生物技术.核酸目标序列量化方法性能评估要求.qPCR和dPCR

[中文适用范围]： 本文件提供了评估特定核酸序列（目标）定量方法的性能和确保其质量的通用要求。 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)，例如基因组 DNA (gDNA) 和质粒 DNA、单链 DNA (ssDNA)、互补 DNA (cDNA) 和单链 RNA (ssRNA)，包括核糖体 RNA (rRNA)、信使 RNA (mRNA) 和长短非编码 RNA [microRNA (miRNA) 和短干扰 RNA (siRNA)] 以及双链 RNA (dsRNA)。 本文件适用于源自生物来源的核酸，例如病毒、原核和真核细胞、无细胞生物液体（例如血浆或细胞培养基）或体外来源[例如寡核苷酸、合成基因构建体和体外转录（IVT）RNA ]。 本文件不适用于非常短的 DNA 寡核苷酸的定量（ [外文原描述]： 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).