核酸标准物质测量方法研究进展

陈桂芳; 欧阳艳艳; 杨佳怡; 高运华

doi:10.12338/j.issn.2096-9015.2020.9022

核酸标准物质测量方法研究进展

doi: 10.12338/j.issn.2096-9015.2020.9022

1.
沈阳化工大学，沈阳 110142
2.
中国计量科学研究院，北京 100029

基金项目: 中国计量科学研究院基本科研业务费项目（AKYZZ2023）

详细信息

作者简介:
陈桂芳（1998-），沈阳化工大学硕士研究生，研究方向：生物计量、材料与工程等，邮箱：chenosmanthus@outlook.com

通讯作者:
杨佳怡（1989-），中国计量科学研究院副研究员，研究方向：生物核酸计量，邮箱：yangjy2018@nim.ac.cn

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出版历程
- 网络出版日期: 2021-06-08

Research Progress on Common Measurement Methods of Nucleic Acid Reference Materials

1.
Shenyang Chemical Industry University, Shenyang 110142, China
2.
National Institute of Metrology, Beijing 100029, China

摘要

摘要: 为提高核酸测量的准确性，种类繁多的核酸标准物质被研制出来。标准物质是保证量值准确性与可溯源性的“计量器具”，具有复现、保存和传递量值的功能，可以为核酸定性与定量检测过程的质量控制提供参考。准确可靠的定值方法是标准物质研制的重要基础，详细介绍了几种核酸标准物质测量方法，重点分析了不同测量方法的原理与应用特点，讨论了测量过程中可能存在的影响因素，为核酸标准物质的深入研究提供参考。
- 脱氧核糖核酸 /
- 核糖核酸 /
- 标准物质 /
- 测量方法
Abstract: A great variety of nucleic acid reference materials have been developed to improve the accuracy of nucleic acid measurement. A reference material is a “measuring instrument” that can ensure the accuracy and traceability of measurement, with the functions of reproducing, preserving, and disseminating measurement values. Reference materials can provide a frame of reference for quality control of qualitative and quantitative detection processes for nucleic acid. Accurate and reliable certification methods are an important basis for the development of reference materials. This article provides details on several methods of measuring nucleic acid reference materials, with a focus on the principles and application characteristics of the methods and a discussion on influencing factors that may affect the measurement.
- DNA /
- RNA /
- reference material /
- measurement methods

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图 1 紫外分光光度法测量原理

Figure 1. Measuring principle of ultraviolet spectrophotometry

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图 2 荧光染料法测量原理

Figure 2. Measuring principle of the fluorescent dye method

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图 3 实时荧光定量PCR定量原理

Figure 3. Quantification principle of real-time fluorescence quantitative PCR

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图 4 微滴式数字PCR定量原理

Figure 4. Quantification principle of micro-drop digital PCR

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图 5 基于磷元素测定定量核酸

Figure 5. Quantification of nucleic acid based on the phosphorus element

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图 6 质谱技术核酸定量原理

Figure 6. Quantification principle of nucleic acid by using mass spectrometry

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图 7 流式细胞术定量原理

Figure 7. Quantification principle of flow cytometry

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表 1 核酸标准物质定量技术

Table 1. Quantification techniques for nucleic acid reference materials

定量方法	定量原理	优势	不足	定量范围
紫外分光光度法	利用核酸在260 nm的紫外吸收对标准物质进行定值	快速简单；可用于检验样品纯度	影响因素多(pH缓冲液等) 重复性差	（5～50）μg/mL
荧光染料	已知浓度的标准物质作为外标，通过绘制标准曲线得到未知样品的量值	快速简单；检测仪器方便	需工作标准物质且染料对温度敏感，需避光孵育检测	（0.05～1）μg/mL
实时荧光定量PCR	已知浓度的标准物质作为外标，通过绘制标准曲线得到未知样品的量值	定量范围广；序列特异性	需工作标准物质	（10～10⁹）copies/μL
数字PCR	直接计数目标分子数，不依靠任何校准物或外标	序列特异性；低浓度定量；不依赖标准物质	操作过程较复杂	（10～10⁵）copies/μL
质谱-磷元素定量	定量磷含量，进而定量核酸	高精度定量；量值可溯源国际单位制基本单位	纯度要求极高，操作条件要求高	>5 ng/mL
同位素稀释质谱-核苷酸定量	同位素标记得不同质荷比(m/z)，检测核苷酸等小分子，进而计算核酸含量	可以不依赖标准物质；量值可溯源国际单位制基本单位	前处理复杂且受水解效率影响，纯度要求高	（20～100） ng/mL
流式细胞术	以计数方法定量荧光标记的核酸	适合低浓度定量；不依赖标准物质	检测仪器要求高，需高分辨的成像仪器	～10³ copies/μL

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