific time in s
3.3.7
maximum temperature
T
max
TTtt=max .T t
() ()() ()
max iN
maximum value of all active temperature sensors in °C at a specific time in s
3.3.8
temperature uniformity
T
uniformity
Tt()=TT()tt− ()
uniformity maxmin
homogeneity of the temperature distribution within the reaction block (3.2.2) or chamber, defined as
maximum temperature (3.3.7) minus minimum temperature (3.3.6) in °C at a specific time in s
3.3.9
temperature transition
T
transition
phase of fast temperature change from one set temperature to another set temperature
3.3.10
ramp rate
heat or cool rate of thermal cycler (3.2.1) in °C/s
3.3.11
average ramp rate
V
t
N
TT−
ii,%90 ,%10
V ==
t
∑
tt−
ii,%90 ,%10
i=1
where
ISO 20836:2021(E)
V is ramp rate in °C/s;
t
i is sensor i of N;
N is total number of sensors;
T
is T at 10 % temperature of the ramp slope in °C;
i,%10
i
T
is T at 90 % temperature of the ramp slope in °C;
i,%90
i
t is time in s.
heat or cool rate of thermal cycler (3.2.1) calculated between 10 % and 90 % time of the heating or
cooling slope
Note 1 to entry: The heat rate is a positive ramp rate (3.3.10). The cool rate is a negative ramp rate.
3.3.12
maximum ramp rate
V
t max
maximum heat or cool rate during heating or cooling slope in °C/s
3.3.13
maximum temperature overshoot
T
i,ovs,max
tholds=15
Tt=TT−=tholds30
() ()
ii,ovs,max ,max tholds=0 i
maximum temperature (3.3.7) value in °C of all active temperature sensors during temperature
overshoot above the average temperature (3.3.4) of the reaction block (3.2.2) or chamber temperature at
hold when heating up
Note 1 to entry: The maximum temperature overshoot is calculated between start and end of the overshoot and
is expressed relative to the temperature at 30 s hold time (3.3.18).
Note 2 to entry: The overshoot occurs typically between 0 s and 15 s hold time. See Annex D for an example
thermal cycler temperature profile (3.3.1).
3.3.14
minimum temperature undershoot
T
i,uns,min
tholds=15
Tt=TT−=tholds30
() ()
ii,uns,min ,min tholds=0 i
minimum temperature (3.3.6) value in °C of all active temperature sensors during temperature
undershoot below the average temperature (3.3.4) of reaction block (3.2.2) or chamber temperature at
hold when cooling down
Note 1 to entry: The maximum temperature undershoot is calculated between start and end of the undershoot
and is expressed relative to the temperature at 30 s hold time (3.3.18). An undershoot is an overshoot in negative
direction.
Note 2 to entry: The undershoot occurs typically between 0 s and 15 s hold time. See Annex D for an example
thermal cycler temperature profile (3.3.1).
3.3.15
average temperature overshoot
T
ovs,avg
N
T
i,ovs,max
T =
ovs,avg ∑
N
i=1
average value of maximum temperature overshoots (3.3.13) of all active block temperature sensors in °C
ISO 20836:2021(E)
3.3.16
average temperature undershoot
T
uns,avg
N
T
i,uns,min
T =
uns,avg ∑
N
i=1
average value of minimum temperature undershoots (3.3.14) of all active block temperature sensors in °C
3.3.17
overshoot duration
time elapsed between start and end of the overshoot in s
Note 1 to entry: The start of the overshoot is defined as the moment in time where the average temperature
(3.3.4) exceeds the average hold temperature, calculated at 30 s hold, at the beginning of the overshoot. The
end of the overshoot is defined as the moment in time where the average temperature reaches the average hold
temperature at the finish of the overshoot.
3.3.18
hold time
time elapsed between start and end of a temperature hold in s
Note 1 to entry: See Annex D for an example to determine start and end of hold.
3.4 Temperature measurement
3.4.1
temperature measurement system
temperature measurement and data logging instrument
3.4.2
sampling frequency
number of samples per second taken from a time-continuous signal to make a time-discrete signal
3.4.3
response time
time required for the temperature measurement system (3.4.1), when subjected to a change in
temperature, to react to this change
3.4.4
measurement uncertainty
parameter associated with the result of a measurement that characterizes the dispersion of the values
that could reasonably be attributed to the quantity intended to be measured
[SOURCE: ISO/IEC Guide 98-3:2008, B.2.18, modified — “quantity intended to be measured” and
replaced “measurand” and the notes to entry have been deleted.]
3.4.5
performance test
test procedure that determines the performance of a thermal cycler (3.2.1)
3.4.6
calibration
operation that, under specified conditions, in a first step, establishes a relation between the quantity
values with measurement uncertainties (3.4.4) provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in a second step, uses this information to
establish a relation for obtaining a measurement result from an indication
Note 1 to entry: Calibration should not be confused with adjustment of a measuring system, nor with auto-check,
self-verification test, verification, normalization, installation qualification (IQ), operational qualification (OQ) or
performance qualification (PQ).
ISO 20836:2021(E)
[SOURCE: ISO/IEC Guide 99:2007, 2.39, modified — Note 1 to entry has replaced the original Notes 1, 2
and 3 to entry.]
4 Installation of thermal cyclers
The manufacturer’s instructions shall be followed.
The following should be taken into consideration.
a) Thermal cyclers should be installed and operated at suitable environmental conditions that do not
invalidate the results or adversely affect the required quality of any test.
b) The environmental conditions that should at minimum be taken into account are room temperature
and relative humidity.
See the manual of the thermal cycler for recommended room conditions.
Thermal cyclers shall be located in such a way that free circulation of air is permanently allowed.
See ISO 22174 for guidelines for contamination prevention and separation of incompatible laboratory
activities.
5 Maintenance of thermal cyclers
The laboratory shall establish a maintenance programme, where appropriate, and keep records to
ensure proper functioning and prevent deterioration of the thermal cyclers.
6 Performance testing of thermal cyclers
6.1 General
If the performance testing method of this document is used as a metrological traceable temperature
calibration, as a conformity test or as a reference method, the performance test shall be carried out
with a minimum number of sensors that represent at least 12,5 % of wells for reaction blocks or
chambers < 96 wells or 12 wells for reaction blocks or chambers > 96 wells (see 6.4.5.1) and metrological
traceability (see 6.3) shall be provided up to the level of the thermal cycler. If the performance testing
method is used for other purposes, such as supplier’s quality control or supplier’s after sales service,
the number of sensors may be reduced to a minimum number of sensors that represent at least 8 % of
wells for reaction blocks or chambers < 96 wells or 8 wells for reaction blocks or chambers > 96 wells
and metrological traceability shall be provided up to the level of the temperature measurement system.
In case of individual reaction chambers, each of the individual reaction chambers shall be tested.
The decision chart in Figure 1 can be us
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