ISO DIS 20395-1
Biotechnology - Requirements for evaluating the performance of quantification methods for nucleic acid target sequences - Part 1: qPCR and dPCR
|Publication Date:||14 September 2018|
|ICS Code (Biology. Botany. Zoology):||07.080|
This document provides generic guidelines for evaluating the performance and ensuring the quality of methods used for the quantification of specific nucleic acid sequences (targets). This will serve to ensure confidence in the data generated and demonstrate that a selected method is fit for its intended purpose.
The document is applicable to the quantification of DNA and RNA 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).
The 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 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, optimisation, in silico and in vitro specificity testing;
- Data quality control and analysis including acceptance criteria, threshold setting and normalisation;
- 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 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). Several sector/application-s