Molecular biomarker analysis — Isothermal polymerase chain reaction (isoPCR) methods — Part 1: General requirements

This document specifies general criteria for development, validation and use of nucleic acid analytical methods based on the isothermal polymerase chain reaction (isoPCR). It provides additional information and guidance for specific isoPCR technologies. This document is applicable to food, feed, plant matrices and their propagules, plant pathogens, and animals in which amplification of a specific biomolecular target sequence is required.

Analyse de biomarqueurs moléculaires — Méthodes de réaction de polymérisation en chaîne isotherme (isoPCR) — Partie 1: Exigences générales

Le présent document spécifie des critères généraux pour la mise au point, la validation et l’utilisation de méthodes d’analyse d’acide nucléique fondées sur la réaction de polymérisation en chaîne isotherme (isoPCR). Il délivre des informations supplémentaires et des recommandations pour des technologies isoPCR particulières. Le présent document est applicable aux aliments, aux aliments pour animaux, aux matrices végétales et à leurs propagules, aux agents phytopathogènes, et aux animaux pour lesquels une amplification d’une séquence biomoléculaire cible spécifique est requise.

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Status
Published
Publication Date
23-Mar-2022
Current Stage
6060 - International Standard published
Start Date
24-Mar-2022
Due Date
06-Oct-2021
Completion Date
24-Mar-2022
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INTERNATIONAL ISO
STANDARD 22942-1
First edition
2022-03
Molecular biomarker analysis —
Isothermal polymerase chain reaction
(isoPCR) methods —
Part 1:
General requirements
Analyse de biomarqueurs moléculaires — Méthodes de réaction de
polymérisation en chaîne isotherme (isoPCR) —
Partie 1: Exigences générales
Reference number
ISO 22942-1:2022(E)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO 22942-1:2022(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2022

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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© ISO 2022 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22942-1:2022(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

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

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

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

4 Principle ........................................................................................................................................................................................................................ 3

5 Development of an isoPCR method ................................................................................................................................................... 3

5.1 General ........................................................................................................................................................................................................... 3

5.2 Intended purpose ................................................................................................................................................................................. 3

5.3 Scientific basis ........................................................................................................................................................................................ 3

5.4 Units of measurement ...................................................................................................................................................................... 6

5.5 Method validation ................................................................................................................................................................................ 6

5.6 Performance criteria ......................................................................................................................................................................... 6

5.6.1 General ........................................................................................................................................................................................ 6

5.6.2 Sensitivity ................................................................................................................................................................................. 6

5.6.3 Nucleic acid extract quality ...................................................................................................................................... 7

5.6.4 Applicability ........................................................................................................................................................................... 7

5.6.5 Nucleic acid sequence specificity ......................................................................................................................... 7

5.6.6 Precision .................................................................................................................................................................................... 7

5.6.7 Accuracy .................................................................................................................................................................................... 8

5.6.8 Selectivity ................................................................................................................................................................................. 8

5.6.9 Linearity .................................................................................................................................................................................... 8

5.6.10 Limit of detection (LOD) .............................................................................................................................................. 8

5.6.11 Limit of quantification (LOQ) ................................................................................................................................. 9

5.6.12 Range ......................................................................................................................................................................................... 10

5.6.13 Robustness ................................... ........................................................................................................ ................................. 10

6 General laboratory and procedural requirements ......................................................................................................11

6.1 Competence ............................................................................................................................................................................................ 11

6.2 Sample preparation ......................................................................................................................................................................... 11

6.2.1 General ..................................................................................................................................................................................... 11

6.2.2 Obtaining a representative sample ................................................................................................................. 11

6.2.3 Preparation of the test portion ........................................................................................................................... 11

6.2.4 Nucleic acid extraction ...............................................................................................................................................12

6.3 Use of controls ......................................................................................................................................................................................12

6.3.1 General .....................................................................................................................................................................................12

6.3.2 Environmental controls ............................................................................................................................................12

6.3.3 Positive controls ..............................................................................................................................................................12

6.3.4 Negative controls ............................................................................................................................................................12

6.3.5 Extraction controls .......................................................................................................................................................12

6.4 Workspace organization .............................................................................................................................................................13

6.4.1 General .....................................................................................................................................................................................13

6.4.2 Design of the workspace — Laboratory design ...................................................................................13

6.4.3 Design of non-laboratory workspaces ......................................................................................................... 13

6.4.4 Personnel ................................................................................................................................................................................ 13

6.4.5 Apparatus and equipment ...................................................................................................................................... 14

7 Materials and reagents ..............................................................................................................................................................................14

8 Interpretation of results ...........................................................................................................................................................................14

8.1 General ........................................................................................................................................................................................................ 14

8.2 Interpretation of controls .......................................................................................................................................................... 14

8.3 Expression of results ...................................................................................................................................................................... 15

8.3.1 General .....................................................................................................................................................................................15

iii
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ISO 22942-1:2022(E)

8.3.2 Expression of a negative result .......................................................................................................................... 15

8.3.3 Expression of a positive result ............................................................................................................................ 16

8.3.4 Expression of quantitative results .................................................................................................................. 16

8.3.5 Expression of ambiguous results ................................... ................................................................................... 16

9 Test report ...............................................................................................................................................................................................................16

Annex A (informative) Minimum information for an isoPCR experiment (MIIPCRE) .................................18

Annex B (normative) Use of controls ................................................................................................................................................................21

Annex C (informative) Examples of isothermal nucleic acid isoPCR amplification results ...................22

Annex D (informative) Loop mediated isothermal amplification (LAMP) ..............................................................23

Annex E (informative) Rolling circle amplification (RCA).........................................................................................................26

Annex F (informative) Helicase dependent amplification (HDA) ......................................................................................27

Annex G (informative) Recombinase polymerase amplification (RPA) ......................................................................29

Annex H (informative) Strand displacement amplification (SDA) ....................................................................................31

Annex I (informative) Nucleic acid sequence based amplification (NASBA) .........................................................33

Annex J (informative) Cas9nAR amplification .......................................................................................................................................36

Bibliography .............................................................................................................................................................................................................................38

© ISO 2022 – All rights reserved
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ISO 22942-1:2022(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

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

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee 34, Food products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 22942 series can be found on the ISO website.

