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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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  • [1] SOMANATH B, NATALIE C, THOMAS M, et al. Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units[J]. Analytical Chemistry, 2010, 82(17): 7185-7192. doi: 10.1021/ac100845m
    [2] HOLDEN M J, HAYNES R J, RABB S A, et al. Factors Affecting Quantification of Total DNA by UV Spectroscopy and PicoGreen Fluorescence[J]. J Agric Food Chem, 2009, 57(16): 7221-7226. doi: 10.1021/jf901165h
    [3] WILKINSON D E, BAYLIS S A, PADLEY D, et al. Establishment of the 1st World Health Organization international standards for human papillomavirus type 16 DNA and type 18 DNA[J]. International Journal of Cancer, 2010, 126(12): 2969-2983.
    [4] DUAN Y, CHEN R, Wu X, et al. Preparation of Reference Materials Used for Detecting Nucleic Acids of Nervous Necrosis Virus[J]. China Animal Health Inspection, 2018, 35(2): 102-107.
    [5] 李春, 刘建涛, 高运华, 等. 紫外分光光度法测定核酸含量的影响因素分析[J]. 化学试剂, 2020, 42(1): 53-57.
    [6] XIA C S, FEN Z Y. Application of Molecular Absorption Spectrophotometric Method to the Determination of Biologic Macromolecular Structures[J]. Spectroscopy Spectral Analysis, 2004(10): 1197-1201.
    [7] OKAMOTO T, OKABE S. Ultraviolet absorbance at 260 and 280 nm in RNA measurement is dependent on measurement solution[J]. International Journal of Molecular Medicine, 2000, 5(6): 657-666.
    [8] LI Z X, YANG R, JIN S S, et al. Factor analysis of effect on purity of RNA extracted by TRIzol[J]. Journal of Xinxiang Medical University, 2016, 33(8): 653-656, 661.
    [9] RENGARAJAN K, CRISTOL S M, MEHTA M, et al. Quantifying DNA concentrations using fluorometry: a comparison of fluorophores[J]. Molecular Vision, 2002(8): 416-421.
    [10] SINGER V L, JONES L J, YUE S T, et al. Characterization of PicoGreen Reagent and Development of a Fluorescence-Based Solution Assay for Double-Stranded DNA Quantitation[J]. Anal Biochem, 1997, 249(2): 228-238. doi: 10.1006/abio.1997.2177
    [11] L J J. RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization[J]. Analytical Biochemistry, 1998, 265(2): 368-374. doi: 10.1006/abio.1998.2914
    [12] XIN L, XU G M, GUO J F, et al. A Method for Quantification of Double Strand DNA Using SYBR Green I Dye[J]. China Biotechnology, 2008, 28(1): 55-60.
    [13] HUGGETT J, BUSTIN S A. Standardisation and reporting for nucleic acid quantification[J]. Accreditation & Quality Assurance, 2011, 16(8-9): 399-405.
    [14] BUSTIN, S. Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems[J]. Journal of Molecular Endocrinology, 2002, 29(1): 23-39. doi: 10.1677/jme.0.0290023
    [15] JIANG L L, WANG H L, WANG X L, et al. Establishment of a national grade Ⅱ reference material for HBV DNA[J]. Laboratory Medicine, 31(8): 703-708.
    [16] YAO L, ZHANG Q, LI F L, et al. Construction, Purification and Quantification of Multiplex Armored RNA Containing Targets for Detection of 4 Foodborne Viruses[J]. Food Science, 2019, 40(8): 293-299.
    [17] Pavšič J, Žel J, Milavec M. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification[J]. Analytical and Bioanalytical Chemistry, 2016, 408(1): 107-121. doi: 10.1007/s00216-015-9107-2
    [18] PINHEIRO L B, COLEMEN V A, HINDSON C M, et al. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification[J]. Analytical Chemistry, 2011, 84(2): 1003-1011.
    [19] ZHU Q Y, YANG W X, GAO Y B, et al. Microfluidic Digital Chip for Absolute Quantification of Nucleic Acid Amplification[J]. Chemical Journal of Chinese Universities, 2013, 34(3): 545-350.
    [20] BHAT S, EMSLIE K R. Digital polymerase chain reaction for characterisation of DNA reference materials[J]. Biomolecular Detection and Quantification, 2016(10): 47-49.
    [21] HAYNES R J, KLINE M C, TOMAN B, et al. Standard Reference Material 2366 for Measurement of Human Cytomegalovirus DNA[J]. Journal of Molecular Diagnostics, 2013, 15(2): 177-185. doi: 10.1016/j.jmoldx.2012.09.007
    [22] YUAN L, LI E H, DONG H, et al. Establishment of the national nucleic acid reference material of HP-PRRSV[J]. Animal Husbandry Veterinary Medicine, 2018, 39(5): 826-829.
    [23] NOLAN T, HANDS R E, BUSTIN S A. Quantification of mRNA using real-time RT-PCR[J]. Nat Protoc 2006, 1(3): 1559-1582.
    [24] YAN N C, YI Y J, JING W, et al. Effect of Reverse Transcription Process on Quantitive Detection of Porcine Reproductive and Respiratory Syndrome virus[J]. Chinese Journal of Animal Infectious Diseases, 2020, 28(2): 80-85.
