ISO 20836:2021
(Main)Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms — Thermal performance testing of thermal cyclers
Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms — Thermal performance testing of thermal cyclers
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).
Microbiologie de la chaîne alimentaire — Réaction de polymérisation en chaîne (PCR) pour la recherche de micro-organismes — Essais de performance thermique des thermocycleurs
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).
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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 2021
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ISO 20836:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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.
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
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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
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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.
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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.
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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).
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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
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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
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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
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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).
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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 used to determine if the performance test shall be a metrological
traceable calibration or a performance test.
NOTE 1 Calibrations that meet the requirements of the ISO/IEC 17025 are considered to be metrological
traceable. ISO/IEC 17025 describes when metrological traceability is required and how metrological traceability
is established.
NOTE 2 The chemistry- or biochemistry-based normalization and verification kits available at the time of
publication of this document offer no traceability to SI and are associated with high measurement uncertainties,
and are therefore inapt as a performance testing method. The normalization kits are developed to optimize the
optical detection and related software to enable the collection of fluorescent data in a real-time thermal cycler.
These kits are not developed for calibration purposes.
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ISO 20836:2021(E)
Figure 1 — Decision chart to determine the requirement for metrological traceable
temperature calibration of thermal cyclers
6.2 Performance testing programme
The laboratory shall establish a performance testing programme, where appropriate, and keep records
to ensure that the thermal cycler is capable of achieving the accuracy required and complies with the
specifications relevant to the intended use.
ISO/IEC 17025-compliant laboratories are required to establish a planned calibration programme in
order to maintain confidence in the status of calibration (see ISO/IEC 17025:2017, 6.4).
NOTE Guidance on how to determine calibration intervals, particularly when setting up a calibration
programme, is provided in, for example, ILAC G24.
6.3 Metrological traceability
Performance testing shall be traceable to the International System of Units (SI).
Metrological traceability is established by considering, and then ensuring, the following:
a) the specification of the measurand (quantity to be measured);
b) a documented unbroken chain of calibrations going back to stated and appropriate references
(appropriate references include national or international standards, and intrinsic standards);
c) measurement uncertainty for each step in the traceability chain is evaluated according to agreed
methods;
d) each step of the chain is performed in accordance with appropriate methods, and the measurement
results and associated, recorded measurement uncertainties;
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ISO 20836:2021(E)
e) the laboratories performing one or more steps in the chain supply evidence for their technical
competence.
NOTE Calibration laboratories fulfilling the requirements of the ISO/IEC 17025 are considered to be
competent. A thermal cycler calibration certificate bearing an accreditation body logo from a calibration
laboratory accredited to the ISO/IEC 17025 is sufficient evidence of traceability of the calibration data reported.
6.4 Temperature performance testing method
6.4.1 General
This performance testing method is intended to determine the thermal cycler temperature parameters
that influence the outcome of the PCR and RT PCR. It can be used to perform a temperature performance
test on both PCR and real-time PCR thermal cyclers.
An example of a test and compliancy report is given in Annex E.
6.4.2 Principle
The temperature is measured by temperature sensors directly in the reaction block in block-based
thermal cyclers or inside the reaction vials in reaction-chamber-based thermal cyclers, in order to
achieve an adequately low measurement uncertainty. The measurement is performed over the complete
reaction temperature range, including at least a minimum, maximum and middle temperature. If the
thermal cycler has a heated lid, the measurement shall be performed with the heated lid closed and
operating, when physically possible.
6.4.3 Equipment
6.4.3.1 Thermal cycler
The thermal cycler shall be checked, before the performance test, to be functional.
The ventilation openings shall be clean and not obstructed, allowing free air circulation.
