ISO 20395:2019
(Main)Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic acid target sequences — qPCR and dPCR
Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic acid target sequences — qPCR and dPCR
This document provides generic requirements for evaluating the performance and ensuring the quality of methods used for the quantification of specific nucleic acid sequences (targets). This document is applicable to the quantification of DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) target sequences using either digital (dPCR) or quantitative real-time PCR (qPCR) amplification technologies. It applies to target sequences present in nucleic acid molecules including double-stranded DNA (dsDNA) such as genomic DNA (gDNA) and plasmid DNA, single stranded DNA (ssDNA), complementary DNA (cDNA), and single stranded RNA (ssRNA) including ribosomal RNA (rRNA), messenger RNA (mRNA), and long and short non-coding RNA [microRNAs (miRNAs) and short interfering RNAs (siRNAs)], as well as double-stranded RNA (dsRNA). This document applies to nucleic acids derived from biological sources such as viruses, prokaryotic and eukaryotic cells, cell-free biological fluids (e.g. plasma or cell media) or in vitro sources [e.g. oligonucleotides, synthetic gene constructs and in vitro transcribed (IVT) RNA]. This document is not applicable to quantification of very short DNA oligonucleotides ( This document covers: — analytical design including quantification strategies (nucleic acid copy number quantification using a calibration curve as in qPCR or through molecular counting as in dPCR, quantification relative to an independent sample and ratio measurements) and use of controls; — quantification of total nucleic acid mass concentration and quality control of a nucleic acid sample including assessment of nucleic acid quality (purity and integrity); — PCR assay design, optimization, in silico and in vitro specificity testing; — data quality control and analysis including acceptance criteria, threshold setting and normalization; — method validation (precision, linearity, limit of quantification, limit of detection, trueness and robustness) with specific requirements for qPCR and dPCR; — approaches to establishing metrological traceability and estimating measurement uncertainty. This document does not provide requirements or acceptance criteria for the sampling of biological materials or processing of biological samples (i.e. collection, preservation, transportation, storage, treatment and nucleic acid extraction). Nor does it provide requirements and acceptance criteria for specific applications (e.g. food or clinical applications where specific matrix issues can arise).
Biotechnologie — Exigences relatives à l'évaluation de la performance des méthodes de quantification des séquences d'acides nucléiques cibles — qPCR et dPCR
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INTERNATIONAL ISO
STANDARD 20395
First edition
2019-08
Biotechnology — Requirements
for evaluating the performance of
quantification methods for nucleic acid
target sequences — qPCR and dPCR
Biotechnologie — Exigences relatives à l'évaluation de la
performance des méthodes de quantification des séquences d'acides
nucléiques cibles — qPCR et dPCR
Reference number
©
ISO 2019
© ISO 2019
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Design of measurement procedure . 8
4.1 General . 8
4.2 Quantification method . 8
4.2.1 General. 8
4.2.2 qPCR determination of nucleic acid concentrations using a calibration curve . 8
4.2.3 dPCR determination of copy number concentration using molecular counting . 9
