ISO 20395:2019

Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
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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 20395:2019(E)
©
ISO 2019

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Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
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ISO 20395:2019(E)

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© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
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ISO 20395:2019(E)

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
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ISO 20395:2019(E)

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 .28
Annex A (informative) Spectrophotometry .29
Annex B (informative) Nucleic acid integrity .31
Annex C (informative) PCR efficiency .33
Annex D (informative) Measurement uncertainty .36
Annex E (informative) MIQE and dMIQE checklists .39
Bibliography .45
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ISO 20395:2019(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 ISO/TC 276, Biotechnology.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
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ISO 20395:2019(E)

Introduction
This document has been developed to specifically support the analytical requirements with
respect to quantification of specific nucleic acid sequences (targets). It can also benefit the broader
biomanufacturing, bioscience research and development, industrial biotechnology, engineering
biology and advanced therapeutics industries which need to demonstrate product quality based on
measurement and quantification of specific nucleic acid targets.
Quantification of nucleic acid target sequences is a cross-cutting fundamental measurement
that broadly impacts many aspects of biotechnology. For example, quantification of nucleic acid
biomarkers for monitoring bioprocess efficiency and conformity with quality by design parameters for
biopharmaceutical manufacture and industrial biotechnology, characterization of purity and quality
of cell-derived advanced therapy medicinal products (ATMPs); assessment of gene copy number for
evaluating the potency and efficacy of gene-based therapies and process control assays for gene editing
and engineering biology applications.
The underpinning technique of polymerase chain reaction (PCR) has transformed the field of nucleic
acid analysis, due to its robustness and simplicity. Technological advances in instrumentation have
resulted in a wide range of PCR-based nucleic acid quantification approaches/instruments with
subsequent developments such as:
— quantitative real-time PCR (qPCR) which offers methods for quantification of DNA and RNA
molecules relative to a calibration material or independent sample, and
— digital PCR (dPCR) which offers the ability to perform SI traceable quantification through the
concept of molecular enumeration without the need for a calibration curve.
However, performing nucleic acid quantification assays to a high standard of analytical quality can be
challenging. For example, it is well known that impure or degraded nucleic acid extracts can affect the
accuracy of quantification. Similarly, a poorly designed qPCR or dPCR assay with poor amplification
efficiency and primer specificity will have an impact on accuracy of quantification. In addition, aspects
such as calibrators, standard curves, data normalization and processing can have a large influence on
the accuracy of quantitative measurement of nucleic acid targets.
This document is expected to improve confidence in the data produced, support selection and
optimization procedures and provide supporting performance parameters that may be utilized during
performance qualification of a particular measurement procedure for quantification of nucleic acid
target sequences. Biotechnology and bioscience industry data with higher measurement confidence
will enable data interoperability, improved product quality, reduced risks and costs and facilitate
international trade.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
Further details can be found in the ISO/IEC Directives, Part 2.
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INTERNATIONAL STANDARD ISO 20395:2019(E)
Biotechnology — Requirements for evaluating the
performance of quantification methods for nucleic acid
target sequences — qPCR and dPCR
1 Scope
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 (<50 bases).
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).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/IEC Guide 98-3:2008, Uncertainty of measurement — Part 3: Guide to the expression of uncertainty in
me a s ur ement (GUM: 1995)
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ISO 20395:2019(E)

ISO/IEC Guide 99, International vocabulary of metrology — Basic and general concepts and associated
terms (VIM)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/IEC Guide 99 and the
following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
amplicon
specific DNA fragment produced by a DNA-amplification technology, such as the polymerase chain
reaction (PCR)
[SOURCE: ISO 13495:2013, 3.3.1]
3.2
amplification plot
graph representing the generation of a reporter (usually fluorescent) signal during a qPCR or dPCR
reaction
Note 1 to entry: For qPCR and some dPCR systems, the amplification plot shows the relationship between cycle
number (x-axis) and fluorescence signal (y-axis).
Note 2 to entry: For end point dPCR, the fluorescent signal of each dPCR partition is displayed. For a single
fluorophore, a one-dimensional amplification plot shows partition number (x-axis) against end point fluorescent
signal (y-axis). A multi-dimensional amplification plot shows fluorescent signal for each detector channel on
each axis.
3.3
calibration curve
standard curve
expression of the relation between indication and corresponding measured quantity value
[SOURCE: ISO/IEC Guide 99:2007, 4.31, modified — The notes have been deleted.]
3.4
calibrator
measurement standard used in calibration
Note 1 to entry: The term “calibrator” is only used in certain fields.
EXAMPLE A qPCR interplate calibrator sample is often included on each qPCR plate in a study comprising
multiple qPCR plates or experiments to compensate for variations across plates due to instrument measurement
factors such as baseline and threshold setting. The interplate calibrator contains the target sequence(s) detected
by the PCR assay and is measured with the same PCR assays as the studied samples.
[SOURCE: ISO/IEC Guide 99:2007, 5.12, modified — The example has been added.]
3.5
cDNA
complementary DNA
single-stranded DNA, complementary to a given RNA and synthesised in the presence of reverse
transcriptase to serve as a template for DNA amplification
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ISO 20395:2019(E)

3.6
copy number
number of molecules (copies) containing a specific nucleic acid sequence
[SOURCE: ISO 16577:2016, 3.28, modified — "of a DNA sequence" replaced with "containing a specific
nucleic acid sequence"]
3.7
copy number concentration
number of molecules (copies) containing a specific nucleic acid sequence in a defined volume
3.8
quantification cycle
C
q
cycle at which the fluorescence from the reaction crosses a specified threshold
...