6.4.3.2 Temperature measurement system
The temperature measurement system shall meet at least the following criteria:
a) multi-sensor system with an adequate number of temperature sensors to measure simultaneously
in at least the number of required wells (see 6.4.5.1), allowing to measure uniformity;
b) capable of recording the heated lid temperature with at least one temperature sensor (when
physically possible);
c) capable of recording temperatures dynamically with a sampling frequency of at least one time per
second in order to measure correctly the ramp rate and overshoot; for thermal cyclers with heat
rates above 4 °C/s, a sampling frequency of at least four times per second per temperature sensor is
recommended;
d) capable of recording temperature over the complete reaction temperature range;
e) capable of being calibrated, traceable to SI, over at least the complete reaction temperature range;
f) resolution ≤ 0,1 °C;
g) expanded combined measurement uncertainty (k = 2) ≤ 0,15 °C/sensor (determined in accordance
with ISO/IEC Guide 98-3:2008);
h) total mass of the sensor head approximately equal to load of thermal cycler when the heating block
or heating chamber is completely filled with tubes containing reagents;
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ISO 20836:2021(E)
i) response time of the sensors ≤ 1 s.
The temperature performance test unit shall be calibrated in a metrological traceable manner at
regular intervals according to a predefined programme.
6.4.4 Environmental conditions
T
...
NORME ISO
INTERNATIONALE 20836
Première édition
2021-11
Microbiologie de la chaîne
alimentaire — Réaction de
polymérisation en chaîne (PCR) pour
la recherche de micro-organismes —
Essais de performance thermique des
thermocycleurs
Microbiology of the food chain — Polymerase chain reaction (PCR)
for the detection of microorganisms — Thermal performance testing
of thermal cyclers
Numéro de référence
ISO 20836:2021(F)
© ISO 2021
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ISO 20836:2021(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2021
Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, aucune partie de cette
publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,
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Publié en Suisse
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ISO 20836:2021(F)
Sommaire Page
Avant-propos .iv
Introduction .v
1 Domaine d’application . 1
2 Références normatives .1
3 Termes et définitions . 1
3.1 Réaction de polymérisation en chaîne . 1
3.2 Thermocycleur . 2
3.3 Caractéristiques thermiques . 2
3.4 Mesurage de la température . 5
4 Installation des thermocycleurs . 6
5 Maintenance des thermocycleurs . .6
6 Essai de performance des thermocycleurs . 6
6.1 Généralités . 6
6.2 Programme d’essais de performance . 7
6.3 Traçabilité métrologique . 7
6.4 Méthode d’essai de performance thermique . 8
6.4.1 Généralités . 8
6.4.2 Principe. 8
6.4.3 Matériel . 8
6.4.4 Conditions environnementales . 9
6.4.5 Mode opératoire . 9
6.4.6 Résultats de l’essai de performance . 11
6.4.7 Rapport d’essai de performance . 11
6.4.8 Essai de conformité .12
6.5 Méthode d’essai de performance optique .12
Annexe A (informative) Emplacement des sondes .13
Annexe B (informative) Protocole de température universel .18
Annexe C (informative) Essai de conformité .19
Annexe D (informative) Exemple de profil thermique d’un thermocycleur .23
Annexe E (informative) Exemple d’essai de performance et d’essai de conformité .24
Bibliographie .28
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ISO 20836:2021(F)
Avant-propos
L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes
nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est
en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude
a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,
gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.
L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui
concerne la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents
critères d'approbation requis pour les différents types de documents ISO. Le présent document a
été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir
www.iso.org/directives).
L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de
droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable
de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant
les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de
l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de
brevets reçues par l'ISO (voir www.iso.org/brevets).
Les appellations commerciales éventuellement mentionnées dans le présent document sont données
pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un
engagement.
Pour une explication de la nature volontaire des normes, la signification des termes et expressions
spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion
de l'ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles
techniques au commerce (OTC), voir www.iso.org/avant-propos.
Le présent document a été élaboré par le comité technique ISO/TC 34, Produits alimentaires, sous-comité
SC 9, Microbiologie, en collaboration avec le comité technique CEN/TC 463, Microbiologie de la chaîne
alimentaire, du Comité européen de normalisation (CEN) conformément à l’Accord de coopération
technique entre l’ISO et le CEN (Accord de Vienne).
