FprEN ISO 20836

Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of microorganisms - Thermal performance testing of thermal cyclers (ISO/FDIS 20836:2021)

2020-12-08: Additional 6 months granted based on CEN/BT decision 036/2020. Dispatch of FV draft is due on 2021-06-08.

Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Leistungsprüfung für PCR-Geräte (ISO/FDIS 20836:2021)

Dieses Dokument legt Anforderungen für die Installation, Instandhaltung, Temperaturkalibrierung und Temperaturleistungsprüfung von Standard-PCR Geräten und Real-time-PCR Geräten 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 PCR Geräte 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/FDIS 20836:2021)

Mikrobiologija živil in krme - Polimerazna verižna reakcija (PCR) za ugotavljanje prisotnosti mikroorganizmov - Preskus toplotnega delovanja cikličnih termostatov (ISO/FDIS 20836:2021)

General Information

• Status: Not Published
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463/WG 1 - General requirements relating to PCR methods
• Current Stage: 6055 - CEN Ratification completed (DOR) - Publishing
• Due Date: 19-Nov-2021
• Completion Date: 19-Nov-2021
• Ref Project: kSIST FprEN ISO 20836:2021 - Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of microorganisms - Thermal performance testing of thermal cyclers (ISO/FDIS 20836:2021)

RELATIONS

• Revised: CEN ISO/TS 20836:2005 - Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Performance testing for thermal cyclers (ISO/TS 20836:2005)
• Effective Date: 13-Jun-2018

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 20836:2020
01-marec-2020

Mikrobiologija živil in krme - Polimerazna verižna reakcija (PCR) za ugotavljanje

prisotnosti povzročiteljev zastrupitev s hrano - Preskus izvedbe preiskave s
pomnoževalnikom (ISO/DIS 20836:2020)

Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of

food-borne pathogens - Thermal performance testing of thermal cyclers (ISO/DIS
20836:2020)

Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR)

zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Leistungsprüfung
für PCR-Geräte (ISO/DIS 20836:2020)

Microbiologie de la chaîne alimentaire - Réaction de polymérisation en chaîne (PCR)

pour la recherche de micro-organismes pathogènes dans les aliments - Essais de
performance des thermocycleurs (ISO/DIS 20836:2020)
Ta slovenski standard je istoveten z: prEN ISO 20836
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 20836:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 20836:2020
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oSIST prEN ISO 20836:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20836
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2020-01-09 2020-04-02
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of food-borne pathogens
— Thermal performance testing of thermal cyclers

Microbiologie de la chaîne alimentaire — Réaction de polymérisation en chaîne (PCR) pour la recherche de

micro-organismes pathogènes dans les aliments — Essais de performance des thermocycleurs

ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 20836:2020(E)
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 SUPPORTING DOCUMENTATION. ISO 2020
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oSIST prEN ISO 20836:2020
ISO/DIS 20836:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
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Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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oSIST prEN ISO 20836:2020
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Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Installation of thermal cyclers ............................................................................................................................................................... 5

5 Maintenance of thermal cyclers............................................................................................................................................................ 6

6 Performance testing of thermal cyclers ....................................................................................................................................... 6

6.1 General ........................................................................................................................................................................................................... 6

6.2 Performance testing program .................................................................................................................................................... 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 ........................................................................................................................................................12

Annex B (informative) Universal temperature protocol ...............................................................................................................17

Annex C (informative) Compliancy testing ..................................................................................................................................................18

Annex D (informative) Example of thermal cycler temperature profile ......................................................................21

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

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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 on 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 the following

URL: www .iso .org/ iso/ foreword .html.

This document was prepared by Technical Committee CEN/TC 275, Food analysis, Working Group

6, Microbiology, in collaboration with ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in

accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).

This second edition cancels and replaces the first edition (ISO/TS 20836:2005), which has been

technically revised.
The main changes compared to the previous edition are as follows:

— the scope has been extended to include both thermal cyclers and real-time thermal cyclers;

— the document type has been changed from ISO/TS to an ISO-Standard;

— the physical performance testing method has been described in more detail, the biochemical

performance testing method has been taken out;
— information for laboratories regarding ISO/IEC 17025 have 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 2020 – All rights reserved
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Introduction

This Standard is part of a series of International Standards under the general title Microbiology of the

food chain – Polymerase chain reaction (PCR) for the detection of food borne pathogens:

— General requirements and definitions (ISO 22174);
— Requirements for sample preparation for qualitative detection (ISO 20837);
— Performance testing for thermal cyclers (ISO 20836);

— Requirements for amplifications and detection for qualitative methods (ISO 20838).

