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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

陈桂芳,欧阳艳艳,杨佳怡,等. 核酸标准物质测量方法研究进展[J]. 计量科学与技术,2021, 65(6): 25-33 doi: 10.12338/j.issn.2096-9015.2020.9022
引用本文: 陈桂芳,欧阳艳艳,杨佳怡,等. 核酸标准物质测量方法研究进展[J]. 计量科学与技术,2021, 65(6): 25-33 doi: 10.12338/j.issn.2096-9015.2020.9022
CHEN Guifang, OUYANG Yanyan, YANG Jiayi, GAO Yunhua. Research Progress on Common Measurement Methods of Nucleic Acid Reference Materials[J]. Metrology Science and Technology, 2021, 65(6): 25-33. doi: 10.12338/j.issn.2096-9015.2020.9022
Citation: CHEN Guifang, OUYANG Yanyan, YANG Jiayi, GAO Yunhua. Research Progress on Common Measurement Methods of Nucleic Acid Reference Materials[J]. Metrology Science and Technology, 2021, 65(6): 25-33. 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
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

Research Progress on Common Measurement Methods of Nucleic Acid Reference Materials

  • 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。
  • 图  1  紫外分光光度法测量原理

    Figure  1.  Measuring principle of ultraviolet spectrophotometry

    图  2  荧光染料法测量原理

    Figure  2.  Measuring principle of the fluorescent dye method

    图  3  实时荧光定量PCR定量原理

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

    图  4  微滴式数字PCR定量原理

    Figure  4.  Quantification principle of micro-drop digital PCR

    图  5  基于磷元素测定定量核酸

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

    图  6  质谱技术核酸定量原理

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

    图  7  流式细胞术定量原理

    Figure  7.  Quantification principle of flow cytometry

    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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  • 网络出版日期:  2021-06-08
  • 刊出日期:  2021-07-08

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