一种模拟游离DNA阳性质控品的制备方法及表征

doi:10.3969/j.issn.1000-1158.2023.03.07

摘要
图/表
参考文献(18)
相关文章 (15)

全文: PDF (3512 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要通过优化一种新型的机理未明确的类酶解作用体系(reaction system,RS),直接作用带有KRAS G12C,KRAS G12D,EGFR L858R和EGFR T790M突变的肿瘤细胞系SW1573,SNU-C2B和NCI-H1975,通过对RS体系作用时间及作用的细胞数量的优化,可以裂解细胞基因组DNA制备得到一种可以高度模拟人体血浆游离DNA(cfDNA)阳性质控品。经芯片电泳检测获得的模拟cfDNA片段长度集中于(166±16)BP,与正常人体血浆cfDNA大小一致;通过微滴数字PCR检测显示RS制备的DNA片段和gDNA在目标突变位点处具有一致的突变丰度。表明经RS体系制备得到的DNA片段可以作为一种cfDNA的阳性质控品。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	方艳
	王霞
	董莲华
	杨靖亚

关键词 ：计量学, 游离DNA, 阳性质控品, 模拟cfDNA;突变丰度, 数字PCR

Abstract：By optimizing a new type of enzymatic hydrolysis system with unknown mechanism (reaction system, RS), which directly acts on tumor cell lines SW1573,SNU-C2B and NCI-H1975, with KRAS G12C,KRAS G12D,EGFR L858R and EGFR T790M mutations, by optimizing the action time and cell number of RS system, a kind of positive quality control substance which can highly mimic human plasma free DNA (cfDNA) can be prepared by cleavage of cell genomic DNA. The length of simulated cfDNA fragments detected by chip electrophoresis is (166±16)BP, which is the same as that of normal human plasma cfDNA. Microdrop digital PCR detection show that the cfDNA fragments prepared by RS and gDNA have the same mutation abundance at the target mutation site. The results show that the DNA fragment prepared by RS system can be used as a positive quality control substance of cfDNA.

Key words： metrology cell free DNA positive quality control substance simulated cfDNA mutation abundance ddPCR

收稿日期: 2021-03-17 发布日期: 2023-03-08

PACS:

TB99

基金资助:战略性国际科技创新合作重点专项(2018YFE0201602)

通讯作者: 董莲华(1981-),河北涞水人,中国计量科学研究院副研究员,博士,主要从事生物计量方面的研究工作。Email: donglh@nim.ac.cn 杨靖亚(1976-),陕西宝鸡人,上海海洋大学副教授,主要从事药理及分子生物学研究工作。Email: jyyang@shou.edu.cn E-mail: lianhuadong@126.com

作者简介: 方艳(1995-),江苏泗洪人,上海海洋大学在读硕士研究生,研究方向为核酸计量。Email: 18521708541@163.com

引用本文:

方艳,王霞,董莲华,杨靖亚. 一种模拟游离DNA阳性质控品的制备方法及表征[J]. 计量学报, 2023, 44(3): 361-367.
FANG Yan,WANG Xia,DONG Lian-hua,YANG Jing-ya. Preparation and Characterization of a Simulated Cell Free DNA Positive Quality Control Material. Acta Metrologica Sinica, 2023, 44(3): 361-367.

链接本文:

http://jlxb.china-csm.org:81/Jwk_jlxb/CN/10.3969/j.issn.1000-1158.2023.03.07 或 http://jlxb.china-csm.org:81/Jwk_jlxb/CN/Y2023/V44/I3/361