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.
© ISO 2022 – All rights reserved
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ISO 22942-1:2022(E)
Introduction

Isothermal nucleic acid amplification describes methods that use constant temperature polymerase-

[1][2][3][4][5][6]

catalysed reactions to amplify a nucleic acid target sequence . In contrast to thermal-

cycler based polymerase chain reactions, isothermal nucleic acid amplification does not require

variable temperature cycling for denaturation, annealing, and polymerization although, in some cases,

primer binding requires a single high temperature denaturation and an annealing step. Isothermal

amplification methods can be described by the term “isothermal PCR (isoPCR)”.

Naturally, living organisms isothermally replicate DNA during cell division and transcribe RNA

to produce structural, and regulatory components. IsoPCR leverages both natural and synthetic

isothermal enzymatic processes. The enzymes include DNA and RNA polymerase, helicase,

recombinase, exonuclease and nickase. Because isoPCR does not require variable temperature

cycling for denaturation, polymerization and annealing there is no need for precision thermal cycling

instruments. Reactions are run at a single temperature, except in cases where a nickase or displacing

enzyme is not present in the reaction and an initial denaturation is required. In addition, various non-

enzymatic nucleic acid binding proteins can be necessary. IsoPCR amplification in many applications

can be performed on cell lysates without nucleic acid extraction. Some examples of amplification

[7]

strategies are loop-mediated isothermal amplification (LAMP) , rolling circle amplification (RCA)

[8] [9] [10]

, helicase dependent amplification (HDA) , recombinase polymerase amplification (RPA) , strand

[11] [12]

displacement amplification (SDA) , nucleic acid sequence-based amplification (NASBA) and

[13]

Cas9 nickase-based amplification reaction (Cas9nAR) . The LAMP, RCA, HDA, RPA, SDA and NASBA

strategies can incorporate both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) into amplified

nucleic acids. Cas9nAR can only use DNA as the starting template for amplification.

IsoPCR methods can be used for amplification, detection, identification, quantification, and analysis of

specific low concentration nucleic acids in food and food products. These methods can, in most cases,

amplify nucleic acids from un-purified nucleotide extracts. Detection of the target sequence is achieved

through real-time or end-point techniques using one of several different amplification strategies and

detection chemistries. Detection chemistries include turbidimetry, chromatography, gel electrophoresis

and fluorescence, and can, in some applications, be achieved in a closed lateral flow device system.

Key features of isoPCR methods are constant temperature nucleic acid amplification, use of crude

extracts, simple detection methods, and short reaction times without the need for precision thermal

cycling instruments.

Because isoPCR methods are gaining in popularity and applicability, standardization of the acceptance

criteria for these methods in food products is important.
© ISO 2022 – All rights reserved
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INTERNATIONAL STANDARD ISO 22942-1:2022(E)
Molecular biomarker analysis — Isothermal polymerase
chain reaction (isoPCR) methods —
Part 1:
General requirements
1 Scope

This document specifies general criteria for development, validation and use of nucleic acid analytical

methods based on the isothermal polymerase chain reaction (isoPCR). It provides additional

information and guidance for specific isoPCR technologies.

This document is applicable to food, feed, plant matrices and their propagules, plant pathogens, and

animals in which amplification of a specific biomolecular target sequence is required.

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/TS 16393, Molecular biomarker analysis — Determination of the performance characteristics of

qualitative measurement methods and validation of methods

ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in

agriculture and food production
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
extraction blank control

negative control reaction generated by performing all required steps in an extraction procedure except

for the addition of the test portion
EXAMPLE By substitution of water for the test portion.

Note 1 to entry: This control is used to demonstrate the absence of contamination during extraction.

3.2
extraction control

positive control reaction generated by performing all required steps in an extraction procedure except

with a known test portion containing a known amount of target nucleic acid or tissue

Note 1 to entry: This control is used to demonstrate the performance of the extraction process.

© ISO 2022 – All rights reserved
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ISO 22942-1:2022(E)
3.3
isothermal polymerase chain reaction
isoPCR
isothermal nucleic acid amplification
isothermal nucleic acid amplification technology
isothermal amplification

polymerase chain reaction that polymerizes nucleic acids without thermal cycling (3.10), e.g., at constant

temperature

Note 1 to entry: In some isoPCR applications, nucleic acids are denatured at a higher temperature prior to the

start of the amplification reaction.

Note 2 to entry: Seven isoPCR strategies are described in this document. These strategies can be applied to a

number of different methods consisting of DNA extraction, amplification and detection chemistries.

3.4
isoPCR method
analytical method that applies an isoPCR (3.3) strategy
3.5
non-laboratory field setting

workspace lacking conditions controlled for environmental aerosol contamination and sophisticated

nucleic acid purification apparatus
3.6
nucleotide sequence specificity

capacity to exclusively recognize a specific nucleic acid sequence target to be amplified, distinguishing

it from other nucleic acids and contaminants
3.7
percentage dynamic range
percentage applicability range
percentage range of quantification

ratio as a percentage of upper and lower limits of quantification as expressed by a set of reference

materials (or dilutions) with a suitable level of precision and accuracy
3.8
representative sample

sampling units (samples or groups) that have been extracted from the lot with a process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
[SOURCE: ISO 22753:2021, 3.15]
3.9
selectivity

extent to which a method can determine particular analyte(s) in a mixture(s) or matrix(matrices)

without interferences from other components of similar behaviour

Note 1 to entry: The selectivity of an isoPCR method (3.4) for RNA or DNA or both can be determined with respect

to inhibitors such as polyamines, polysaccharides and polyphenols, since these interfere with the ability of the

reaction to amplify and disclose a specific target sequence.

Note 2 to entry: Selectivity is differentiated from nucleotide sequence specificity (3.6) which measures the

recognition of the target sequence by the assay at the molecular or taxonomic levels.

© ISO 2022 – All rights reserved
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ISO 22942-1:2022(E)
3.10
thermal cycling
thermocycling

process including numerous heating and cooling steps of a pre-determined temperature regime used to

denature, anneal, and elongate nucleic acids in a polymerase chain reaction
4 Principle

Detection of the target sequence is achieved through real-time or end-point techniques that apply

a specific amplification strategy and leverage several different detection chemistries. Detection

chemistries include turbidimetry, chromatography, gel electrophoresis and fluorescence. In some

isoPCR applications, a product can be detected in a closed lateral flow device system or fluorescence

detection instrument.

A general overview for seven examples of isoPCR amplification strategies is provided in Table 1.