4.2.4 Relative quantification by qPCR .10
4.2.5 dPCR determination of ratio between two targets .11
4.3 Normalization strategy .11
4.4 Controls .12
5 Sample QC — Total nucleic acid quantity, integrity and purity .13
5.1 General .13
5.2 Total nucleic acid quantification .13
5.2.1 General.13
5.2.2 Spectrophotometry . .14
5.2.3 Fluorometry .14
5.2.4 Assessment of total DNA concentration using qPCR/dPCR .14
5.3 Nucleic acid integrity .15
5.4 Nucleic acid purity .15
6 Assay design and optimization for quantification of nucleic acid target sequences .16
6.1 Assay design .16
6.1.1 General.16
6.1.2 Amplicon selection .16
6.1.3 Primer and probe design .16
6.1.4 In silico evaluation of specificity .16
6.1.5 RT-qPCR/RT-dPCR design .17
6.2 Assay optimization using purified samples .17
6.2.1 General.17
6.2.2 Optimization of fluorescence signal .17
6.2.3 (RT)-qPCR amplification efficiency .18
6.2.4 RT efficiency.18
6.2.5 Specificity .18
6.3 Method optimization using test samples .19
6.3.1 Effect of PCR inhibitors in sample matrix .19
6.3.2 Presence of nucleic acid contaminants in test sample .19
6.3.3 Validated measurement range .20
6.4 No template controls .20
7 Data quality control (QC) and analysis .20
7.1 General .20
7.2 Acceptance criteria .20
7.2.1 qPCR .20
7.2.2 dPCR .20
7.3 Threshold setting .21
7.3.1 qPCR .21
7.3.2 dPCR .21
7.4 Data pre-processing .21
7.4.1 qPCR using calibration curve .21
7.4.2 Relative quantification (qPCR) .21
7.5 Identification of outliers .22
8 Nucleic acid quantification measurement method validation .22
8.1 General .22
8.2 Precision .22
8.3 LOQ .23
8.4 LOD .23
8.5 Linearity . .24
8.6 Trueness .24
8.7 Robustness .24
8.8 Specific considerations for qPCR method validation .25
8.8.1 Repeatability of qPCR- or RT-qPCR .25
8.8.2 Intermediate precision and reproducibility of qPCR- or RT-qPCR .25
8.9 Specific considerations for dPCR method validation .25
9 Nucleic acid quantification measurement traceability and comparability .25
9.1 Metrological traceability .25
9.2 Use of reference materials .26
9.3 Instrument calibration .26
10 Measurement uncertainty (MU) in qPCR and dPCR measurements .26
10.1 General requirements for MU calculations .26
10.2 qPCR measurement uncertainty .27
10.3 Ratio-based measurements .27
10.4 dPCR measurement uncertainty .27
11 Reporting .
...
INTERNATIONAL ISO
STANDARD 20395
First edition
2019-08
Biotechnology — Requirements
for evaluating the performance of
quantification methods for nucleic acid
target sequences — qPCR and dPCR
Biotechnologie — Exigences relatives à l'évaluation de la
performance des méthodes de quantification des séquences d'acides
nucléiques cibles — qPCR et dPCR
Reference number
©
ISO 2019
© ISO 2019
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Design of measurement procedure . 8
4.1 General . 8
4.2 Quantification method . 8
4.2.1 General. 8
4.2.2 qPCR determination of nucleic acid concentrations using a calibration curve . 8
4.2.3 dPCR determination of copy number concentration using molecular counting . 9
4.2.4 Relative quantification by qPCR .10
4.2.5 dPCR determination of ratio between two targets .11
4.3 Normalization strategy .11
4.4 Controls .12
5 Sample QC — Total nucleic acid quantity, integrity and purity .13
5.1 General .13
5.2 Total nucleic acid quantification .13
5.2.1 General.13
5.2.2 Spectrophotometry . .14
5.2.3 Fluorometry .14
5.2.4 Assessment of total DNA concentration using qPCR/dPCR .14
5.3 Nucleic acid integrity .15
5.4 Nucleic acid purity .15
6 Assay design and optimization for quantification of nucleic acid target sequences .16
6.1 Assay design .16
6.1.1 General.16
6.1.2 Amplicon selection .16
6.1.3 Primer and probe design .16
6.1.4 In silico evaluation of specificity .16
6.1.5 RT-qPCR/RT-dPCR design .17
6.2 Assay optimization using purified samples .17
6.2.1 General.17
6.2.2 Optimization of fluorescence signal .17
6.2.3 (RT)-qPCR amplification efficiency .18
6.2.4 RT efficiency.18
6.2.5 Specificity .18
6.3 Method optimization using test samples .19
6.3.1 Effect of PCR inhibitors in sample matrix .19
6.3.2 Presence of nucleic acid contaminants in test sample .19
6.3.3 Validated measurement range .20
6.4 No template controls .20
7 Data quality control (QC) and analysis .20
7.1 General .20
7.2 Acceptance criteria .20
7.2.1 qPCR .20
7.2.2 dPCR .20
7.3 Threshold setting .21
7.3.1 qPCR .21
7.3.2 dPCR .21
7.4 Data pre-processing .21
7.4.1 qPCR using calibration curve .21
7.4.2 Relative quantification (qPCR) .21
7.5 Identification of outliers .22
8 Nucleic acid quantification measurement method validation .22
8.1 General .22
8.2 Precision .22
8.3 LOQ .23
8.4 LOD .23
8.5 Linearity . .24
8.6 Trueness .24
8.7 Robustness .24
8.8 Specific considerations for qPCR method validation .25
8.8.1 Repeatability of qPCR- or RT-qPCR .25
8.8.2 Intermediate precision and reproducibility of qPCR- or RT-qPCR .25
8.9 Specific considerations for dPCR method validation .25
9 Nucleic acid quantification measurement traceability and comparability .25
9.1 Metrological traceability .25
9.2 Use of reference materials .26
9.3 Instrument calibration .26
10 Measurement uncertainty (MU) in qPCR and dPCR measurements .26
10.1 General requirements for MU calculations .26
10.2 qPCR measurement uncertainty .27
10.3 Ratio-based measurements .27
10.4 dPCR measurement uncertainty .27
11 Reporting .
...
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