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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Published
Publication Date
07-Aug-2019
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6060 - International Standard published
Start Date
04-Jul-2019
Completion Date
08-Aug-2019
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ISO 20395:2019 - Biotechnology -- Requirements for evaluating the performance of quantification methods for nucleic acid target sequences -- qPCR and 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 20395:2019(E)
ISO 2019
---------------------- Page: 1 ----------------------
ISO 20395:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© 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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Published in Switzerland
ii © ISO 2019 – All rights reserved
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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

iv © ISO 2019 – All rights reserved
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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

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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).

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.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.
© ISO 2019 – All rights reserved v
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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.
vi © ISO 2019 – All rights reserved
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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
2 © ISO 2019 – All rights reserved
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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

cycle at which the fluorescence from the reaction crosses a specified threshold level at which

the signal can be distinguished from background levels

Note 1 to entry: Quantification cycle is a generic term which includes cycle threshold (C ), crossing point (C ),

t p

take off point and all other instrument specific terms referring to the fractional cycle used to quantify the

concentration of target in the qPCR assay.

Note 2 to entry: The quantification cycle is based either on a threshold applied to all samples or on a regression

analysis of the signal, for each sample.
[1]

[SOURCE: ISO 16577:2016, 3,32 modified according to MIQE Guidelines — Notes 1 and 2 to entry have

been added.]
3.9
delta C
difference between two C values, ΔC = C − C
q q q(1) q(2)
3.10
digital PCR
dPCR

procedure in which nucleic acid templates are distributed across multiple partitions of nominally

equivalent volume, such that some partitions contain template and others do not, followed by

PCR amplification of target sequences and detection of specific PCR products, providing a count of the

number of partitions with a positive and negative signal for the target template

Note 1 to entry: Nucleic acid target sequences are assumed to be randomly and independently distributed across

the partitions during the partitioning process.

Note 2 to entry: The count of positive and negative partitions is normally based on end point detection of

PCR products following thermal cycling, however real-time qPCR monitoring of PCR product accumulation is

additionally possible for some dPCR platforms.
3.11
gene of interest
GOI
gene target sequence under investigation
3.12
lambda value

mean number of targets per dPCR partition based on the fraction of droplets where amplification has

occurred

Note 1 to entry: Essential dPCR quantities for calculation of lambda are the number of positive partitions (N )

and the total number of partitions (N ).
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ISO 20395:2019(E)
3.13
limit of quantification
LOQ
lowest concentration or quantity of the nu
...

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