Descriptions of each isoPCR strategy, their applications, advantages and disadvantages can be found in

Annexes D to J.
5 Development of an isoPCR method
5.1 General

A DNA or RNA isoPCR method can be used to detect, identify and, as required, quantify an intended

specific nucleic acid target(s). A method consists of:
— a matrix-specific extraction (where required);
— any further purification step(s);
— the enzymatic components and reagents;

— a description of the oligonucleotide primers and probes (labelled and non-labelled) that will be used

(including how the target and oligonucleotide sequences were chosen);
— a description of how the amplified products will be detected;

— a protocol describing the conditions under which the isoPCR method is used including the use of

controls and example calculations.

Guidelines for the minimum information for publication of isoPCR experiments (MIIPCRE) are provided

in Annex A. MIIPCRE are guidelines for the minimum information necessary for evaluating isoPCR

experiments. Annex A is a checklist for laboratories.
5.2 Intended purpose

Information regarding the intended purpose and the limitations of a method shall be provided.

Specifically, the method shall be evaluated for fitness for purpose based on the criteria and requirements

described in this document.
5.3 Scientific basis

An overview of the principles and application of the method shall be provided. Appropriate references

to relevant scientific publications should be included.
© ISO 2022 – All rights reserved
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ISO 22942-1:2022(E)
© ISO 2022 – All rights reserved
Table 1 — General overview for seven isoPCR amplification strategies

IsoPCR Target Enzymes Initial Time Amplification Meas- LOD Analyte Detection Equipment needed

strategy nucleic involved heating (h) urement (copies) method(s)
Power Tempera-
acid method(s)
ture ( C)

LAMP DNA, RNA Polymerase Yes < 1 Exponen- 60 to 65 Qualitative, ~5 DNA, RNA, Turbidimetry of Visual detection, tur-

tial quantitative Small pyrophosphate, bidimeter (real-time);
molecules fluorescent dye, elec- isothermal fluorom-
trochemistry, eter; electrochemical
single-stranded LAMP microfluidic
nucleotide tag chip, lateral flow
hybridization detection strips or
printed array strip

RCA DNA, RNA Polymerase Yes 1 to 4 Linear 30 to 65 Qualitative, 10 DNA, RNA, Fluorescent tags, Spectrophotometer,

quantitative Protein, fluorometry isothermal
Methylated fluorometer
DNA, Small
molecules,
Cells

HDA DNA, RNA Helicase, No 0,5 to 2 Exponen- 64 Qualitative, 1 DNA, Gel electrophoresis; Electrophoresis cham-

polymerase tial quantitative Protein immunohistochemis- ber, UV transillumina-
try, fluorescent dyes tor; closed lateral flow
device system, isother-
mal fluorometer

RPA DNA, RNA Recombi- No < 1 Exponen- 37 to 42 Qualitative, 1 DNA, RNA, Gel electrophoresis; Electrophoresis cham-

nase, tial quantitative Protein immunohistochemis- ber, UV Transillumina-
polymerase try, fluorometry tor; closed lateral flow
device system, isother-
mal fluorometer

SDA DNA, RNA Polymerase, Yes 1 to 2 Exponen- 30 to 55 Qualitative 10 DNA, RNA, Gel electrophoresis; Electrophoresis cham-

restriction tial small pH indicator dyes; ber, UV transillumina-
enzyme molecules fluorescence tor; visual detection;
spectrophotometer or
isothermal fluorom-
eter
NOTE Adapted from Reference [19].
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ISO 22942-1:2022(E)
© ISO 2022 – All rights reserved
Table 1 (continued)

IsoPCR Target Enzymes Initial Time Amplification Meas- LOD Analyte Detection Equipment needed

strategy nucleic involved heating (h) urement (copies) method(s)
Power Tempera-
acid method(s)
ture ( C)

NASBA RNA, DNA Reverse No 1 to 3 Exponen- 41 Qualitative, 1 DNA, RNA, Gel electrophoresis; Electrophoresis

tran- tial quantitative miRNA, fluorescent probes; chamber, UV transil-
scriptase, Protein ELISA, fluorometry luminator; microplate
RNA pol- reader, isothermal
ymerase, fluorometer
RNase H

Cas9nAR DNA Cas9 No Exponen- 37 Qualitative DNA Gel electrophoresis; Electrophoresis

polymerase tial fluorescent dyes chamber, UV transil-
luminator; isothermal
fluorometer or visual
NOTE Adapted from Reference [19].
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ISO 22942-1:2022(E)
5.4 Units of measurement

Qualitative (binary) measurement with isoPCR methods provides a binary result based on a

predetermined probability of detection (POD). Qualitative measurements are used to determine the

presence or absence of molecular biomarkers in food or food products (including seeds and propagules

of food crops). The performance characterization of a qualitative method shall be carried out as

described in ISO/TS 16393.

Quantitative methods determine the amount of the target analyte present in a sample. Quantitative

units of measurement (e.g. target copy number), performance and da
...

NORME ISO
INTERNATIONALE 22942-1
Première édition
2022-03
Analyse de biomarqueurs
moléculaires — Méthodes de
réaction de polymérisation en chaîne
isotherme (isoPCR) —
Partie 1:
Exigences générales
Molecular biomarker analysis — Isothermal polymerase chain
reaction (isoPCR) methods —
Part 1: General requirements
Numéro de référence
ISO 22942-1:2022(F)
© ISO 2022
---------------------- Page: 1 ----------------------
ISO 22942-1:2022(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2022

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publication ne peut être reproduite ni utilisée sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique,

y compris la photocopie, ou la diffusion sur l’internet ou sur un intranet, sans autorisation écrite préalable. Une autorisation peut

être demandée à l’ISO à l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.

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Publié en Suisse
© ISO 2022 – Tous droits réservés
---------------------- Page: 2 ----------------------
ISO 22942-1:2022(F)
Sommaire Page

Avant-propos ...............................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ..................................................................................................................................................................................1

3 Termes et définitions ...................................................................................................................................................................................... 1

4 Principe.......................................................................................................................................................................................................................... 3

5 Mise au point d’une méthode isoPCR ............................................................................................................................................. 3

5.1 Généralités ................................................................................................................................................................................................. 3

5.2 Finalité prévue........................................................................................................................................................................................ 4

5.3 Fondement scientifique ........................................................................................................................................... ........................ 4

5.4 Unités de mesure .................................................................................................................................................................................. 7

5.5 Validation de la méthode ............................................................................................................................................................... 7

5.6 Critères de performance ................................................................................................................................................................ 7

5.6.1 Généralités ............................................................................................................................................................................... 7

5.6.2 Sensibilité ................................................................................................................................................................................. 8

5.6.3 Qualité de l’extrait d’acide nucléique ................................................................................................................ 8

