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新型十万目六边形微腔芯片数字PCR对肺癌基因突变的绝对定量分析
Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer
- 第三军医大学学报 2021年43卷第8期 页码：675-682
- 作者机构：
1.400037 重庆，陆军军医大学(第三军医大学)第二附属医院检验医学中心
2.400044 重庆，重庆大学光电技术及系统教育部重点实验室，新型微纳器件与系统技术国防重点学科实验室
- 作者简介：
[蒲晓允，E-mail: puxiaoyong63@sina.com PU Xiaoyun, E-mail: puxiaoyong63@sina.com
- 基金信息：国家自然科学基金面上项目(81772284);重庆市技术创新应用示范项目(CSTC2018jscx-mszdX0073)
DOI：10.16016/j.1000-5404.202101070
中图分类号： R446.61;R730.43;R734.2收稿：2021-01-07，
修回：2021-02-05，
纸质出版：2021-04-30
- 稿件说明：
移动端阅览
冯攀, 郭铭静, 李刚, 等. 新型十万目六边形微腔芯片数字PCR对肺癌基因突变的绝对定量分析[J]. 第三军医大学学报, 2021,43(8):675-682.
Pan FENG, Mingjing GUO, Gang LI, et al. Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer[J]. Journal of Third Military Medical University, 2021, 43(8): 675-682.
冯攀, 郭铭静, 李刚, 等. 新型十万目六边形微腔芯片数字PCR对肺癌基因突变的绝对定量分析[J]. 第三军医大学学报, 2021,43(8):675-682. DOI： 10.16016/j.1000-5404.202101070.
Pan FENG, Mingjing GUO, Gang LI, et al. Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer[J]. Journal of Third Military Medical University, 2021, 43(8): 675-682. DOI： 10.16016/j.1000-5404.202101070.
摘要
目的
2
以表皮生长因子受体(epidermal growth factor receptor，EGFR)G719S突变为对象，建立用于肺癌基因突变绝对定量分析的新型十万目六边形微腔芯片(novel 100 000 hexagonal microchamber chip，NHMC)数字PCR(digital PCR，dPCR)平台。
方法
2
首先构建并优化适用于EGFR G719S突变的PCR检测体系；然后用NHMC-dPCR平台检测8个浓度梯度的EGFR G719S标准品质粒和4种干扰物质，评估该方法的灵敏度、特异性和抗干扰能力；最后分别用NHMC-dPCR、液滴式数字PCR(droplet digital PCR，ddPCR)和DNA测序法检测6例EGFR G719X突变的肺癌组织样本，验证NHMC-dPCR在临床样本检测中的可靠性。
结果
2
NHMC-dPCR对EGFR G719S的最低检测浓度为3.01 copies/μL，检测值与期望值的线性方程为Y=0.725X- 0.581，相关系数
R
2
=0.984。该平台检测4种干扰物未发现阳性拷贝，检测G719S突变及其与4种干扰物混合的结果分别为23 194.4 copies和22 095.7 copies。NHMC-dPCR、ddPCR和DNA测序3种方法检测6例临床样本的定性结果符合率为100%，其中2例由NHMC-dPCR定量的结果为1 822.4 copies和451.7 copies，其对应由ddPCR定量的结果为1 581.8 copies和218.3 copies。
结论
2
本研究建立的核酸绝对定量技术——NHMC-dPCR具有成本低、操作简单、速度快、抗干扰能力强等优点，可用于肺癌基因突变的绝对定量分析。
Abstract
Objective
2
To construct a novel 100 000 hexagonal microchamber chip (NHMC) digital PCR (dPCR) platform for absolute quantification analysis of gene mutations in lung cancer based on epidermal growth factor receptor (EGFR) G719S mutation as an object.
Methods
2
First
a PCR detection system for EGFR G719S mutation was constructed and optimized. And then 8 concentration gradients of EGFR G719S standard plasmids and 4 interfering substances were detected by the NHMC-dPCR platform
and the sensitivity
specificity and anti-interference ability of the platform were evaluated. Finally
6 tissue samples of lung cancer with EGFR G719X mutation were detected by NHMC-dPCR
droplet digital PCR (ddPCR) and DNA sequencing
and the reliability of NHMC-dPCR in clinical sample testing was verified.
Results
2
The minimum detection concentration of EGFR G719
S detected by NHMC-dPCR is 3.01 copies/μL. The linear equation between the detected value and the expected value is Y=0.725X-0.581
and the correlation coefficient is
R
2
=0.984. Four interfering substances were tested by this platform and no positive holes were found. The results of the G719S mutant plasmids and the mixture of 4 interferences were 23 194.4 copies and 22 095.7 copies
respectively. The qualitative results of 6 clinical samples tested by NHMC-dPCR
ddPCR and DNA sequencing had a coincidence rate of 100%. Among them
the quantitative results of 2 cases by NHMC-dPCR were 1 822.4 copies and 451.7 copies
and their corresponding quantitative results by ddPCR were 1 581.8 copies and 218.3 copies
respectively.
Conclusion
2
The nucleic acid absolute quantification technology established in this study
namely NHMC-dPCR
has the advantages of low cost
simple operation
fast speed
and strong anti-interference ability
and can be used for absolute quantification analysis of gene mutations in lung cancer.
关键词
Keywords
references
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