    [25] GRIFFITHS K R, BURKE D G, EMSLIE K R. Quantitative polymerase chain reaction: A framework for improving the quality of results and estimating uncertainty of measurement[J]. Analytical Methods, 2011, 3(10): 2201-2211. doi: 10.1039/c1ay05069a
    [26] HUGGETT J F, NOVAK T, GARSON J A, et al. Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon[J]. BMC Research Notes, 2008, 1(1): 70-70. doi: 10.1186/1756-0500-1-70
    [27] INCHUL Y. A strategy for establishing accurate quantitation standards of oligonucleotides: quantitation of phosphorus of DNA phosphodiester bonds using inductively coupled plasma-optical emission spectroscopy[J]. Analytical Biochemistry, 2004, 335(1): 150-161. doi: 10.1016/j.ab.2004.08.038
    [28] HOLDEN M J, RABB S A, TEWARI Y B, et al. Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of DNA[J]. Analytical Chemistry, 2007, 79(4): 1536-1541. doi: 10.1021/ac061463b
    [29] BRENNAN R G, RABB S A, HOLDEN M J, et al. Potential Primary Measurement Tool for the Quantification of DNA[J]. Analytical Chemistry, 2009, 81(9): 3414-3420. doi: 10.1021/ac802688x
    [30] LECLERC O, FRAISSE P O, LABARRAQUE G, et al. Method development for genomic Legionella pneumophila DNA quantification by inductively coupled plasma mass spectrometry[J]. Anal Biochem, 2013, 435(2): 153-158. doi: 10.1016/j.ab.2012.12.023
    [31] GAO Y H, LI H F, LI J X, et al. Quantitative Analysis of Fluorescent Dye-labeled DNA by High Resolution Inductively Coupled Plasma Mass Spectrometry[J]. Chemical Journal of Chinese Universities, 2010, 31(12): 2360-2365.
    [32] WEN L, YAN L I, LI X U, et al. Quantification of Plasmid DNA Reference Material for Vibrio Cholerae by Using ICP-MS and Digital PCR[J]. Chemical Reagents, 2018, 40(12): 1179-1182.
    [33] KUNG A W, KILBY P M, PORTWOOD D E, et al. Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry[J]. Rapid Commun Mass Spectrom, 2018, 32(7): 590-596. doi: 10.1002/rcm.8074
    [34] DONG L, ZANG C, WANG J, et al. Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry[J]. Analytical Bioanalytical Chemistry, 2012, 402(6): 2079-2088. doi: 10.1007/s00216-011-5644-5
    [35] O'CONNOR G, DAWSON C, WOOLFORD A, et al. Quantitation of Oligonucleotides by Phosphodiesterase Digestion Followed by Isotope Dilution Mass Spectrometry:   Proof of Concept[J]. Anal Chem, 2002, 74(15): 3670-3676. doi: 10.1021/ac0255375
    [36] MENG Z, LIMBACH P A. Quantitation of Ribonucleic Acids Using18O Labeling and Mass Spectrometry[J]. Analytical Chemistry, 2005, 77(6): 1891-1895. doi: 10.1021/ac048801y
    [37] SHIBAYAMA S, FUJII S-I, INAGAKI K, et al. Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification[J]. Journal of Chromatography A, 2016, 1468: 109-115. doi: 10.1016/j.chroma.2016.09.031
    [38] SACHIE, SHIBAYAMA, SHIN I, et al. Development of certified reference material NMIJ CRM 6205-a for the validation of DNA quantification methods: accurate mass concentrations of 600-bp DNA solutions having artificial sequences[J]. Analytical & Bioanalytical Chemistry, 2019, 411(23): 6091-6100.
    [39] Frédéric R, PIROTTE S, PAUW E D, et al. Positive and negative ion mode ESI-MS and MS/MS for studying drug–DNA complexes[J]. International Journal of Mass Spectrometry, 2006, 253(3): 156-171. doi: 10.1016/j.ijms.2005.11.027
    [40] LI H, CHANG Y. The Principle of Flow Cytometer and Clinical Application[J]. China Medical Device, 2011, 17(5): 37-39.
    [41] ZHENG J, YEUNG E S. Counting Single DNA Molecules in a Capillary with Radial Focusing[J]. Australian Journal of Chemistry, 2003, 56(3): 149-153. doi: 10.1071/CH02192
    [42] LIM H-M, YOO H B, HONG N S, et al. Count-based quantitation of trace level macro-DNA molecules[J]. Metrologia, 2009, 46(3): 375-387. doi: 10.1088/0026-1394/46/3/028
    [43] YOO H B, OH D, SONG J Y, et al. A candidate reference method for quantification of low concentrations of plasmid DNA by exhaustive counting of single DNA molecules in a flow stream[J]. Metrologia, 2014, 51(5): 491-502. doi: 10.1088/0026-1394/51/5/491
    [44] YOO H B, LEE C, HONG K S, et al. Quantification of single-strand DNA by sequence-specific counting in capillary flow cytometry[J]. Metrologia, 2020, 57(6): 065019. doi: 10.1088/1681-7575/abb113
    [45] YOO H, PARK S R, DONG L, et al. International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction[J]. Anal Chem, 2016, 88(24): 12169. doi: 10.1021/acs.analchem.6b03076
    [46] HUSSELS M, ENGEL S, BOCK N. Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method[J]. Biomolecular Detection and Quantification, 2019, 17: 2214-7535.
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  • 网络出版日期:  2021-06-08
  • 刊出日期:  2021-07-08

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