5.6.4 Applicabilité ............................................................................................................................................................................ 8

5.6.5 Spécificité vis-à-vis de la séquence d’acide nucléique ....................................................................... 8

5.6.6 Fidélité ......................................................................................................................................................................................... 8

5.6.7 Exactitude................................................................................................................................................................................. 9

5.6.8 Sélectivité ................................................................................................................................................................................. 9

5.6.9 Linéarité ..................................................................................................................................................................................... 9

5.6.10 Limite de détection (LOD) .......................................................................................................................................... 9

5.6.11 Limite de quantification (LOQ) .......................................................................................................................... 10

5.6.12 Gamme ...................................................................................................................................................................................... 11

5.6.13 Robustesse ............................................................................................................................................................................ 12

6 Exigences générales du laboratoire et du mode opératoire ...............................................................................12

6.1 Compétence ............................................................................................................................................................................................ 12

6.2 Préparation des échantillons .................................................................................................................................................. 13

6.2.1 Généralités ............................................................................................................................................................................13

6.2.2 Obtention d’un échantillon représentatif ..................................................................................................13

6.2.3 Préparation de la prise d’essai ............................................................................................................................ 13

6.2.4 Extraction de l’acide nucléique ........................................................................................................................... 13

6.3 Utilisation de témoins ................................................................................................................................................................... 14

6.3.1 Généralités ............................................................................................................................................................................ 14

6.3.2 Témoins environnementaux ................................................................................................................................. 14

6.3.3 Témoins positifs ............................................................................................................................................................... 14

6.3.4 Témoins négatifs ............................................................................................................................................................. 14

6.3.5 Témoins d’extraction ................................................................................................................................................... 14

6.4 Organisation de l’espace de travail.................................................................................................................................... 15

6.4.1 Généralités ............................................................................................................................................................................15

6.4.2 Conception de l’espace de travail — Conception du laboratoire ..........................................15

6.4.3 Conception des espaces de travail hors du laboratoire ................................................................15

6.4.4 Personnel ................................................................................................................................................................................ 15

6.4.5 Appareillage et équipement .................................................................................................................................. 16

7 Matériaux et réactifs ....................................................................................................................................................................................16

8 Interprétation des résultats .................................................................................................................................................................16

8.1 Généralités .............................................................................................................................................................................................. 16

8.2 Interprétation des témoins....................................................................................................................................................... 16

8.3 Expression des résultats ............................................................................................................................................................. 17

8.3.1 Généralités ............................................................................................................................................................................ 17

iii
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ISO 22942-1:2022(F)

8.3.2 Expression d’un résultat négatif ....................................................................................................................... 17

8.3.3 Expression d’un résultat positif ......................................................................................................................... 18

8.3.4 Expression des résultats quantitatifs........................................................................................................... 18

8.3.5 Expression des résultats ambigus ................................................................................................................... 19

9 Rapport d’essai ...................................................................................................................................................................................................19

Annexe A (informative) Informations minimales à fournir pour une expérience isoPCR

(MIIPCRE) .................................................................................................................................................................................................................20

Annexe B (normative) Utilisation de témoins.........................................................................................................................................23

Annexe C (informative) Exemples de résultats d’amplification isotherme isoPCR d’acide

nucléique ...................................................................................................................................................................................................................24

Annexe D (informative) LAMP (loop mediated isothermal amplification) .............................................................25

Annexe E (informative) RCA (rolling circle amplification) .......................................................................................................28

Annexe F (informative) HDA (helicase dependent amplification) ....................................................................................29

Annexe G (informative) RPA (recombinase polymerase amplification) ....................................................................32

Annexe H (informative) SDA (strand displacement amplification) .................................................................................34

Annexe I (informative) NASBA (nucleic acid sequence based amplification) .......................................................36

Annexe J (informative) Amplification Cas9nAR ...................................................................................................................................40

Bibliographie ...........................................................................................................................................................................................................................42

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ISO 22942-1:2022(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 16, Méthodes horizontales pour l'analyse moléculaire de biomarqueurs.

Une liste de toutes les parties de la série ISO 22942 se trouve sur le site web de l’ISO.

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.
© ISO 2022 – Tous droits réservés
---------------------- Page: 5 ----------------------
ISO 22942-1:2022(F)
Introduction

L’amplification isotherme d’acide nucléique désigne des méthodes qui utilisent des réactions catalysées

[1][2][3][4][5]

par polymérase à température constante pour amplifier une séquence cible d’acide nucléique

[6]

. Contrairement aux réactions de polymérisation en chaîne reposant sur l’emploi d’un thermocycleur,

l’amplification isotherme d’acide nucléique ne nécessite pas de cycle de variation de la température pour

la dénaturation, l’hybridation et la polymérisation même si, dans certains cas, la liaison de l’amorce

nécessite une dénaturation à une seule température élevée et une étape d’hybridation. Les méthodes

d’amplification isotherme peuvent être désignées par le terme «PCR isotherme (isoPCR)».

Les organismes vivants répliquent naturellement l’ADN lors de la division cellulaire et transcrivent

l’ARN pour produire des éléments structurels et régulateurs de manière isotherme. L’isoPCR s’appuie

sur des processus enzymatiques isothermes aussi bien naturels que synthétiques. L’ADN polymérase,

l’ARN polymérase, l’hélicase, la recombinase, l’exonucléase et la nickase comptent parmi les enzymes

mises en jeu. L’isoPCR ne nécessitant pas de cycle de variation de la température pour la dénaturation,

la polymérisation et l’hybridation, elle ne nécessite pas de thermocycleur de précision. Les réactions

sont menées à une seule température, sauf lorsqu’une nickase ou une enzyme de déplacement n’est

pas présente dans la réaction et qu’une dénaturation initiale est requise. De plus, diverses protéines

de liaison d’acide nucléique non enzymatiques peuvent être nécessaires. L’amplification isoPCR peut

dans bon nombre d’applications être réalisée sur des lysats cellulaires sans extraction de l’acide

[7]

nucléique. La LAMP (de l’anglais, «loop-mediated isothermal amplification ») , la RCA (« rolling circle

[8] [9]

amplification ») , la HDA (« helicase dependent amplification ») , la RPA (« recombinase polymerase

[10] [11]

amplification ») , la SDA (« strand displacement amplification ») , la NASBA (« nucleic acid sequence-

[12] [13]

based amplification ») et la Cas9nAR (« Cas9 nickase-based amplification reaction ») sont

quelques exemples de stratégies d’amplification. Les stratégies LAMP, RCA, HDA, RPA, SDA et NASBA

peuvent intégrer aussi bien de l’acide désoxyribonucléique (ADN) que de l’acide ribonucléique (ARN)

dans les acides nucléiques amplifiés. La Cas9nAR n’utilise que de l’ADN comme matrice de départ pour

l’amplification.

