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

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

中国计量科学研究院前沿中心，北京 100029

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

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出版历程
- 网络出版日期: 2022-04-11

Measurement Comparison of Plasmid DNA Capability by Digital PCR

National Institute of Metrology, Beijing 100029, China

摘要

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。
- 数字PCR /
- 转基因 /
- 质粒DNA /
- 核酸 /
- 拷贝数比值 /
- 拷贝数浓度
Abstract: Digital PCR (dPCR) is an absolute quantitative technique of nucleic acid, which is widely used in transgenic and gene detection. In order to ensure the accuracy and reliability of digital PCR measurement results in China, 16 laboratories were tested using transgenic plasmid DNA samples. The comparison results showed that the difference between the ratio of foreign gene to internal copy number (<25%) to evaluate the measuring ability of the laboratories. The 16 laboratories were all within the acceptable range, and there was no significant difference among different digital PCR platforms. Due to the influence of plasmid conformation, the absolute copy number content of plasmid DNA measured in 7 laboratories was significantly lower than the reference value. It is suggested that when using digital PCR for quantitative measurement of plasmid DNA copy number, it should be confirmed whether the conformation of plasmid has an effect on amplification.
- digital PCR /
- transgene /
- plasmid DNA /
- nucleic acid /
- copy number ratio /
- copy number concentration

HTML全文

图 1 转基因样品均匀性结果

Figure 1. Homogeneity results of Sample

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图 2 不同平台测量结果分布

Figure 2. Distribution of measurement results on different platforms

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图 3 9家实验室的Z’比分数

Figure 3. Z 'scores for nine laboratories

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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	NK603	ZSSIIB	NK603/ZSSIIB比值
F_0.05(6，14)	2.85	2.85	2.85
F	1.55	1.42	2.27
$ {s}_{\mathrm{b}\mathrm{b}} $	140	107	0.019
$ {s}_{\mathrm{r}} $	328	285	0.030
${u_{{\text{bb}}}}$	101	86	0.009
结论	F <F_0.05(6，14)，均匀	F <F_0.05(6，14)，均匀	F <F_0.05(6，14)，均匀

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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
0	10158	9053	1.12
0.1	10201	9207	1.11
6	9667	8657	1.05
$ \left\| {{b_1}} \right\| $	86.1	79.1	0.011
$ s $	36	115	0.009
$ s\left(b1\right)*{t}_{\mathrm{0.95,1}} $	94.2	300.8	0.024
结论	稳定	稳定	稳定

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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	8159	7945	1.00
标准偏差	2178	2088	/
u_a	7.8×10²	7.4×10²	/
$u{\text{r(}}v{\text{)}}$	1.8×10²	1.7×10²	/
$u{\text{r(}}v{\text{p)}}$	65	64	/
u_定值	8.0×10²	7.6×10²	0.020
u_bb	81	77	0.0082
u_stab	36	1.3×10²	0.0105
u_c	8.0×10²	7.8×10²	0.024
U（k=2）	1.6×10³	1.6×10³	0.048
$u{\text{r(}}v{\text{)}}$：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。

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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
15	QX200	5.00
14	QX200	5.00
2	BioDigital青	0.83
12	Quantstudio 3D	1.00
11	QX200	0.50
16	QX200	2.00
7	Drop Maker M1、Chip Reader R1	2.12
13	QX200	0.75
9	QX200	1.67
1	QX200	0.14
6	Accu ONE	4.00
3	QX200	2.00
4	QX200	2.00
5	SG-2000、Dscanner4-1000	0.00
10	QX200	0.00
8	MicroDrop -100	0.00

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