This Standard 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.

Attention is drawn to the possibility that some methods described in this document may be subject to

patent rights. ISO shall not be held responsible for identifying any or all such patent rights.

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oSIST prEN ISO 20836:2020
DRAFT INTERNATIONAL STANDARD ISO/DIS 20836:2020(E)
Microbiology of the food chain — Polymerase chain
reaction (PCR) for the detection of food-borne pathogens
— Thermal performance testing of thermal cyclers
1 Scope

This International Standard provides requirements for the installation, maintenance, temperature

calibration and temperature performance testing of standard thermal cyclers and real-time thermal

cyclers and is applicable to the detection of food-borne pathogens as well as any other applications in

food and feeding stuffs using polymerase chain reaction (PCR) based methods.

This standard has been established for food testing, but can also be applied in other domains using

thermal cyclers (e.g. environmental, human health, animal health and forensic testing). There can be

other requirements in specific documents.
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 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the

detection of food-borne pathogens — General requirements and definitions

ISO/IEC Guide 99, International vocabulary of metrology — Basic and general concepts and associated

terms (VIM)

ISO/IEC Guide 98-3, 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:

— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at https:// www .iso .org/ obp
3.1 PCR
3.1.1
polymerase chain reaction
PCR
enzymatic procedure which allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005 3.4.1]
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3.1.2
PCR method
test method based on the PCR technique

Note 1 to entry: Examples include, but are not limited to, PCR, quantitative real-time PCR (qPCR), reverse

transcriptase PCR (RT PCR) and reverse transcriptase quantitative real-time PCR (RT qPCR).

3.2 Thermal cycler
3.2.1
thermal cycler

automatic device which performs defined heating and cooling cycles necessary for PCR or real-time PCR

[SOURCE: Adapted from ISO 22174:2005 3.4.20]

Note 1 to entry: The thermal cycler can be a block based or (individual) reaction chamber based thermal cycler.

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 thermal cycler which is applied in block based thermal cyclers onto reaction tubes to

prevent condensation of reaction mix to cap of reaction tube and evaporation from reaction tube and

applies pressure onto the tubes to ensure proper thermal contact
3.2.5
PCR temperature protocol

heating and cooling cycles required for PCR, typically consisting of denaturation, annealing and

extension temperature steps which are repeated typically 30-45 times

Note 1 to entry: In certain PCR methods 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 (see Annex D

for an example graph of thermal cycler temperature profile)
3.3.2
t time
temperature in °C of sensor i at time stamp time in s
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3.3.3
set temperature
set
target temperature programmed to be reached in °C
3.3.4
average temperature
t time
t ()time =
avg ∑
i=1
where
tt()ime is average temperature in °C at time stamp time;
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 stamp in s

3.3.5
temperature deviation
t timet= timet−
() ()
devavg set
average temperature minus set temperature in °C at a specific time stamp in s
3.3.6
minimum temperature
t ()timem= in()t ()timet… ()time
mini N

minimum value of all active temperature sensors in °C at a specific time stamp in s

3.3.7
maximum temperature
t ()timem= ax()t ()timet… ()time
maxi N

maximum value of all active temperature sensors in °C at a specific time stamp in s

3.3.8
temperature uniformity
t timet= timet− time
() () ()
uniformity maxmin

homogeneity of the temperature distribution within the reaction block or chamber, defined as maximum

temperature minus minimum temperature in °C at a specific time stamp in s
3.3.9
average ramp rate
tt−
 
i%,,90 i10%
V ==
 
t ∑
 
time −time
i,,90%i 10%
 
i=1
where
V is ramp rate in °C/s
is sensor i of N
N is total number of sensors.
t is t at 10 % time of the ramp slope in °C
i,%10 i
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t is t at 90 % time of the ramp slope in °C
i,%90
time
is time in s

heat or cool rate of thermal cycler calculated between 10 % and 90 % time of the heating or cool-

ing slope.

Note 1 to entry: The heat rate is a positive ramp rate. The cool rate is a negative ramp rate.