Les méthodes isoPCR peuvent être utilisées pour l’amplification, la détection, l’identification,

la quantification et l’analyse d’acides nucléiques spécifiques en faible concentration dans les aliments

et les produits alimentaires. Ces méthodes peuvent, dans la plupart des cas, amplifier des acides

nucléiques d’extraits nucléotidiques non purifiés. La détection de la séquence cible est réalisée

au moyen de techniques en temps réel ou au point final utilisant l’une des différentes stratégies

d’amplification et différentes chimies de détection. Les chimies de détection incluent la turbidimétrie,

la chromatographie, l’électrophorèse sur gel et la fluorescence, et peuvent, dans certaines applications,

être réalisées dans un système de dispositif à débit latéral fermé.

Les caractéristiques clés des méthodes isoPCR sont l’amplification d’acide nucléique à température

constante, l’utilisation d’extraits bruts, des méthodes de détection simples, et des temps de réaction

courts sans nécessiter de thermocycleur de précision.

Les méthodes isoPCR gagnant en popularité et ayant une applicabilité de plus en plus diversifiée, il est

important de normaliser les critères d’acceptation de ces méthodes pour les produits alimentaires.

© ISO 2022 – Tous droits réservés
---------------------- Page: 6 ----------------------
NORME INTERNATIONALE ISO 22942-1:2022(F)
Analyse de biomarqueurs moléculaires — Méthodes
de réaction de polymérisation en chaîne isotherme
(isoPCR) —
Partie 1:
Exigences générales
1 Domaine d’application

Le présent document spécifie des critères généraux pour la mise au point, la validation et l’utilisation

de méthodes d’analyse d’acide nucléique fondées sur la réaction de polymérisation en chaîne isotherme

(isoPCR). Il délivre des informations supplémentaires et des recommandations pour des technologies

isoPCR particulières.

Le présent document est applicable aux aliments, aux aliments pour animaux, aux matrices végétales

et à leurs propagules, aux agents phytopathogènes, et aux animaux pour lesquels une amplification

d’une séquence biomoléculaire cible spécifique est requise.
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).

ISO/TS 16393, Analyse de biomarqueurs moléculaires — Détermination des caractéristiques de

performance des méthodes de mesure qualitatives et validation des méthodes

ISO 16577, Analyse de biomarqueurs moléculaires — Vocabulaire pour les méthodes d’analyse de

biomarqueurs moléculaires dans l’agriculture et la production agroalimentaire
3 Termes et définitions

Pour les besoins du présent document, les termes et les définitions de l'ISO 16577 ainsi que les 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
témoin négatif d’extraction

témoin négatif obtenu après avoir effectué toutes les étapes requises d’un mode opératoire d’extraction

ne comprenant toutefois pas l’ajout de la prise d’essai
EXEMPLE En remplaçant la prise d’essai par de l’eau.

Note 1 à l'article: Ce témoin permet de démontrer l’absence de contamination durant l’extraction.

© ISO 2022 – Tous droits réservés
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ISO 22942-1:2022(F)
3.2
témoin d’extraction

témoin positif obtenu après avoir effectué toutes les étapes requises d’un mode opératoire d’extraction

sur une prise d’essai toutefois connue dont la quantité d’acide nucléique ou de tissu cible est connue

Note 1 à l'article: Ce témoin permet de démontrer la performance du processus d’extraction.

3.3
réaction de polymérisation en chaîne isotherme
isoPCR
amplification isotherme d’acide nucléique
technologie d’amplification isotherme d’acide nucléique
amplification isotherme

réaction de polymérisation en chaîne qui polymérise des acides nucléiques sans cycle thermique (3.10),

par exemple à température constante

Note 1 à l'article: Dans certaines applications isoPCR, les acides nucléiques sont dénaturés à une température

plus élevée avant de commencer la réaction d’amplification.

Note 2 à l'article: Sept stratégies isoPCR sont décrites dans le présent document. Ces stratégies peuvent être

appliquées à un certain nombre de méthodes différentes intégrant une extraction d’ADN, une amplification et des

chimies de détection.
3.4
méthode isoPCR
méthode d’analyse qui applique une stratégie isoPCR (3.3)
3.5
conditions de terrain hors du laboratoire

espace de travail ne bénéficiant ni de conditions maîtrisées concernant la contamination par un aérosol

environnemental, ni d’un appareillage sophistiqué de purification de l’acide nucléique

3.6
spécificité vis-à-vis d’une séquence nucléotidique

aptitude à ne reconnaître exclusivement qu’une séquence cible d’acide nucléique à amplifier, en la

différenciant des autres acides nucléiques et contaminants
3.7
gamme dynamique de pourcentage
gamme d’applicabilité de pourcentage
domaine de quantification de pourcentage

rapport exprimé sous la forme d’un pourcentage des limites supérieure et inférieure de quantification

présentées par un groupe de matériaux (ou dilutions) de référence avec un niveau approprié de fidélité

et d’exactitude
3.8
échantillon représentatif

unités d’échantillonnage (échantillons ou groupes) qui ont été extraites du lot au moyen d’un processus

garantissant que toutes les unités d’échantillonnage ont les mêmes chances d’être sélectionnées et n’ont

pas été modifiées au point de fausser le résultat d’analyse

Note 1 à l'article: Le processus d’extraction peut être un processus à multiples étapes.

[SOURCE: ISO 22753:2021, 3.15]
© ISO 2022 – Tous droits réservés
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ISO 22942-1:2022(F)
3.9
sélectivité

limite jusqu’à laquelle une méthode peut déterminer la présence d’un ou plusieurs analytes particuliers

en mélange ou dans une ou plusieurs matrices sans interférences d’autres composants de comportement

similaire

Note 1 à l'article: La sélectivité d’une méthode isoPCR (3.4) destinée à l’ARN ou à l’ADN ou aux deux peut être

déterminée par rapport à des inhibiteurs tels que les polyamines, les polysaccharides et les polyphénols, car

ceux-ci interfèrent sur l’aptitude de la réaction à amplifier et déceler une séquence cible spécifique.