Cette première édition de Norme internationale annule et remplace la première édition de la
Spécification technique (ISO/TS 20836:2005), qui a fait l’objet d’une révision technique. Les principales
modifications apportées par rapport à la précédente édition sont les suivantes:
— extension du Domaine d’application pour inclure les thermocycleurs classiques, ainsi que les
thermocycleurs en temps réel;
— ajout de précisions concernant la méthode d’essai physique de performance et suppression de la
méthode d’essai biochimique de performance;
— ajout d’informations destinées aux laboratoires concernant l’ISO/IEC 17025;
— alignement de la méthode d’essai de performance sur l’ISO/IEC 17025;
— ajout d’un essai de conformité;
— ajout de deux modes opératoires visant à définir des spécifications à partir de la méthode par PCR,
donnés à l’Annexe C.
Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent
document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes
se trouve à l’adresse www.iso.org/fr/members.html.
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ISO 20836:2021(F)
Introduction
Le présent document fait partie d’une famille de Normes internationales regroupées sous le titre
Microbiologie de la chaîne alimentaire — Réaction de polymérisation en chaîne (PCR) pour la recherche de
micro-organismes pathogènes dans les aliments:
— ISO 22174, Exigences générales et définitions;
— ISO 20837, Exigences relatives à la préparation des échantillons pour la détection qualitative;
— ISO 20836, Essais de performance thermique des thermocycleurs;
— ISO 20838, Exigences relatives à l’amplification et à la détection pour les méthodes qualitatives.
Le présent document décrit une méthode d’essai de performance pour les thermocycleurs classiques,
ainsi que les thermocycleurs en temps réel permettant aux laboratoires de déterminer si le
thermocycleur utilisé est adapté à l’usage prévu et satisfait aux spécifications définies par le laboratoire.
La méthode décrite s’appuie sur une méthode physique visant à réaliser des mesurages directement
dans le bloc des thermocycleurs à bloc et dans les tubes des thermocycleurs à chambre de réaction. La
méthode décrite offre une incertitude de mesure suffisamment faible pour effectuer des comparaisons
pertinentes avec les spécifications.
De plus, il est à noter pour les laboratoires soumis à la norme ISO/IEC 17025 que la méthode satisfait
aux critères d’une méthode d’étalonnage métrologiquement traçable.
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NORME INTERNATIONALE ISO 20836:2021(F)
Microbiologie de la chaîne alimentaire — Réaction de
polymérisation en chaîne (PCR) pour la recherche de
micro-organismes — Essais de performance thermique des
thermocycleurs
1 Domaine d’application
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).
2 Références normatives
Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur
contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.
Pour les références non datées, la dernière édition du document de référence s'applique (y compris les
éventuels amendements).
Guide ISO/IEC 98-3:2008, Incertitude de mesure — Partie 3: Guide pour l’expression de l’incertitude de
me s ur e (GUM: 1995)
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s’appliquent.
L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en
normalisation, consultables aux adresses suivantes:
— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp
— IEC Electropedia: disponible à l’adresse https:// www .electropedia .org/
3.1 Réaction de polymérisation en chaîne
3.1.1
réaction de polymérisation en chaîne
PCR
méthode enzymatique permettant l’amplification in vitro de l’ADN
[SOURCE: ISO 22174:2005, 3.4.1]
3.1.2
méthode par PCR
méthode d’essai fondée sur la technique PCR (3.1.1)
Note 1 à l'article: Des exemples incluent, sans s’y limiter, la PCR, la PCR quantitative en temps réel (qPCR), la PCR
à transcription inverse (RT PCR) et la PCR quantitative en temps réel à transcription inverse (RT qPCR).
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ISO 20836:2021(F)
3.2 Thermocycleur
3.2.1
thermocycleur
appareil automatique qui réalise les cycles de chauffage et de refroidissement nécessaires à la PCR
(3.1.1) ou à la PCR en temps réel
Note 1 à l'article: Le thermocycleur peut être un thermocycleur à bloc ou à chambre de réaction (individuelle).
