核酸提取仪校准技术案例分析及探讨

王志栋; 沈海滢; 吴枭; 高运华

doi:10.12338/j.issn.2096-9015.2023.0316

核酸提取仪校准技术案例分析及探讨

doi: 10.12338/j.issn.2096-9015.2023.0316

中国计量科学研究院，北京 100029

基金项目: 国家基础科研条件与重大科学仪器设备重点研发计划（2022YFF0710403）；国家市场监督管理总局技术保障专项项目(2022YJ13)。

详细信息

作者简介:
王志栋（1987-），中国计量科学研究院工程师，研究方向：核酸计量技术及标准研究，邮箱：wangzhd@nim.ac.cn

通讯作者:
高运华（1972-），中国计量科学研究院研究员，研究方向：核酸计量技术及标准研究，邮箱： gaoyh@nim.ac.cn

中图分类号: TB99
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出版历程
- 收稿日期: 2023-11-27
- 录用日期: 2023-11-29
- 修回日期: 2023-12-01
- 网络出版日期: 2023-12-08
- 刊出日期: 2023-12-18

Case Analysis and Discussion on Nucleic Acid Extractor Calibration Techniques

National Institute of Metrology, Beijing 100029, China

摘要

摘要: 针对核酸提取仪校准过程中存在的问题，依据JJF 1874-2020 《(自动)核酸提取仪校准规范》，解析了核酸提取仪的关键计量特性和量传溯源体系。以常见核酸提取仪为例，开展了核酸提取仪温度、振动频率和核酸提取回收率等四类计量特性的计量校准方法和不确定度评定案例分析，温度示值误差0.5 ℃(55 ℃点)和0.7 ℃(70 ℃点)，温度均匀性0.3 ℃(55 ℃点)和0.5 ℃(70 ℃点)，温度稳定性±0.1 ℃，振动频率稳定性±0.1 Hz，核酸提取回收率82.9%，回收率一致性6.3%，回收率重复性4.5%。评定了温度示值误差和核酸提取回收率的不确定度。相比JJF 1874-2020《(自动)核酸提取仪校准规范》中核酸提取回收率的不确定度评定示例，增加了微量分光光度计测量不确定度、移液器和电子天平引入的不确定度分量。总结了核酸提取仪温度测量装置、振动频率测量装置的类型和优缺点，分析了核酸回收率的影响因素，以及核酸回收率测量过程使用恒一提取方法和提取试剂的重要性。对校准过程中的问题和难点，提出了针对性的解决方案和优化方法，为核酸提取仪校准工作的开展提供方法参考。探讨了核酸提取仪交叉污染率的概念和检测方法，实验测得交叉污染率为0.0%。
- 计量学 /
- 核酸提取仪 /
- 校准技术 /
- 交叉污染率 /
- 核酸回收率校准
Abstract: Addressing the challenges in the calibration process of nucleic acid extractors, this study explores the key metrological characteristics and measurement traceability system based on the JJF 1874-2020 standard, "Calibration Specification for (Automatic) Nucleic Acid Extractors." Using common nucleic acid extractors as examples, this paper analyzes the calibration methods and uncertainty assessments for four metrological characteristics: temperature, vibration frequency, and nucleic acid extraction efficiency. The results include temperature indication errors of 0.5 ℃ at 55 ℃ and 0.7 ℃ at 70 ℃, temperature uniformity of 0.3 ℃ at 55 ℃ and 0.5 ℃ at 70 ℃, temperature stability of ±0.1 ℃, vibration frequency stability of ±0.1 Hz, nucleic acid extraction efficiency of 82.9%, efficiency homogeneity of 6.3%, and repeatability of 4.5%. The study evaluates the uncertainty of temperature indication errors and nucleic acid extraction efficiency. Compared to the JJF 1874-2020 standard, additional uncertainty components from the micro spectrophotometer, pipettor, and electronic balance are included. The study summarizes the types and pros and cons of temperature and vibration frequency measuring devices, analyzes factors affecting nucleic acid recovery rate, and highlights the importance of using consistent extraction methods and reagents. It proposes solutions and optimization methods for calibration challenges and discusses the concept and detection methods of cross-contamination rates in nucleic acid extractors, with an experimental finding of 0.0% cross-contamination rate.
- metrology /
- nucleic acid extractor /
- calibration techniques /
- cross-contamination rate /
- nucleic acid extraction efficiency calibration

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图 1 核酸提取仪量传溯源层级示意图

Figure 1. Schematic diagram of the traceability system for nucleic acid extractors

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图 2 旋转式光电振动频率测量装置示意图

Figure 2. Schematic diagram of the rotating photoelectric vibration frequency measurement device

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图 3 交叉污染率样本排布示意图

Figure 3. Schematic diagram of sample arrangement for testing cross-contamination rates

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表 1 被检核酸提取仪

Table 1. The nucleic acid extractor under test

仪器名称	仪器型号	生产厂家
核酸提取仪	711	Thermo Fisher

下载: 导出CSV

表 2 标准器和标准物质

Table 2. Standard instruments and reference materials

仪器名称	型号	生产厂家
温度巡检仪	TQC96L	波普新创
振动频率测量装置	PVF01	中国计量科学研究院
电子天平	XP56	MettlerToledo
移液器	(100～1000)μL	Eppendorfr
微量分光光度计	NanoDrop 2000	Thermo Fisher
小牛胸腺DNA含量标准物质	GBW09801	中国计量科学研究院

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表 3 核酸提取仪的温度示值误差和均匀性数据

Table 3. Temperature indication error and uniformity data for nucleic acid extractors /°C

设定温度	测量温度							平均温度	示值误差	均匀性
55.0	54.36	54.54	54.51	54.59	54.47	54.38	54.62	54.5	0.5	0.3
70.0	69.44	69.38	69.15	69.54	69.37	69.02	69.25	69.3	0.7	0.5

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表 4 核酸提取仪的温度稳定性数据

Table 4. Temperature stability data for nucleic acid extractors /°C

测量平均值					稳定性
69.30	69.04	69.28	69.15	69.18	±0.1
69.28	69.27	69.17	69.13	69.07	±0.1

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表 5 振动频率稳定性数据

Table 5. Stability data for vibration frequency /Hz

测量值					稳定性
6.1	6.1	6.2	6.1	6.2	±0.1
6.2	6.3	6.1	6.2	6.2	±0.1

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表 6 核酸提取回收率一致性数据

Table 6. Homogeneity data for nucleic acid extraction efficiency

项目	测量数据
洗脱液体积V_i(μL)	95.5	94.8	97.8	96.2	93.7	95.3	95.7
洗脱液核酸浓度C_i(ng/μL)	25.1	24.8	22.8	24.0	24.8	24.3	25.3
回收的核酸质量m_i(ng)	2397.1	2351.0	2210.3	2308.8	2323.8	2315.8	2421.2
核酸提取回收率一致性∆C_u(%)	6.3

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表 7 核酸提取回收率重复性数据

Table 7. Repeatability data for nucleic acid extraction efficiency %

项目	测量数据
核酸回收率R_i	82.9	78.3	84.5
回收率的重复性RSD	4.5

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表 8 各样本Ct值数据

Table 8. Ct value data for each sample

样本编号	Ct值
B2	17.39	17.02	17.11
A2	No Ct	No Ct	No Ct
C2	No Ct	No Ct	No Ct
E2	No Ct	No Ct	No Ct
G2	No Ct	No Ct	No Ct
A8	No Ct	No Ct	No Ct
C8	No Ct	No Ct	No Ct
E8	No Ct	No Ct	No Ct
G8	No Ct	No Ct	No Ct

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