Note 2 à l'article: Il est nécessaire de bien faire la distinction entre sélectivité et spécificité vis-à-vis d’une séquence

nucléotidique (3.6), la spécificité caractérisant le degré de reconnaissance de la séquence cible par l’analyse aux

niveaux moléculaires ou taxonomiques.
3.10
cycle thermique
thermocycle

processus incluant de nombreuses étapes de chauffage et de refroidissement d’un programme de

température prédéterminé utilisé pour dénaturer, hybrider et allonger des acides nucléiques dans une

réaction de polymérisation en chaîne
4 Principe

La détection de la séquence cible est réalisée au moyen de techniques en temps réel ou au point final

qui appliquent une stratégie d’amplification donnée et s’appuient sur différentes chimies de détection.

Les chimies de détection incluent la turbidimétrie, la chromatographie, l’électrophorèse sur gel et la

fluorescence. Dans certaines applications isoPCR, un produit peut être détecté dans un système de

dispositif à débit latéral fermé ou dans un instrument de détection de fluorescence.

Une vue d’ensemble de sept exemples de stratégies d’amplification isoPCR est donnée dans le Tableau 1.

Les Annexes D à J fournissent une description et précisent les domaines d’application, les avantages et

les inconvénients de chaque stratégie isoPCR.
5 Mise au point d’une méthode isoPCR
5.1 Généralités

Une méthode isoPCR sur ADN ou ARN peut être utilisée pour détecter, identifier et, si besoin, quantifier

un ou plusieurs acides nucléiques cibles spécifiques voulus. Une méthode comprend:

— une extraction propre à la matrice (si besoin);
— une ou plusieurs éventuelles étapes de purification complémentaire;
— les constituants enzymatiques et réactifs;

— une description des amorces et sondes oligonucléotidiques (marquées et non marquées) qui seront

utilisées (notamment la façon dont les séquences oligonucléotidiques et cibles ont été choisies);

— une description de la méthode de détection des produits amplifiés;

— un protocole décrivant les conditions selon lesquelles la méthode isoPCR est utilisée notamment

l’utilisation de témoins et des exemples de calcul.

Un cadre directeur pour les informations minimales à fournir en vue d’une publication d’expériences

isoPCR (MIIPCRE, de l’anglais «minimum information for publication of isoPCR experiments ») est donné

à l’Annexe A. Le MIIPCRE constitue un cadre directeur pour les informations minimales nécessaires

à l’évaluation des expériences isoPCR. L’Annexe A est une liste de points de contrôle destinée aux

laboratoires.
© ISO 2022 – Tous droits réservés
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ISO 22942-1:2022(F)
5.2 Finalité prévue

Des informations concernant la finalité prévue et les limites d’une méthode doivent être fournies.

L’adéquation de la méthode quant à la finalité doit notamment être évaluée sur la base des critères et

exigences spécifiés dans le présent document.
5.3 Fondement scientifique

Une vue d’ensemble des principes et de l’application de la méthode doit être fournie. Il convient d’inclure

des références appropriées à des publications scientifiques pertinentes.
© ISO 2022 – Tous droits réservés
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ISO 22942-1:2022(F)
© ISO 2022 – Tous droits réservés
Tableau 1 — Vue d’ensemble générale des sept stratégies d’amplification isoPCR

Stra- Acide nu- Enzymes Chauf- Durée Amplification Méthode(s) LOD Analyte Méthode(s) de Matériel nécessaire

tégie cléique impliquées fage (h) de mesure (co- détection
Type Tempéra-
isoPCR cible initial pies)
ture (°C)

LAMP ADN, Polymérase Oui < 1 Exponen- 60 à 65 Qualita- ~5 ADN, Turbidimétrie de Détection visuelle, turbi-

ARN tielle tive, quan- ARN, pyrophosphate, dimètre (en temps réel);
titative petites marqueur fluores- fluorimètre isot
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22942-1
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Isothermal polymerase chain reaction
Voting begins on:
2021-12-24 (isoPCR) methods —
Voting terminates on:
Part 1:
2022-02-18
General requirements
Analyse de biomarqueurs moléculaires — Méthodes de réaction de
polymérisation en chaîne isotherme (isoPCR) —
Partie 1: Exigences générales
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 22942-1:2021(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 22942-1:2021(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
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 22942-1:2021(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction .............................................................................................................................................................................................................................. vi

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

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

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

4 Principle ........................................................................................................................................................................................................................ 3

5 Development of an isoPCR method ................................................................................................................................................... 3

5.1 General ........................................................................................................................................................................................................... 3

5.2 Intended purpose ................................................................................................................................................................................. 3

5.3 Scientific basis ........................................................................................................................................................................................ 3

5.4 Units of measurement ...................................................................................................................................................................... 6

5.5 Method validation ................................................................................................................................................................................ 6

5.5.1 General ........................................................................................................................................................................................ 6

5.6 Performance criteria ......................................................................................................................................................................... 6

5.6.1 General ........................................................................................................................................................................................ 6

5.6.2 Sensitivity ................................................................................................................................................................................. 7

5.6.3 Nucleic acid extract quality ...................................................................................................................................... 7

5.6.4 Applicability ........................................................................................................................................................................... 7

5.6.5 Nucleic acid sequence specificity ......................................................................................................................... 7

5.6.6 Precision .................................................................................................................................................................................... 7

5.6.7 Accuracy .................................................................................................................................................................................... 8

5.6.8 Selectivity ................................................................................................................................................................................. 8

5.6.9 Linearity .................................................................................................................................................................................... 8

5.6.10 Limit of detection (LOD) .............................................................................................................................................. 8

5.6.11 Limit of quantification (LOQ) ................................................................................................................................. 9

5.6.12 Range ......................................................................................................................................................................................... 10

5.6.13 Robustness .................. .................................................... ...................................................................................................... 11

6 General laboratory and procedural requirements ......................................................................................................11

6.1 Competence ............................................................................................................................................................................................ 11

6.2 Sample preparation ......................................................................................................................................................................... 11

6.2.1 General ..................................................................................................................................................................................... 11

6.2.2 Obtaining a representative sample ................................................................................................................. 11

6.2.3 Preparation of the test portion ...........................................................................................................................12

6.2.4 Nucleic acid extraction ...............................................................................................................................................12

6.3 Use of controls ......................................................................................................................................................................................12

6.3.1 General .....................................................................................................................................................................................12

6.3.2 Environmental controls ............................................................................................................................................12

6.3.3 Positive controls .............................................................................................................................................................. 12

6.3.4 Negative controls ............................................................................................................................................................12

