Case Analysis and Discussion on Nucleic Acid Extractor Calibration Techniques
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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%。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.
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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 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 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 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 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 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Vi(μL) 95.5 94.8 97.8 96.2 93.7 95.3 95.7 洗脱液核酸浓度Ci(ng/μL) 25.1 24.8 22.8 24.0 24.8 24.3 25.3 回收的核酸质量mi(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∆Cu(%) 6.3 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Ri 82.9 78.3 84.5 回收率的重复性RSD 4.5 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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