本文件提供了评估性能和确保特定核酸序列（目标）量化方法质量的一般要求。 本文件适用于使用数字（dPCR）或定量实时PCR（qPCR）扩增技术对DNA（脱氧核糖核酸）和RNA（核糖核酸）靶序列进行定量。它适用于核酸分子中存在的靶序列，包括双链DNA（dsDNA），如基因组DNA（gDNA）和质粒DNA，单链DNA（ssDNA），互补DNA（cDNA），以及单链RNA（ssRNA），包括核糖体RNA（rRNA），信使RNA（mRNA），以及长和短非编码RNA[微RNA（miRNA）和短干扰RNA（siRNA）]，以及双链RNA- 单链RNA（dsRNA）。 本文件适用于源于生物来源的核酸，如病毒、原核细胞和真核细胞、无细胞生物液体（如血浆或细胞培养基）或体外来源[如寡核苷酸、合成基因结构和体外转录（IVT）RNA]。 本文件不适用于非常短的DNA寡核苷酸（<；50碱基）的定量。 本文件包括: -分析设计，包括量化策略（如qPCR中使用校准曲线进行核酸拷贝数量化，或如dPCR中使用分子计数进行核酸拷贝数量化，相对于独立样本和比率测量进行量化）和对照的使用； -核酸总质量浓度的量化和核酸样品的质量控制，包括核酸质量（纯度和完整性）的评估； -PCR检测设计、优化、电子和体外特异性检测； -数据质量控制和分析，包括验收标准、阈值设置和规范化； -方法验证（精密度、线性、定量限、检测限、真实性和鲁棒性）以及qPCR和dPCR的具体要求； -建立计量溯源性和估计测量不确定度的方法。 本文件未提供生物材料取样或生物样品处理（即收集、保存、运输、储存、处理和核酸提取）的要求或验收标准。它也没有提供特定应用的要求和验收标准（例如:。 g、 可能出现特定基质问题的食品或临床应用）。

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).