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).<\/p>\n
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).<\/p>\n
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].<\/p>\n
This document is not applicable to quantification of very short DNA oligonucleotides (<50 bases).<\/p>\n
This document covers:<\/p>\n
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;<\/p>\n<\/li>\n
quantification of total nucleic acid mass concentration and quality control of a nucleic acid sample including assessment of nucleic acid quality (purity and integrity);<\/p>\n<\/li>\n
PCR assay design, optimization, in silico and in vitro specificity testing;<\/p>\n<\/li>\n
data quality control and analysis including acceptance criteria, threshold setting and normalization;<\/p>\n<\/li>\n
method validation (precision, linearity, limit of quantification, limit of detection, trueness and robustness) with specific requirements for qPCR and dPCR;<\/p>\n<\/li>\n
approaches to establishing metrological traceability and estimating measurement uncertainty.<\/p>\n<\/li>\n<\/ul>\n
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).<\/p>\n
| PDF Pages<\/th>\n | PDF Title<\/th>\n<\/tr>\n | ||||||
|---|---|---|---|---|---|---|---|
| 2<\/td>\n | National foreword <\/td>\n<\/tr>\n | ||||||
| 7<\/td>\n | Foreword <\/td>\n<\/tr>\n | ||||||
| 8<\/td>\n | Introduction <\/td>\n<\/tr>\n | ||||||
| 9<\/td>\n | 1 Scope 2 Normative references <\/td>\n<\/tr>\n | ||||||
| 10<\/td>\n | 3 Terms and definitions <\/td>\n<\/tr>\n | ||||||
| 16<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 17<\/td>\n | 4.2.3 dPCR determination of copy number concentration using molecular counting <\/td>\n<\/tr>\n | ||||||
| 18<\/td>\n | 4.2.4 Relative quantification by qPCR <\/td>\n<\/tr>\n | ||||||
| 19<\/td>\n | 4.2.5 dPCR determination of ratio between two targets 4.3 Normalization strategy <\/td>\n<\/tr>\n | ||||||
| 20<\/td>\n | 4.4 Controls <\/td>\n<\/tr>\n | ||||||
| 21<\/td>\n | 5 Sample QC \u2014 Total nucleic acid quantity, integrity and purity 5.1 General 5.2 Total nucleic acid quantification 5.2.1 General <\/td>\n<\/tr>\n | ||||||
| 22<\/td>\n | 5.2.2 Spectrophotometry 5.2.3 Fluorometry 5.2.4 \u200bAssessment of total DNA concentration using qPCR\/dPCR <\/td>\n<\/tr>\n | ||||||
| 23<\/td>\n | 5.3 Nucleic acid integrity 5.4 Nucleic acid purity <\/td>\n<\/tr>\n | ||||||
| 24<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 25<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 26<\/td>\n | 6.2.3 (RT)-qPCR amplification efficiency 6.2.4 RT efficiency 6.2.5 Specificity <\/td>\n<\/tr>\n | ||||||
| 27<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 28<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 29<\/td>\n | 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) <\/td>\n<\/tr>\n | ||||||
| 30<\/td>\n | 7.5 Identification of outliers 8 Nucleic acid quantification measurement method validation 8.1 General 8.2 Precision <\/td>\n<\/tr>\n | ||||||
| 31<\/td>\n | 8.3 LOQ 8.4 LOD <\/td>\n<\/tr>\n | ||||||
| 32<\/td>\n | 8.5 Linearity 8.6 Trueness 8.7 Robustness <\/td>\n<\/tr>\n | ||||||
| 33<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 34<\/td>\n | 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 <\/td>\n<\/tr>\n | ||||||
| 35<\/td>\n | 10.2 qPCR measurement uncertainty 10.3 Ratio-based measurements 10.4 dPCR measurement uncertainty <\/td>\n<\/tr>\n | ||||||
| 36<\/td>\n | 11 Reporting <\/td>\n<\/tr>\n | ||||||
| 37<\/td>\n | Annex\u00a0A (informative) Spectrophotometry <\/td>\n<\/tr>\n | ||||||
| 39<\/td>\n | Annex\u00a0B (informative) Nucleic acid integrity <\/td>\n<\/tr>\n | ||||||
| 41<\/td>\n | Annex\u00a0C (informative) PCR efficiency <\/td>\n<\/tr>\n | ||||||
| 44<\/td>\n | Annex\u00a0D (informative) Measurement uncertainty <\/td>\n<\/tr>\n | ||||||
| 47<\/td>\n | Annex\u00a0E (informative) MIQE and dMIQE checklists <\/td>\n<\/tr>\n | ||||||
| 53<\/td>\n | Bibliography <\/td>\n<\/tr>\n<\/table>\n","protected":false},"excerpt":{"rendered":" Biotechnology. Requirements for evaluating the performance of quantification methods for nucleic acid target sequences. qPCR and dPCR<\/b><\/p>\n |