6.3.5 Extraction controls ....................................................................................................................................................... 13

6.4 Workspace organization .............................................................................................................................................................13

6.4.1 General .....................................................................................................................................................................................13

6.4.2 Design of the workspace — Laboratory design ...................................................................................13

6.4.3 Design of non-laboratory workspaces ......................................................................................................... 13

6.4.4 Personnel ................................................................................................................................................................................ 14

6.4.5 Apparatus and equipment ...................................................................................................................................... 14

7 Materials and reagents ..............................................................................................................................................................................14

8 Interpretation of results ...........................................................................................................................................................................14

8.1 General ........................................................................................................................................................................................................ 14

8.2 Interpretation of controls .......................................................................................................................................................... 14

8.3 Expression of results ...................................................................................................................................................................... 15

iii
© ISO 2021 – All rights reserved
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ISO/FDIS 22942-1:2021(E)

8.3.1 General .....................................................................................................................................................................................15

8.3.2 Expression of a negative result .......................................................................................................................... 15

8.3.3 Expression of a positive result ............................................................................................................................ 16

8.3.4 Expression of quantitative results .................................................................................................................. 16

8.3.5 Expression of ambiguous results ...................................................................................................................... 16

9 Test report ...............................................................................................................................................................................................................17

Annex A (informative) Minimum information for an isoPCR experiment (MIIPCRE) .................................18

Annex B (normative) Use of controls ................................................................................................................................................................21

Annex C (informative) Examples of isothermal nucleic acid isoPCR amplification results ...................22

Annex D (informative) Loop mediated isothermal amplification (LAMP) ..............................................................23

Annex E (informative) Rolling circle amplification (RCA).........................................................................................................26

Annex F (informative) Helicase dependent amplification (HDA) ......................................................................................27

Annex G (informative) Recombinase polymerase amplification (RPA) ......................................................................29

Annex H (informative) Strand displacement amplification (SDA) ....................................................................................31

Annex I (informative) Nucleic acid sequence based amplification (NASBA) .........................................................33

Annex J (informative) Cas9nAR amplification .......................................................................................................................................36

Bibliography .............................................................................................................................................................................................................................38

© ISO 2021 – All rights reserved
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ISO/FDIS 22942-1:2021(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 34, Food products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.
A list of all parts in the ISO 22942 series can be found on the ISO website.

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.
© ISO 2021 – All rights reserved
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ISO/FDIS 22942-1:2021(E)
Introduction

Isothermal nucleic acid amplification describes methods that use constant temperature polymerase-

[1][2][3][4][5][6]

catalysed reactions to amplify a nucleic acid target sequence . In contrast to thermal-

cycler based polymerase chain reactions, isothermal nucleic acid amplification does not require

variable temperature cycling for denaturation, annealing, and polymerization although, in some cases,

primer binding requires a single high temperature denaturation and an annealing step. Isothermal

amplification methods can be described by the term “isothermal PCR (isoPCR)”.

Naturally, living organisms isothermally replicate DNA during cell division and transcribe RNA

to produce structural, and regulatory components. IsoPCR leverages both natural and synthetic

isothermal enzymatic processes. The enzymes include DNA and RNA polymerase, helicase,

recombinase, exonuclease and nickase. Because isoPCR does not require variable temperature

cycling for denaturation, polymerization and annealing there is no need for precision thermal cycling

instruments. Reactions are run at a single temperature, except in cases where a nickase or displacing

enzyme is not present in the reaction and an initial denaturation is required. In addition, various non-

enzymatic nucleic acid binding proteins can be necessary. IsoPCR amplification in many applications

can be performed on cell lysates without nucleic acid extraction. Some examples of amplification

[7]

strategies are loop-mediated isothermal amplification (LAMP) , rolling circle amplification (RCA)

[8] [9] [10]

, helicase dependent amplification (HDA) , recombinase polymerase amplification (RPA) , strand

[11] [12]

displacement amplification (SDA) , nucleic acid sequence-based amplification (NASBA) and

[13]

Cas9 nickase-based amplification reaction (Cas9nAR) . The LAMP, RCA, HDA, RPA, SDA and NASBA

strategies can incorporate both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) into amplified

nucleic acids. Cas9nAR can only use DNA as the starting template for amplification.

IsoPCR methods can be used for amplification, detection, identification, quantification, and analysis of

specific low concentration nucleic acids in food and food products. These methods can, in most cases,

amplify nucleic acids from un-purified nucleotide extracts. Detection of the target sequence is achieved

through real-time or end-point techniques using one of several different amplification strategies and

detection chemistries. Detection chemistries include turbidimetry, chromatography, gel electrophoresis

and fluorescence, and can, in some applications, be achieved in a closed lateral flow device system.

Key features of isoPCR methods are constant temperature nucleic acid amplification, use of crude

extracts, simple detection methods, and short reaction times without the need for precision thermal

cycling instruments.

Because isoPCR methods are gaining in popularity and applicability, standardization of the acceptance

criteria for these methods in food products is important.
© ISO 2021 – All rights reserved
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 22942-1:2021(E)
Molecular biomarker analysis — Isothermal polymerase
chain reaction (isoPCR) methods —
Part 1:
General requirements
1 Scope

This document specifies general criteria for development, validation and use of nucleic acid analytical

methods based on the isothermal polymerase chain reaction (isoPCR). It provides additional

information and guidance for specific isoPCR technologies.

This document is applicable to food, feed, plant matrices and their propagules, plant pathogens, and

animals in which amplification of a specific biomolecular target sequence is required.

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/TS 16393, Molecular biomarker analysis — Determination of the performance characteristics of

qualitative measurement methods and validation of methods

ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in

agriculture and food production
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
extraction blank control

negative control reaction generated by performing all required steps in an extraction procedure except

for the addition of the test portion
EXAMPLE By substitution of water for the test portion.

Note 1 to entry: This control is used to demonstrate the absence of contamination during extraction.

3.2
extraction control

positive control reaction generated by performing all required steps in an extraction procedure except

with a known test portion containing a known amount of target nucleic acid or tissue

Note 1 to entry: This control is used to demonstrate the performance of the extraction process.

© ISO 2021 – All rights reserved
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ISO/FDIS 22942-1:2021(E)
3.3
isothermal polymerase chain reaction
isoPCR
isothermal nucleic acid amplification
isothermal nucleic acid amplification technology
isothermal amplification

polymerase chain reaction that polymerizes nucleic acids without thermal cycling (3.10), e.g., at constant

temperature

Note 1 to entry: In some isoPCR applications, nucleic acids are denatured at a higher temperature prior to the

start of the amplification reaction.

