Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer

Pan FENG; Mingjing GUO; Gang LI; Tengbao XIE; Liqun ZHANG; Fei LIU; Xiaoyun PU

doi:10.16016/j.1000-5404.202101070

您当前的位置：

首页 >

文章列表页 >

Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer

Basic Medicine | 更新时间：2026-01-09

- Novel 100 000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer
- Journal of Third Military Medical University Vol. 43, Issue 8, Pages: 675-682(2021)
- 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：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81772284);the Technical Innovation and Application Demonstration Program of Chongqing(CSTC2018jscx-mszdX0073)
- DOI：10.16016/j.1000-5404.202101070
  CLC： R446.61;R730.43;R734.2
- Received：07 January 2021，
  
  Revised：2021-02-05，
  
  Published：30 April 2021
- 稿件说明：
移动端阅览
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：

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

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

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

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

=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

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

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

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

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

=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

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

SIEGEL R L, MILLER K D, FUCHS H E, et al . Cancer statistics, 2021[J ] . CA Cancer J Clin, 2021, 71(1): 7-33. DOI: 10.3322/caac.21654.

IMYANITOV E N, IYEVLEVA A G, LEVCHENKO E V. Molecular testing and targeted therapy for non-small cell lung cancer: Current status and perspectives[J]. Crit Rev Oncol Hemat, 2021, 157: 103194. DOI: 10.1016/j.critrevonc.2020.103194.

SIGGILLINO A, ULIVI P, PASINI L, et al . Detection of EGFR mutations in plasma cell-free tumor DNA of TKI-treated advanced-NSCLC patients by three methodologies: scorpion-ARMS, PNAClamp, and digital PCR[J ] . Diagnostics, 2020, 10(12): 1062. DOI: 10.3390/diagnostics10121062.

SHIN S, KIM J, KIM Y, et al . Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients[J ] . Clin Chem Lab Med, 2017, 55(12): 1962-1969. DOI: 10.1515/cclm-2016-0851.

JOY R A, THELAKKATTUSSERRY S K, VIKKATH N, et al . Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing[J ] . BMC cancer, 2020, 20(1): 902. DOI: 10.1186/s12885-020-07411-1.

SUGIMORI M, SUGIMORI K, TSUCHIYA H, et al . Quantitative monitoring of circulating tumor DNA in patients with advanced pancreatic cancer undergoing chemotherapy[J ] . Cancer Sci, 2020, 111(1): 266-278. DOI: 10.1111/cas.14245.

CUI X, WU L, WU Y, et al . Fast and robust sample self-digitization for digital PCR[J ] . Anal Chim Acta, 2020, 1107: 127-134. DOI: 10.1016/j.aca.2020.02.010.

OSOEGAWA A, HASHIMOTO T, TAKUMI Y, et al . Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation[J ] . Invest New Drug, 2018, 36(6): 999-1005. DOI: 10.1007/s10637-018-0592-y.

WOOD-BOUWENS C M, HASLEM D, MOULTON B, et al . Therapeutic monitoring of circulating DNA mutations in metastatic cancer with personalized digital PCR[J ] . J Mol Diagn, 2020, 22(2): 247-261. DOI: 10.1016/j.jmoldx.2019.10.008.

LIU X M, NOLL L, SHI X R, et al . Single-cell-based digital PCR detection and association of shiga toxin-producing Escherichia coli serogroups and major virulence genes[J ] . J Clin Microbiol, 2020, 58(3): e01684-19. DOI: 10.1128/jcm.01684-19..

POMARI E, PIUBELLI C, PERANDIN F, et al . Digital PCR: a new technology for diagnosis of parasitic infections[J ] . Clin Microbiol Infec, 2019, 25(12): 1510-1516. DOI: 10.1016/j.cmi.2019.06. 009.

STALEY Z R, BOYD R J, SHUM P, et al . Microbial source tracking using quantitative and digital PCR to identify sources of fecal contamination in stormwater, river water, and beach water in a great lakes area of concern[J ] . Appl Environ Microb, 2018, 84(20): e01634-18. DOI: 10.1128/aem.01634-18.

AHRBERG C D, CHOI J W, LEE J M, et al . Plasmonic heating-based portable digital PCR system[J ] . Lab Chip, 2020, 20(19): 3560-3568. DOI: 10.1039/d0lc00788a.

NING Y, CUI X, YANG C, et al . A self-digitization chip integrated with hydration layer for low-cost and robust digital PCR[J ] . Anal Chim Acta, 2019, 1055: 65-73. DOI: 10.1016/j.aca.2018.12.029.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Genome-wide screening of tumor suppressor genes in oncogenic transformation of normal lung epithelial cells and preliminary validation in sample databases

Differences in CT findings between solid solitary mucinous lung invasive adenocarcinoma and non-mucinous lung invasive adenocarcinoma

Analysis of gene mutations and clinical phenotypes in children with retinoblastoma

Effect of TET1 gene expression on migration and invasion in lung cancer cells

Role of leptin up-regulated GRP78 expression in migration of lung cancer cells

Related Author

Binyu ZHAO

Junlei ZHANG

Jiaqi WANG

Yan RUAN

Yue ZHANG

Lianlian LIU

Rui JIAN

Jiaxiang XIONG

Related Institution

Department of Ophthalmology, First Affiliated Hospital, Army Medical University (Third Military Medical University)

Teaching Experiment Center, College of Basic Medical Sciences, Army Medical University (Third Military Medical University)

Department of Histology and Embryology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University)

Department of Physiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University)

Department of Radiology, the First Affiliated Hospital of Chongqing Medical University

⁰