1.1)上海海洋大学食品学院,上海 201306;2.2)中国计量科学研究院前沿计量科学中心,北京 100029;3.3)火箭军特色医学中心,北京 100088
国家质量基础的共性技术研究与应用专项课题(2017YFF0204605)和人类生殖遗传资源服务管理云平台开发与安全标准规范体系建设 (2016YFC1000301-3)资助项目.
1.1)College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China;2.2)National Institute of Metrology, Beijing 100029, China;3.3)Center for Advanced Measurement Science, PLA Rocket Force Characteristic Medical Center, Beijing 100088, China
This work was supported by grants from Research and Application of Common Technology of National Quality Foundation(2017YFF0204605) and Development of Human Reproductive Genetic Resources Service Management Cloud Platform and Construction of Safety Standard System(2016YFC1000301-3).
采用实验室建立的数字PCR方法(ddPCR)和荧光定量PCR(qPCR)方法研究HER2基因组DNA标准物质的互通性,评价HER2基因组DNA标准物质与临床样本的互通性,为临床实验室检测提供具有互通性的可溯源标准物质. 将研制的5个水平标准物质随机穿插于29例临床样本间,使用ddPCR方法与qPCR方法同时进行检测. 参考美国临床实验室标准化协会(CLSI)指南文件EP30和中华人民共和国卫生行业标准基质效应与互通性评估指南WS/T356-2011的推荐,采用Deming回归法评价标准物质的互通性. 结果显示,5种标准物质与临床样本之间具有良好的互通性,可以用于临床实验室对HER2基因拷贝数变异检测方法的方法验证和质量控制.
To evaluate the commutability between HER2 genomic DNA reference materials (RM) and clinical samples, ddPCR and qPCR were used to study the commutability of HER2 genomic DNA RM, which can provide traceable RM with commutability for clinical laboratory testing. 29 clinical samples were randomly measured among the 5 levels of RM by real time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). The averaged copy number ratio of HER2 to RPPH1 was calculated. According to the guideline EP30 of American Society for clinical laboratory standardization (CLSI) and the guideline WS/t356-2011 for Guideline for Evaluation of Matrix Effects and Commutability of the People’s Republic of China, a regression curve was drawn with the result of ddPCR as abscissa and the result of qPCR as ordinate, and the commutability of RM was evaluated by Deming regression method. If the test results of the proposed RM fall within the 95% confidence interval of its predicted, it is considered that the analyzed RM is communtable; if it falls outside the range, it is considered that the analyzed RM is not communtable. In addition, the results were compared with the ratio of copy number concentration when CEP17 gene was used as reference gene. HER2/RPPH1 of the five RMs determined by ddPCR and qPCR were: 1.91, 1.86; 5.70, 4.45; 16.94, 12.21; 22.38, 17.19; 35.38, 28.84, respectively, which fall in the prediction range, indicating the five RMs are communtable. Moreover, this was confirmed by the ratio of HER2/CEP17 determined by the two methods. However, the regression coefficient of HER2/RPPH1 (R2 = 0.97) was better than that of HER2/CEP17 (R2 = 0.78) and even one group of clinical sample fell outside the 95% confident interval. We used FISH to detect the cell lines used to prepare HER2 genomic DNA RM. The results showed that in the same nucleus, part of chromosome 17 showed HER2 amplification in the short arm, and part of chromosome 17 showed no HER2 amplification. Additionally, CEP17 was amplified in some cells, which indicates it is not suitable to be used as reference gene as this will cause false negative results. This confirms the necessity of using RPPH1 gene as reference gene when diagnostic of HER2. In conclusion, the five RMs are communtable, which can be used for the method validation and quality control in analyzing of HER2 copy number variation in clinical laboratories.
邢德纯,程波,王霞,高颖,刘争,孙锁柱,董莲华,杨靖亚.HER2基因组DNA标准物质互通性研究[J].生物化学与生物物理进展,2021,48(9):1104-1110
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