3.3.10
maximum ramp rate
tmax
maximum heat or cool rate during heating or cooling slope in °C/s
3.3.11
maximum temperature overshoot
timmehold=enndovershoot
tt= ()time −=t ()timeholds30
io,,vs maxi,max timeholde=b ginovershoot i

maximum temperature value in °C of all active temperature sensors during temperature overshoot

above the average temperature of the reaction block or chamber temperature at hold when heating up

Note 1 to entry: The maximum temperature overshoot is calculated between begin and end of the overshoot and

is expressed relative to the temperature at 30 s hold time.

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.
3.3.12
minimum temperature undershoot
timmehold=eendundershoot
tt= ()time −=t ()timeholds30
i,,uns mini,min timeholde=b ginundershoot i

minimum temperature value in °C of all active temperature sensors during temperature undershoot

below the average temperature of reaction block or chamber temperature at hold when cooling down.

Note 1 to entry: The maximum temperature undershoot is calculated between begin and end of the undershoot

and is expressed relative to the temperature at 30 s hold time. An undershoot is an overshoot in negative

direction.

Note 2 to entry: to entry The undershoot occurs typically between 0 s and 15 s hold time. See Annex D for an

example thermal cycler temperature profile
3.3.13
average temperature overshoot
 
io,,vs max
t =
 
ovsa, vg
 
i=1

average value of maximum temperature overshoots of all active block temperature sensors in °C

3.3.14
average temperature undershoot
 
i,,uns min
t =
 
uns,avg ∑
 
i=1

average value of maximum temperature undershoot of all active block temperature sensors in °C

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3.3.15
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 time stamp where the average temperature 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 time stamp where the average temperature reaches the average hold temperature at the finish

of the overshoot.
3.3.16
hold time
time elapsed between start and end of a temperature hold in s.
Note 1 to entry: See Annex D for 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, when subjected to a change in temperature, to

react to this change
3.4.4
measurement uncertainty

parameter associated with the result of the measurement that characterizes the dispersion of the

values that could reasonably be attributed to the quantity intended to be measured

[SOURCE: Adapted from ISO/IEC Guide 99:2007, 2.26]
3.4.5
performance test
test procedure which determines the performance of a thermal cycler
3.4.6
calibration

operation that, under specified conditions, in a first step, establishes a relation between the quantity

values with measurement uncertainties 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, normalisation, installation qualification (IQ), operational qualification (OQ) or

performance qualification (PQ)
[SOURCE: ISO/IEC Guide 99:2007, 2.39]
4 Installation of thermal cyclers
The manufacturer’s instructions shall be followed.
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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) Environmental conditions which should at minimum be taken into account are room temperature

and relative humidity.

c) Thermal cyclers shall be located in such a way that free circulation of air is permanently allowed.

5 Maintenance of thermal cyclers

The laboratory shall establish a maintenance program, 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 standard 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 of 12 sensors (6.4.5.1) and metrological traceability (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 of 8 sensors 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 measured.

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. The ISO/IEC 17025 describes when metrological traceability is required and how metrological

traceability is established.

NOTE 2 The chemistry or biochemistry based normalisation and verification kits, which do exist at the

moment of publication of this standard, offer no traceability to SI and are associated with high measurement

uncertainties and therefore inapt as performance testing method.
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Figure 1 — Decision chart to determine requirement for metrological traceable temperature

calibration of thermal cyclers
6.2 Performance testing program

The laboratory shall establish a performance testing program, 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.

NOTE ISO/IEC 17025 compliant laboratories are required to establish a planned calibration program in

order to maintain confidence in the status of calibration.
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; and

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.

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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. It can be used to perform a temperature performance test on

both PCR and real-time PCR thermal cyclers.
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 (6.4.5.1), allowing to measure uniformity;

b) capable of recording the heated lid temperature with at least 1 temperature sensor (when physically

possible);

c) capable of recording temperatures dynamically with a sampling frequency of a least 2 times per

second in order to measure correctly the ramp rate and overshoot;

NOTE For thermal cyclers with heat rates above 4 °C/s a sampling frequency of at least 4 times per

second per temperature sensor is recommended.

a) capable of recording temperature over the complete reaction temperature range;

b) capable of being calibrated, traceable to SI, over at least the complete reaction

...