[SOURCE: ISO 22174:2005, 3.4.20, modifié — L’expression «ou à la PCR en temps réel» et la Note 1 à
l’article ont été ajoutées.]
3.2.2
bloc de réaction
bloc en métal chauffé et refroidi dans lequel les tubes à réaction PCR, contenant le mélange réactionnel
PCR, peuvent être insérés
Note 1 à l'article: Le bloc peut être chauffé et refroidi à l’aide de différentes techniques, parmi lesquelles le
chauffage et refroidissement à effet Peltier est la plus couramment utilisée.
3.2.3
chambre de réaction
chambre en métal chauffée et refroidie dans laquelle les tubes à réaction PCR, contenant le mélange
réactionnel PCR, peuvent être insérés directement ou dans un carrousel
Note 1 à l'article: La chambre peut être chauffée et refroidie à l’aide de différentes techniques, parmi lesquelles le
chauffage et refroidissement à air est la plus couramment utilisée.
3.2.4
couvercle chauffant
couvercle chauffé d’un thermocycleur (3.2.1), qui est installé sur les tubes de réaction dans les
thermocycleurs à bloc afin d’empêcher un dépôt par condensation du mélange réactionnel dans le
bouchon du tube de réaction ou sur le joint, ainsi que l’évaporation à partir du tube de réaction, et qui
applique une pression sur les tubes afin de garantir un bon contact thermique
3.2.5
protocole de température PCR
cycles de chauffage et de refroidissement requis pour la PCR (3.1.1), composés en général d’étapes de
dénaturation, d’hybridation et d’élongation qui sont, le plus souvent, répétées 30 à 45 fois
Note 1 à l'article: Certaines méthodes par PCR (3.1.2) utilisent un protocole de température en deux étapes dans
lequel les phases d’hybridation et d’élongation sont combinées en une seule étape.
3.3 Caractéristiques thermiques
3.3.1
profil thermique du thermocycleur
graphique de l’évolution de la température à partir de mesurages effectués à des intervalles définis
Note 1 à l'article: Voir l’Annexe D pour obtenir un exemple de graphique de profil thermique de thermocycleur.
3.3.2
Tt
i
()
température en °C de la sonde i au temps t en s
3.3.3
température de consigne
T
cons
température cible programmée en °C à atteindre
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ISO 20836:2021(F)
3.3.4
température moyenne
T
moy
N
T ()t
i
T ()t =
moy ∑
N
i=1
où
Tt
() est la température moyenne en °C au temps t;
moy
i est la sonde i sur N;
N est le nombre total de sondes;
moyenne des valeurs mesurées par toutes les sondes de température actives en °C à un temps donné en
s
3.3.5
écart de température
T
écart
Tt()=TT()t −
écartmoy cons
température moyenne (3.3.4) moins la température de consigne (3.3.3) en °C à un temps donné en s
3.3.6
température minimale
T
min
Tt()=min()TT()tt. ()
min iN
valeur minimale de toutes les sondes de température actives en °C à un temps donné en s
3.3.7
température maximale
T
max
TTtt=max .T t
() ()() ()
max iN
valeur maximale de toutes les sondes de température actives en °C à un temps donné en s
3.3.8
uniformité de la température
T
uniformité
Tt =TTtt−
() () ()
uniformité maxmin
homogénéité de la répartition de la température au sein du bloc de réaction (3.2.2) ou de la chambre de
réaction, définie comme la température maximale (3.3.7) moins la température minimale (3.3.6) en °C à
un temps donné en s
3.3.9
transition de température
T
transition
phase de passage rapide d’une température de consigne à une autre température de consigne
3.3.10
vitesse de montée/descente
vitesse de chauffe ou de refroidissement du thermocycleur (3.2.1) en °C/s
3.3.11
vitesse de montée moyenne
V
t
N
TT−
ii,%90 ,%10
V ==
t
∑
tt−
ii,%90 ,%10
i=1
où
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ISO 20836:2021(F)
V est la vitesse de montée en °C/s;
t
i est la sonde i sur N;
N est le nombre total de sondes;
T
est T à 10 % de la température de la pente de la montée en °C;
i,%10
i
T
est T à 90 % de la température de la pente de la montée en °C;
i,%90
i
t est le temps en s;
vitesse de chauffe ou de refroidissement du thermocycleur (3.2.1) calculée entre 10 % et 90 % du temps
de la pente de chauffage ou de refroidissement
Note 1 à l'article: La vitesse de chauffe correspond à une vitesse de montée (3.3.10) positive. La vitesse de
refroidissement correspond à une vitesse de montée négative.
