National foreword

This British Standard is the UK implementation of ISO 20395:2019.

The UK participation in its preparation was entrusted to Technical Committee BTI/1, Biotechnologies.

A list of organizations represented on this committee can be obtained on request to its secretary.

This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.

Compliance with a British Standard cannot confer immunity from legal obligations.

This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2019.

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).

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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.

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.

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

3 Terms and definitions

4 Design of measurement procedure

4.1 General

4.2 Quantification method

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

5.3 Nucleic acid integrity

5.4 Nucleic acid purity

6 Assay design and optimization for quantification of nucleic acid target sequences

6.1 Assay design

6.2 Assay optimization using purified samples

6.3 Method optimization using test samples

6.4 No template controls

7 Data quality control (QC) and analysis

7.1 General

7.2 Acceptance criteria

7.3 Threshold setting

7.4 Data pre-processing

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

8.9 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

Bibliography

BS ISO 20395:2019

Biotechnology. Requirements for evaluating the performance of quantification methods for nucleic acid target sequences. qPCR and dPCR

Current, Under Review

Published:

31 Aug 2019