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A Perspective on Nucleic Acid Reference Materials
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摘要: 核酸是遗传信息的载体，随着以核酸扩增为基础的体外诊断技术的快速发展，核酸检测被广泛应用于医学诊断、法医鉴定、进出口检验检疫以及物种进化研究等领域。核酸标准物质是保证核酸检测结果准确性和溯源性的“金标准”，WHO和我国国家市场监督管理总局均规定，体外诊断试剂需使用国际、国家标准物质或厂级标准品对其准确性进行溯源。HER2、BRAF、EGFR等基因突变致癌机制的解析促使曲妥珠单抗、威罗菲尼、美罗华等一系列靶向治疗药物上市，导致我国核酸标准物质面临很大缺口。由于核酸具有结构复杂、易降解和分子量大等特点，难以满足均匀性和稳定性要求，因此核酸标准物质的研制进展缓慢。本文对核酸标准物质的制备方法及国内外研究进展进行简要综述，为高效研制体外诊断用核酸标准物质提供参考。Abstract: Nucleic acid is the carrier of genetic information. With the rapid development of in vitro diagnostic technology based on nucleic acid amplification, nucleic acid detection is widely used in medical diagnosis, forensic identification, import and export inspection and quarantine, species evolution research, etc. Nucleic acid reference materials are a gold standard to ensure the accuracy and traceability of nucleic acid test results. Both the WHO and the Chinese Food and Drug Administration stipulate that in vitro diagnostic reagents need to use international and national reference materials or factory-level standards to guarantee the traceability and accuracy. The analysis of the carcinogenic mechanism of HER2, BRAF, EGFR and other gene mutations has prompted the listing of a series of targeted therapies such as trastuzumab, veilofenib, and rituximab, which has caused a large gap in Chinese nucleic acid reference materials. Due to the complex structure, easy degradation and large molecular weight of nucleic acid, it is difficult to meet the requirements of uniformity and stability. Therefore, development of nucleic acid reference materials is slow. This article briefly reviews preparation methods of nucleic acid reference materials and domestic and abroad research progress, for the efficient development of nucleic acid reference materials for in vitro diagnostics.
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Key words:
- in vitro diagnosis /
- nucleic acid assay /
- nucleic acid reference materials /
- DNA /
- RNA
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图 3 BRAF基因V600E突变基因组DNA标准物质的制备流程
Figure 3. The procedure for developing BRAF V600E nucleic acid reference materials
图 4 国家有证核酸标准物质的组成
Figure 4. The constitution of certified nucleicacid reference materials
表 1 在售有证核酸标准物质
Table 1. The certified nucleic acid reference materials on sale
标物名称 编号 标准值 定值方法 猪繁殖与呼吸综合征病毒美洲经典株
（PRRSV CH-1a）质粒核酸标准物质GBW(E)091038 （9.4±0.9）×108 copy/μL 数字PCR 猪繁殖与呼吸综合征病毒美洲变异株
（PRRSV HuN4）质粒核酸标准物质GBW(E)091039 （7.8±1.0）×108 copy/μL 数字PCR 新型冠状病毒核酸标准物质（高浓度） GBW(E)091089 E基因 （7.05±0.54）×105 copy/μL 数字PCR ORF1ab （1.10±0.12）×106 copy/μL N基因 （8.38±0.76）×105 copy/μL 新型冠状病毒核酸标准物质（低浓度） GBW(E)091090 E基因 （7.63±0.66）×102 copy/μL 数字PCR ORF1ab （1.24±0.19）×103 copy/μL N基因 （9.8±1.2）×102 copy/μL 新型冠状病毒核糖核酸基因组标准物质 GBW(E)091098 E基因 （1.29±0.26）×102 copy/μL 数字PCR ORF1ab （9.5±1.8）×103 copy/μL N基因 （2.05±0.31）×102 copy/μL 新型冠状病毒核糖核酸基因组标准物质 GBW(E)091099 E基因 （1.06±0.11）×103 copy/μL 数字PCR ORF1ab （8.96±0.61）×102 copy/μL N基因 （1.73±0.13）×103 copy/μL KRAS（G12A）基因突变丰度标准物质(突变丰度67.3%) GBW09841 G12A突变丰度（67.3±2.0）% 数字PCR KRAS（G12D）基因突变丰度标准物质(突变丰度47.7%) GBW09842 G12D突变丰度（47.7±3.0）% 数字PCR KRAS（G12R）基因突变丰度标准物质(突变丰度51.8%) GBW09843 G12R突变丰度（51.8±3.0）% 数字PCR KRAS（G12C）基因突变丰度标准物质(突变丰度100%) GBW09844 G12C突变丰度（100±2.0）% 数字PCR KRAS（G12S）基因突变丰度标准物质(突变丰度100%) GBW09845 G12S突变丰度（100±2.0）% 数字PCR KRAS（G12V）基因突变丰度标准物质(突变丰度100%) GBW09846 G12V突变丰度（100±2.0）% 数字PCR KRAS（G13D）基因突变丰度标准物质(突变丰度50.4%) GBW09847 G13D突变丰度（50.4±3.0）% 数字PCR KRAS基因7种突变丰度标准物质（1%水平） GBW09848 G12A （1.0±10）% 数字PCR G12D （1.0±10）% G12R （1.0±11）% G12C （1.0±9）% G12S （1.0±9）% G12V （1.0±9）% G13D （1.0±8）% KRAS基因7种突变丰度标准物质（5%水平） GBW09849 G12A （5.0±7）% 数字PCR G12D （5.0±7）% G12R （5.0±7）% G12C （5.1±8）% G12S （4.9±10）% G12V （5.0±8）% G13D （5.0±7）% 转基因玉米NK603质粒分子标准物质[19] GBW10086 测序比值 1.0±0.04 测序法
数字PCRPCR比值 0.97±0.09 分子浓度 （2.40±0.14）×108 copy/μL 转基因水稻BT63质粒分子标准物质[20] GBW10090 测序比值 1.0±0.07 测序法
数字PCRPCR比值 1.0±0.08 分子浓度 8.9×105copy/μL 转基因大豆MON89788质粒分子标准物质[21] GBW10092 测序比值 1.00±0.04 测序法
数字PCRPCR比值 1.01±0.09 分子浓度 4.02×105copy/μL 320bp寡聚DNA含量标准物质 GBW09804 （110.6±7.1）mg/g ICP-MS 转基因BT63水稻种子粉基体标准物质[22] GBW10070 （0.50±0.04）×10−2 mg/g
（0.51±0.07）×10−8 copy/μL重量法
数字PCR转基因BT63水稻种子粉基体标准物质[22] GBW10071 （1.01±0.07）×10−2 mg/g
（1.00±0.13）×10−8 copy/μL重量法
数字PCR转基因BT63水稻种子粉基体标准物质[22] GBW10072 （2.01±0.11）×10−2 mg/g
（1.91±0.25）×10−8 copy/μL重量法
数字PCR转基因BT63水稻种子粉基体标准物质[22] GBW10073 （5.03±0.35）×10−2 mg/g
（4.31±0.58）×10−8 copy/μL重量法
数字PCR
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