3.3.12
vitesse de montée maximale
V
t max
vitesse de chauffe ou de refroidissement maximale sur la pente de chauffage ou de refroidissement
en °C/s
3.3.13
pic maximal de température « overshoot »
T
i,ovs,max
tmaintiens=15
Tt=TT() −=()tmaintien 330s
ii,ovs,max ,max tmaintiens=0 i
valeur de température maximale (3.3.7) en °C de toutes les sondes de température actives au cours du
pic de température « overshoot » au-dessus de la température moyenne (3.3.4) du bloc de réaction (3.2.2)
ou de la température de la chambre maintenue au cours du chauffage
Note 1 à l'article: Le pic maximal de température «overshoot» est calculé entre le début et la fin du pic et est
exprimé par rapport à un temps de maintien (3.3.18) de la température de 30 s.
Note 2 à l'article: Le pic de température «overshoot» survient, en général, entre 0 s et 15 s du temps de maintien.
Voir l’Annexe D pour obtenir un exemple de profil thermique de thermocycleur (3.3.1).
3.3.14
pic minimal de température « undershoot »
T
i,uns,min
tmaintiens=15
Tt=TT() −=()tmaintien 330s
ii,uns,min ,min tmaintiens=0 i
valeur de température minimale (3.3.6) en °C de toutes les sondes de température actives au cours du
pic de température « undershoot » en dessous de la température moyenne (3.3.4) du bloc de réaction
(3.2.2) ou de la température de la chambre maintenue au cours du refroidissement
Note 1 à l'article: Le pic maximal de température «undershoot» est calculé entre le début et la fin du pic
et est exprimé par rapport à un temps de maintien (3.3.18) de la température de 30 s. Un pic de température
«undershoot» est un pic de température négatif.
Note 2 à l'article: Le pic de température «undershoot» survient, en général, entre 0 s et 15 s du temps de maintien.
Voir l’Annexe D pour obtenir un exemple de profil thermique de thermocycleur (3.3.1).
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ISO 20836:2021(F)
3.3.15
pic moyen de température « overshoot »
T
ovs,moy
N
T
i,ovs,max
T =
ovs,moy ∑
N
i=1
valeur moyenne des pics maximaux de température « overshoot » (3.3.13) de toutes les sondes de
température actives du bloc en °C
3.3.16
pic moyen de température « undershoot »
T
uns,moy
N
T
i,uns,min
T =
uns,moy
∑
N
i=1
valeur moyenne des pics minimaux de température « undershoot » (3.3.14) de toutes les sondes de
température actives du bloc en °C
3.3.17
durée du pic de température « overshoot »
temps écoulé entre le début et la fin du pic de température « overshoot » en s
Note 1 à l'article: Le début du pic de température «overshoot» est défini comme le moment où la température
moyenne (3.3.4) dépasse la température de maintien moyenne, calculée à 30 s de maintien, au début du pic.
La fin du pic de température «overshoot» est définie comme le moment où la température moyenne atteint la
température de maintien moyenne à la fin du pic.
3.3.18
temps de maintien
temps écoulé entre le début et la fin du maintien d’une température en s
Note 1 à l'article: Voir l’Annexe D pour obtenir un exemple de détermination du début et de la fin du maintien
d’une température.
