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BS ISO 20395:2019 Biotechnology. Requirements for evaluating the performance of quantification methods for nucleic acid target sequences. qPCR and dPCR >
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BS ISO 20395:2019 Biotechnology. Requirements for evaluating the performance of quantification methods for nucleic acid target sequences. qPCR and dPCR, 2019
- National foreword
- Foreword
- Introduction
- 1 Scope
- 2 Normative references
- 3 Terms and definitions
- 4 Design of measurement procedure
- 4.1 General
- 4.2 Quantification method
- 4.2.1 General
- 4.2.2 qPCR determination of nucleic acid concentrations using a calibration curve
- 4.2.3 dPCR determination of copy number concentration using molecular counting
- 4.2.4 Relative quantification by qPCR
- 4.2.5 dPCR determination of ratio between two targets
- 4.3 Normalization strategy
- 4.4 Controls
- 5 Sample QC — Total nucleic acid quantity, integrity and purity
- 5.1 General
- 5.2 Total nucleic acid quantification
- 5.2.1 General
- 5.2.2 Spectrophotometry
- 5.2.3 Fluorometry
- 5.2.4 Assessment of total DNA concentration using qPCR/dPCR
- 5.3 Nucleic acid integrity
- 5.4 Nucleic acid purity
- 6 Assay design and optimization for quantification of nucleic acid target sequences
- 6.1 Assay design
- 6.1.1 General
- 6.1.2 Amplicon selection
- 6.1.3 Primer and probe design
- 6.1.4 In silico evaluation of specificity
- 6.1.5 RT-qPCR/RT-dPCR design
- 6.2 Assay optimization using purified samples
- 6.2.1 General
- 6.2.2 Optimization of fluorescence signal
- 6.2.3 (RT)-qPCR amplification efficiency
- 6.2.4 RT efficiency
- 6.2.5 Specificity
- 6.3 Method optimization using test samples
- 6.3.1 Effect of PCR inhibitors in sample matrix
- 6.3.2 Presence of nucleic acid contaminants in test sample
- 6.3.3 Validated measurement range
- 6.4 No template controls
- 7 Data quality control (QC) and analysis
- 7.1 General
- 7.2 Acceptance criteria
- 7.2.1 qPCR
- 7.2.2 dPCR
- 7.3 Threshold setting
- 7.3.1 qPCR
- 7.3.2 dPCR
- 7.4 Data pre-processing
- 7.4.1 qPCR using calibration curve
- 7.4.2 Relative quantification (qPCR)
- 7.5 Identification of outliers
- 8 Nucleic acid quantification measurement method validation
- 8.1 General
- 8.2 Precision
- 8.3 LOQ
- 8.4 LOD
- 8.5 Linearity
- 8.6 Trueness
- 8.7 Robustness
- 8.8 Specific considerations for qPCR method validation
- 8.8.1 Repeatability of qPCR- or RT-qPCR
- 8.8.2 Intermediate precision and reproducibility of qPCR- or RT-qPCR
- 8.9 Specific considerations for dPCR method validation
- 9 Nucleic acid quantification measurement traceability and comparability
- 9.1 Metrological traceability
- 9.2 Use of reference materials
- 9.3 Instrument calibration
- 10 Measurement uncertainty (MU) in qPCR and dPCR measurements
- 10.1 General requirements for MU calculations
- 10.2 qPCR measurement uncertainty
- 10.3 Ratio-based measurements
- 10.4 dPCR measurement uncertainty
- 11 Reporting
- Annex A (informative) Spectrophotometry
- Annex B (informative) Nucleic acid integrity
- Annex C (informative) PCR efficiency
- Annex D (informative) Measurement uncertainty
- Annex E (informative) MIQE and dMIQE checklists
- Bibliography [Go to Page]