ISO 20395:2019

Overview

ISO 20395:2019 - Biotechnology - Requirements for evaluating the performance of quantification methods for nucleic acid target sequences - qPCR and dPCR - defines generic, science-based requirements to evaluate and ensure the performance of methods used to quantify specific nucleic acid targets. It covers both quantitative real-time PCR (qPCR) and digital PCR (dPCR) for DNA and RNA targets (gDNA, plasmid DNA, ssDNA, cDNA, ssRNA, dsRNA) from biological and in vitro sources. The standard focuses on analytical design, assay performance, data quality, validation, metrological traceability and reporting, but does not cover biological sampling or application-specific acceptance criteria.

Key topics and technical requirements

ISO 20395:2019 specifies practical technical elements required for robust nucleic acid quantification, including:

• Quantification strategies: qPCR using calibration curves (copy number via calibration) and dPCR via molecular counting; relative quantification and ratio measurements.
• Sample QC: total nucleic acid mass concentration, purity and integrity assessment (spectrophotometry, fluorometry and PCR-based checks).
• Assay design & optimization: amplicon selection, primer/probe design, in silico specificity testing, RT-qPCR/RT-dPCR considerations, fluorescence and efficiency optimization.
• Controls and normalization: use of no-template controls, internal/external controls and strategies for normalization.
• Data quality control: acceptance criteria, threshold setting, preprocessing, outlier identification, and calibration-curve analysis.
• Method validation: precision, linearity, limit of detection (LOD), limit of quantification (LOQ), trueness, robustness, and validation specifics for qPCR and dPCR.
• Metrological traceability & uncertainty: approaches to traceability, use of reference materials, instrument calibration and estimation of measurement uncertainty.
• Reporting: essential information to include in quantitative method reports; Annex E cross-references MIQE/dMIQE checklists.

Practical applications

ISO 20395:2019 is applicable wherever reliable quantification of specific nucleic acid targets is required, such as:

• Biopharmaceutical process control and quality assurance
• Gene therapy and advanced therapy medicinal product (ATMP) characterization
• Viral load and molecular diagnostics workflows (method development/validation)
• Research and development in molecular biology and synthetic biology
• Environmental and industrial biotechnology assays requiring accurate copy-number or ratio measurements

This standard helps labs produce comparable, traceable, and auditable quantitative results across qPCR and dPCR platforms.

Who should use this standard

• Clinical, research and quality laboratories developing or validating qPCR/dPCR assays
• Method validation and regulatory affairs teams in biotech and pharma
• Instrument and reagent manufacturers designing or qualifying assays and workflows
• Metrology institutes and organizations establishing reference materials and traceability

Related standards and guidance

• MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) and dMIQE checklists (referenced in Annex E) - complementary for reporting and experimental metadata.
• Users should consult ISO 20395:2019 alongside sector-specific standards or regulatory guidance when application-specific acceptance criteria (e.g., clinical or food matrices) are required.