3.4 Mesurage de la température
3.4.1
système de mesure de la température
instrument de mesure de la température et d’enregistrement des données
3.4.2
fréquence d’échantillonnage
nombre d’échantillons pris par seconde à partir d’un signal continu pour obtenir un signal discret
3.4.3
temps de réponse
temps nécessaire au système de mesure de la température (3.4.1), lorsqu’il est soumis à un changement
de température, pour réagir à ce changement
3.4.4
incertitude de mesure
paramètre, associé au résultat d’un mesurage, qui caractérise la dispersion des valeurs qui pourraient
raisonnablement être attribuées à la grandeur destinée à être mesurée
[SOURCE: ISO/IEC Guide 98-3:2008, B.2.18, modifié — Le terme «mesurande» a été remplacé par
«grandeur destinée à être mesurée» et les notes à l’article ont été supprimées.]
3.4.5
essai de performance
mode opératoire d’essai qui détermine la performance d’un thermocycleur (3.2.1)
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ISO 20836:2021(F)
3.4.6
étalonnage
opération qui, dans des conditions spécifiées, établit en une première étape une relation entre les
valeurs et les incertitudes de mesure (3.4.4) associées qui sont fournies par des étalons et les indications
correspondantes avec les incertitudes associées, puis utilise en une seconde étape cette information
pour établir une relation permettant d’obtenir un résultat de mesure à partir d’une indication
Note 1 à l'article: Il convient de ne pas confondre l’étalonnage avec l’ajustage d’un système de mesure ni avec
l’autocontrôle, l’essai d’autovérification, la vérification, la normalisation, la qualification d’installation (QI), la
qualification opérationnelle (QO) ou la qualification de performance (QP).
[SOURCE: ISO/IEC Guide 99:2007, 2.39, modifié — Les Notes 1, 2 et 3 à l’article d’origine ont été
remplacées par la Note 1 à l’article.]
4 Installation des thermocycleurs
Les instructions du fabricant doivent être respectées.
Il convient que les points suivants soient pris en compte:
a) il convient d’installer et d’utiliser les thermocycleurs dans des conditions environnementales
adaptées qui n’invalident pas les résultats et ne nuisent pas à la qualité requise de tout essai;
b) la température ambiante et l’humidité relative constituent les conditions environnementales qu’il
convient au minimum de prendre en compte.
Se reporter au manuel du thermocycleur pour connaître les conditions ambiantes recommandées.
Les thermocycleurs doivent être installés de sorte à ne pas entraver la libre circulation de l’air à tout
moment.
Se reporter à l’ISO 22174 pour les lignes directrices en matière de prévention de la contamination et de
séparation des activités de laboratoire incompatibles.
5 Maintenance des thermocycleurs
Le laboratoire doit établir un programme de maintenance, le cas échéant, et conserver des
enregistrements afin de garantir le bon fonctionnement et d’empêcher la détérioration des
thermocycleurs.
6 Essai de performance des thermocycleurs
6.1 Généralités
Si la méthode d’essai de performance du présent document est utilisée comme méthode d’étalonnage
de la température métrologiquement traçable, méthode d’essai de conformité ou méthode de référence,
l’essai de performance doit être réalisé en utilisant un nombre minimal de sondes correspondant à
au moins 12,5 % du nombre de puits pour les blocs ou chambres de réaction de moins de 96 puits ou
à 12 puits pour les blocs ou chambres de réaction de plus de 96 puits (voir 6.4.5.1) et la traçabilité
métrologique (voir 6.3) doit être assurée jusqu’au niveau du thermocycleur. Si la méthode d’essai de
performance est utilisée à d’autres fins, en guise de contrôle qualité du fournisseur ou au titre du
service après-vente du fournisseur par exemple, le nombre de sondes peut être réduit à un minimum
de sondes correspondant à au moins 8 % du nombre de puits pour les blocs ou chambres de réaction de
moins de 96 puits ou à 8 puits pour les blocs ou chambres de réaction de plus de 96 puits et la traçabilité
métrologique doit être assurée jusqu’au niveau du système de mesure de la température.
