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数字PCR测量转基因质粒DNA比对

王霞 牛春艳 刘争 陆琳 董莲华

王霞,牛春艳,刘争,等. 数字PCR测量转基因质粒DNA比对[J]. 计量科学与技术,待出版 doi: 10.12338/j.issn.2096-9015.2021.0590
引用本文: 王霞,牛春艳,刘争,等. 数字PCR测量转基因质粒DNA比对[J]. 计量科学与技术,待出版 doi: 10.12338/j.issn.2096-9015.2021.0590
WANG Xia, NIU Chunyan, LIU Zheng, LU Lin, DONG Lianhua. Measurement Comparison of Plasmid DNA Capability by Digital PCR[J]. Metrology Science and Technology. doi: 10.12338/j.issn.2096-9015.2021.0590
Citation: WANG Xia, NIU Chunyan, LIU Zheng, LU Lin, DONG Lianhua. Measurement Comparison of Plasmid DNA Capability by Digital PCR[J]. Metrology Science and Technology. doi: 10.12338/j.issn.2096-9015.2021.0590

数字PCR测量转基因质粒DNA比对

doi: 10.12338/j.issn.2096-9015.2021.0590
基金项目: 国家质量基础共性关键技术重点研发专项 (2017YFF0204605)。
详细信息
    作者简介:

    王霞(1994-),中国计量科学研究院科研助理,研究方向:核酸计量,邮箱:wangxia@nim.ac.cn;通讯作者:

    通讯作者:

    董莲华(1981-),中国计量科学研究院研究员,研究方向:核酸计量,邮箱:donglh@nim.ac.cn

Measurement Comparison of Plasmid DNA Capability by Digital PCR

  • 摘要: 数字PCR(digital PCR, dPCR)技术是一种核酸绝对定量技术,被广泛应用于转基因检测和基因检测。为保障国内数字PCR测量结果的准确和可靠,采用转基因质粒DNA样品,考查了16家参比实验室的数字PCR测量能力。比对结果表明:以转基因质粒DNA的外源基因与内拷贝数比值的差值(<25%)来评估参比实验室的测量能力,16家参比实验室均在可接受的范围内,且不同数字PCR平台没有显著差异。受质粒构象影响,7个实验室对质粒DNA的绝对拷贝数含量测量结果显著低于参考值。建议在使用数字PCR进行质粒DNA拷贝数定量测量时,应先确认质粒构象是否对扩增有影响。
  • 图  1  转基因样品均匀性结果

    Figure  1.  Homogeneity results of Sample

    图  2  不同平台测量结果分布

    Figure  2.  Distribution of measurement results on different platforms

    图  3  9家实验室的Z’比分数

    Figure  3.  Z 'scores for nine laboratories

    表  1  样品均匀性检验结果及不确定度

    Table  1.   Homogeneity results of sample and uncertainty

    参数NK603ZSSIIBNK603/ZSSIIB比值
    F0.05(6,14)2.852.852.85
    F1.551.422.27
    $ {s}_{\mathrm{b}\mathrm{b}} $1401070.019
    $ {s}_{\mathrm{r}} $3282850.030
    ${u_{{\text{bb}}}}$101860.009
    结论F <F0.05(6,14),均匀F <F0.05(6,14),均匀F <F0.05(6,14),均匀
    下载: 导出CSV

    表  2  样品稳定性检验结果

    Table  2.   Stability results of sample

    时间(月)ZSSIIB(copy/μL)NK603(copy/μL)NK603/ZSSIIB比值
    01015890531.12
    0.11020192071.11
    6966786571.05
    $ \left| {{b_1}} \right| $86.179.10.011
    $ s $361150.009
    $ s\left(b1\right)*{t}_{\mathrm{0.95,1}} $94.2300.80.024
    结论稳定稳定稳定
    下载: 导出CSV

    表  3  比对样品参考值及不确定度

    Table  3.   Reference value of sample

    参数NK603测量结果(copies/μL)ZSSIIB测量结果(copies/μL)NK603/ZSSIIB比值
    参考值815979451.00
    标准偏差21782088/
    ua7.8×1027.4×102/
    $u{\text{r(}}v{\text{)}}$1.8×1021.7×102/
    $u{\text{r(}}v{\text{p)}}$6564/
    u定值8.0×1027.6×1020.020
    ubb81770.0082
    ustab 361.3×1020.0105
    uc8.0×1027.8×1020.024
    Uk=2)1.6×1031.6×1030.048
    $u{\text{r(}}v{\text{)}}$:移液器体积引入不确定度;$u{\text{r(}}v{\text{p)}}$:微滴体积引入不确定度。
    下载: 导出CSV

    表  4  比对结果的统计分析

    Table  4.   Statistical analysis of results

    实验室编号使用平台编号NK603/ZSSIIB比值
    15QX2005.00
    14QX2005.00
    2BioDigital青0.83
    12Quantstudio 3D1.00
    11QX2000.50
    16QX2002.00
    7Drop Maker M1、Chip Reader R12.12
    13QX2000.75
    9QX2001.67
    1QX2000.14
    6Accu ONE4.00
    3QX2002.00
    4QX2002.00
    5SG-2000、Dscanner4-10000.00
    10QX2000.00
    8MicroDrop -1000.00
    下载: 导出CSV
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  • 网络出版日期:  2022-04-11

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