Note 2 to entry: Seven isoPCR strategies are described in this document. These strategies can be applied to a

number of different methods consisting of DNA extraction, amplification and detection chemistries.

3.4
isoPCR method
analytical method that applies an isoPCR (3.3) strategy
3.5
non-laboratory field setting

workspace lacking conditions controlled for environmental aerosol contamination and sophisticated

nucleic acid purification apparatus
3.6
nucleotide sequence specificity

capacity to exclusively recognize a specific nucleic acid sequence target to be amplified, distinguishing

it from other nucleic acids and contaminants
3.7
percentage dynamic range
percentage applicability range
percentage range of quantification

ratio as a percentage of upper and lower limits of quantification as expressed by a set of reference

materials (or dilutions) with a suitable level of precision and accuracy
3.8
representative sample

sampling units (samples or groups) that have been extracted from the lot with a process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
[SOURCE: ISO 22753:2021, 3.15]
3.9
selectivity

extent to which a method can determine particular analyte(s) in a mixture(s) or matrix(matrices)

without interferences from other components of similar behaviour

Note 1 to entry: The selectivity of an isoPCR method (3.4) for RNA or DNA or both can be determined with respect

to inhibitors such as polyamines, polysaccharides and polyphenols, since these interfere with the ability of the

reaction to amplify and disclose a specific target sequence.

Note 2 to entry: Selectivity is differentiated from nucleotide sequence specificity (3.6) which measures the

recognition of the target sequence by the assay at the molecular or taxonomic levels.

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ISO/FDIS 22942-1:2021(E)
3.10
thermal cycling
thermocycling

process including numerous heating and cooling steps of a pre-determined temperature regime used to

denature, anneal, and elongate nucleic acids in a polymerase chain reaction
4 Principle

Detection of the target sequence is achieved through real-time or end-point techniques that apply

a specific amplification strategy and leverage several different detection chemistries. Detection

chemistries include turbidimetry, chromatography, gel electrophoresis and fluorescence. In some

isoPCR applications, a product can be detected in a closed lateral flow device system or fluorescence

detection instrument.

A general overview for seven examples of isoPCR amplification strategies is provided in Table 1.

Descriptions of each isoPCR strategy, their applications, advantages and disadvantages can be found in

Annexes D to J.
5 Development of an isoPCR method
5.1 General

A DNA or RNA isoPCR method can be used to detect, identify and, as required, quantify an intended

specific nucleic acid target(s). A method consists of:
— a matrix-specific extraction (where required);
— any further purification step(s);
— the enzymatic components and reagents;

— a description of the oligonucleotide primers and probes (labelled and non-labelled) that will be used

(including how the target and oligonucleotide sequences were chosen);
— a description of how the amplified products will be detected;

— a protocol describing the conditions under which the isoPCR method is used including the use of

controls and example calculations.

Guidelines for the minimum information for publication of isoPCR experiments (MIIPCRE) are provided

in Annex A. MIIPCRE are guidelines for the minimum information necessary for evaluating isoPCR

experiments. Annex A is a checklist for laboratories.
5.2 Intended purpose

Information regarding the intended purpose and the limitations of a method shall be provided.

Specifically, the method shall be evaluated for fitness for purpose based on the criteria and requirements

described in this document.
5.3 Scientific basis

An overview of the principles and application of the method shall be provided. Appropriate references

to relevant scientific publications should be included.
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ISO/FDIS 22942-1:2021(E)
© ISO 2021 – All rights reserved
Table 1 — General overview for seven isoPCR amplification strategies

IsoPCR Target Enzymes Initial Time Amplification Meas- LOD Analyte Detection Equipment needed

strategy nucleic involved heating (h) urement (copies) method(s)
Power Tempera-
acid method(s)
ture ( C)

LAMP DNA, RNA Polymerase Yes < 1 Exponen- 60 to 65 Qualitative, ~5 DNA, RNA, Turbidimetry of Visual detection, tur-

tial quantitative Small pyrophosphate, bidimeter (real-time);
molecules fluorescent dye, elec- isothermal fluorom-
trochemistry, eter; electrochemical
single-stranded LAMP microfluidic
nucleotide tag chip, lateral flow
hybridization detection strips or
printed array strip

RCA DNA, RNA Polymerase Yes 1 to 4 Linear 30 to 65 Qualitative, 10 DNA, RNA, Fluorescent tags, Spectrophotometer,

quantitative Protein, fluorometry isothermal
Methylated fluorometer
DNA, Small
molecules,
Cells

HDA DNA, RNA Helicase, No 0,5 to 2 Exponen- 64 Qualitative, 1 DNA, Gel electrophoresis; Electrophoresis cham-

polymerase tial quantitative Protein immunohistochemis- ber, UV transillumina-
try, fluorescent dyes tor; closed lateral flow
device system, isother-
mal fluorometer

RPA DNA, RNA Recombi- No < 1 Exponen- 37 to 42 Qualitative, 1 DNA, RNA, Gel electrophoresis; Electrophoresis cham-

nase, tial quantitative Protein immunohistochemis- ber, UV Transillumina-
polymerase try, fluorometry tor; closed lateral flow
device system, isother-
mal fluorometer

SDA DNA, RNA Polymerase, Yes 1 to 2 Exponen- 30 to 55 Qualitative 10 DNA, RNA, Gel electrophoresis; Electrophoresis cham-

restriction tial small pH indicator dyes; ber, UV transillumina-
enzyme molecules fluorescence tor; visual detection;
spectrophotometer or
isothermal fluorom-
eter
NOTE Adapted from Reference [19].
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ISO/FDIS 22942-1:2021(E)
© ISO 2021 – All rights reserved
Table 1 (continued)

IsoPCR Target Enzymes Initial Time Amplification Meas- LOD Analyte Detection Equipment needed

strategy nucleic involved heating (h) urement (copies) method(s)
Power Tempera-
acid method(s)
ture ( C)

NASBA RNA, DNA Reverse No 1 to 3 Exponen- 41 Qualitative, 1 DNA, RNA, Gel electrophoresis; Electrophoresis

tran- tial quantitative miRNA, fluorescent probes; chamber, UV transil-
scriptase, Protein ELISA, fluorometry luminator; microplate
RNA pol- reader, isothermal
ymerase, fluorometer
RNase H
Cas9nAR DNA Cas9 No Exponen- 37 Qualitative DNA Gel electroph
...

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