En cas de chambres de réaction individuelles, chacune d’elles doit être soumise à essai.
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ISO 20836:2021(F)
L’arbre de décision de la Figure 1 peut être utilisé pour déterminer si l’essai de performance doit être un
essai de performance ou un étalonnage métrologiquement traçable.
NOTE 1 Les étalonnages qui satisfont aux exigences de l’ISO/IEC 17025 sont considérés comme étant
métrologiquement traçables. L’ISO/IEC 17025 explique à quel moment la traçabilité métrologique est requise et
comment elle est établie.
NOTE 2 Les kits de normalisation et de vérification chimiques et biochimiques, disponibles au moment
de la publication du présent document, n’offrent pas de traçabilité au SI et sont associés à des incertitudes de
mesure élevées et, par conséquent, ne constituent pas une méthode d’essai de performance adaptée. Les kits de
normalisation sont conçus pour optimiser la détection optique et le logiciel associé pour permettre le recueil des
données de fluorescence dans un thermocycleur en temps réel. Les kits ne sont pas conçus à des fins d’étalonnage.
Figure 1 — Arbre de décision pour déterminer la nécessité de procéder à un étalonnage
de la température métrologiquement traçable des thermocycleurs
6.2 Programme d’essais de performance
Le laboratoire doit établir un programme d’essais de performance, le cas échéant, et conserver des
enregistrements pour s’assurer que le thermocycleur permet d’atteindre l’exactitude requise et
respecte les spécifications adaptées à l’usage prévu.
Les laboratoires soumis à l’ISO/IEC 17025 sont tenus d’établir un programme d’étalonnage planifié afin
de maintenir la confiance dans l’état d’étalonnage (voir ISO/IEC 17025:2017, 6.4).
NOTE Les recommandations relatives à la méthode de détermination de la fréquence d’étalonnage,
notamment au moment de définir le programme d’étalonnage, sont données par exemple dans ILAC G24.
6.3 Traçabilité métrologique
L’essai de performance doit être traçable par rapport au Système international d’unités (SI).
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ISO 20836:2021(F)
La traçabilité métrologique est établie en tenant compte et en s’assurant de ce qui suit:
a) les spécifications du mesurande (grandeur à mesurer);
b) une chaîne ininterrompue d’étalonnages documentée remontant jusqu’aux références appropriées
et mentionnées (les références appropriées incluent les normes internationales et nationales, ainsi
que les étalons intrinsèques);
c) l’évaluation de l’incertitude de mesure à chaque étape de la chaîne de traçabilité selon des méthodes
convenues;
d) la réalisation de chaque étape de la chaîne selon des méthodes appropriées, ainsi que les résultats
de mesure et les incertitudes de mesure associées et consignées;
e) la fourniture de preuves de la compétence technique des laboratoires effectuant une étape ou plus
dans la chaîne.
NOTE Les laboratoires d’étalonnage qui satisfont aux exigences
...
FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 20836
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Polymerase chain reaction (PCR)
Voting begins on:
20210823 for the detection of microorganisms
— Thermal performance testing of
Voting terminates on:
20211018
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
ISO/CEN PARALLEL PROCESSING
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/FDIS 20836:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN
DARDS TO WHICH REFERENCE MAY BE MADE IN
©
NATIONAL REGULATIONS. ISO 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 20836:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
CH1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 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
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ISO/FDIS 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 nongovernmental, 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 the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in collaboration with ISO Technical Committee
TC 34, Food products, Subcommittee SC 9, Microbiology, 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.
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ISO/FDIS 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.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 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 983:2008, 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).
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ISO/FDIS 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 twostep 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
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ISO/FDIS 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
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ISO/FDIS 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
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ISO/FDIS 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 timecontinuous signal to make a timediscrete 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).
