ISO 20395:2019

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

General Information

• Status: Published
• Technical Committee: COVID 19 - COVID 19 - ISO
• Directive: standardi COVID-19 - UPDATE ON THE AVAILABILITY OF CEN, CENELEC, ISO, IEC STANDARDS AND THEIR NATIONAL ADOPTIONS IN RESPONSE TO THE COVID-19 OUTBREAK

Standards Content (Sample)

Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
Samo za branje. Kopiranje in posredovanje prepovedano. © SIST 2020-04-10
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
Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
Samo za branje. Kopiranje in posredovanje prepovedano. © SIST 2020-04-10
© ISO 2019
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Published in Switzerland
ii © ISO 2019 – All rights reserved

Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
Samo za branje. Kopiranje in posredovanje prepovedano. © SIST 2020-04-10
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
Posebna objava SIST z dovoljenjem CEN, CENELEC, ISO in IEC: Za potrebe zagotavljanja brezplačnega dostopa do vsebin standardov v času epidemije COVID-19.
Samo za branje. Kopiranje in posredovanje prepovedano. © SIST 2020-04-10
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 .
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