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ISO/FDIS 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 used to determine if the performance test shall be a metrological
traceable calibration or a performance test.
NOTE 1 Calibrations that meet the requirements of the ISO/IEC 17025 are considered to be metrological
traceable. ISO/IEC 17025 describes when metrological traceability is required and how metrological traceability
is established.
NOTE 2 The chemistry- or biochemistry-based normalization and verification kits available at the time of
publication of this document offer no traceability to SI and are associated with high measurement uncertainties,
and are therefore inapt as a performance testing method. The normalization kits are developed to optimize the
optical detection and related software to enable the collection of fluorescent data in a real-time thermal cycler.
These kits are not developed for calibration purposes.
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ISO/FDIS 20836:2021(E)
Figure 1 — Decision chart to determine the requirement for metrological traceable
temperature calibration of thermal cyclers
6.2 Performance testing programme
The laboratory shall establish a performance testing programme, where appropriate, and keep records
to ensure that the thermal cycler is capable of achieving the accuracy required and complies with the
specifications relevant to the intended use.
ISO/IEC 17025-compliant laboratories are required to establish a planned calibration programme in
order to maintain confidence in the status of calibration (see ISO/IEC 17025:2017, 6.4).
NOTE Guidance on how to determine calibration intervals, particularly when setting up a calibration
programme, is provided in, for example, ILAC G24.
6.3 Metrological traceability
Performance testing shall be traceable to the International System of Units (SI).
Metrological traceability is established by considering, and then ensuring, the following:
a) the specification of the measurand (quantity to be measured);
b) a documented unbroken chain of calibrations going back to stated and appropriate references
(appropriate references include national or international standards, and intrinsic standards);
c) measurement uncertainty for each step in the traceability chain is evaluated according to agreed
methods;
d) each step of the chain is performed in accordance with appropriate methods, and the measurement
results and associated, recorded measurement uncertainties;
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ISO/FDIS 20836:2021(E)
e) the laboratories performing one or more steps in the chain supply evidence for their technical
competence.
NOTE Calibration laboratories fulfilling the requirements of the ISO/IEC 17025 are considered to be
competent. A thermal cycler calibration certificate bearing an accreditation body logo from a calibration
laboratory accredited to the ISO/IEC 17025 is sufficient evidence of traceability of the calibration data reported.
6.4 Temperature performance testing method
6.4.1 General
This performance testing method is intended to determine the thermal cycler temperature parameters
that influence the outcome of the PCR and RT PCR. It can be used to perform a temperature performance
test on both PCR and real-time PCR thermal cyclers.
An example of a test and compliancy report is given in Annex E.
6.4.2 Principle
The temperature is measured by temperature sensors directly in the reaction block in block-based
thermal cyclers or inside the reaction vials in reaction-chamber-based thermal cyclers, in order to
achieve an adequately low measurement uncertainty. The measurement is performed over the complete
reaction temperature range, including at least a minimum, maximum and middle temperature. If the
thermal cycler has a heated lid, the measurement shall be performed with the heated lid closed and
operating, when physically possible.
6.4.3 Equipment
6.4.3.1 Thermal cycler
The thermal cycler shall be checked, before the performance test, to be functional.
The ventilation openings shall be clean and not obstructed, allowing free air circulation.
6.4.3.2 Temperature measurement system
The temperature measurement system shall meet at least the following criteria:
a) multi-sensor system with an adequate number of temperature sensors to measure simultaneously
in at least the number of required wells (see 6.4.5.1), allowing to measure uniformity;
b) capable of recording the heated lid temperature with at least one temperature sensor (when
physically possible);
c) capable of recording temperatures dynamically with a sampling frequency of at least one time per
second in order to measure correctly the ramp rate and overshoot; for thermal cyclers with heat
rates above 4 °C/s, a sampling frequency of at least four times per second per temperature sensor is
recommended;
d) capable of recording temperature over the complete reaction temperature range;
e) capable of being calibrated, traceable to SI,
...
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