［13］	董莲华, 隋志伟, 王晶, 等. 数字PCR方法准确测量质粒DNA拷贝浓度［J］. 计量学报, 2017, 38(2):247-251.
［14］	Lee J-S, Kim M, Seong M-W, et al. Plasma vs. serum in circulating tumor DNA measurement: characterization by DNA fragment sizing and digital droplet polymerase chain reaction［J］. Clinical Chemistry and Laboratory Medicine, 2020, 58(4): 527-532.
［18］	Warren J D, Xiong W, Bunker A M, et al. Septin 9 methylated DNA is a sensitive and specific blood test for colorectal cancer［J］. BMC Medicine, 2011, 9(133):1-9.
［6］	Stroun M, Lyautey J, Lederrey C, et al. About the possible origin and mechanism of circulating DNA: apoptosis and active DNA release［J］. Clin Chim Acta,2001, 313:139-142.
［7］	Jahr S, Hentze H, Englisch S, et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells［J］. Cancer Res, 2001, 61:1659-1665.
［4］	Jiang P, Chan K C A, Lo Y M D. Liver-derived cell-free nucleic acids in plasma: Biology and applications in liquid biopsies［J］. J Hepatol, 2019, 71(2): 409-421.
	Dong L H,Wang J, Fu B Q, et al. Comparability of Digital PCR and Next Generation Sequencing on Detection of KRAS Mutations Frequency. Acta Metrologica Sinica, 2018, 39(3): 436-441.
	Dong L H, Sui Z W, Wang J, et al. Accurate Plasmid DNA Copy Concentration Quantification by Digital PCR［J］. Acta Metrologica Sinica, 2017, 38(2): 247-251.
［1］	Leon S A, Shapiro B, Sklaroff D M, et al. Free DNA in the serum of cancer patients and the effect of therapy［J］. Cancer Res, 1977, 37(3)646-650.
［8］	谭晓华, 张亚历, 姜泊, 等. 凋亡细胞中小片段DNA的简单快速提取方法［J］. 第一军医大学学报, 1996, 16(3): 227-228.
［12］	董莲华, 王晶, 傅博强, 等. 数字PCR和下一代测序方法用于KRAS基因突变检测中的可比性研究［J］. 计量学报, 2018, 39(3): 436-441.
［2］	Spindler K G, Boysen A K, Pallisgard N, et al. Cell-Free DNA in Metastatic Colorectal Cancer: A Systematic Review and Meta-Analysis［J］. Oncologist, 2017, 22(9): 1049-1055.
［3］	Mehra N, Dolling D, Sumanasuriya S, et al. Plasma Cell-free DNA Concentration and Outcomes from Taxane Therapy in Metastatic Castration-resistant Prostate Cancer from Two Phase III Trials (FIRSTANA and PROSELICA)［J］. Eur Urol, 2018, 74(3): 283-291.
［5］	Quigley D, Alumkal J J, Wyatt A W, et al. Analysis of Circulating Cell-Free DNA Identifies Multiclonal Heterogeneity of BRCA2 Reversion Mutations Associated with Resistance to PARP Inhibitors［J］. Cancer Discov, 2017, 7(9): 999-1005.
［9］	Jung K, Fleischhacker M, Rabien A. Cell-free DNA in the blood as a solid tumor biomarker—a critical appraisal of the literature［J］. Clin Chim Acta, 2010, 411(21-22): 1611-1624.
［10］	Thierry A R, El Messaoudi S, Gahan P B, et al. Origins, structures, and functions of circulating DNA in oncology［J］. Cancer Metastasis Rev, 2016, 35(3): 347-376.
［11］	Nagata S. DNA degradation in development and programmed cell death［J］. Immunology, 2005, 23:853-875.
［15］	Yang Z, LaRiviere M J, Ko J, et al. A Multianalyte Panel Consisting of Extracellular Vesicle miRNAs and mRNAs, cfDNA, and CA19-9 Shows Utility for Diagnosis and Staging of Pancreatic Ductal Adenocarcinoma［J］. Clinical Cancer Research, 2020, 26(13): 3248-3258.
［16］	Hussung S, Follo M, Klar R F U, et al. Development and Clinical Validation of Discriminatory Multitarget Digital Droplet PCR Assays for the Detection of Hot Spot KRAS and NRAS Mutations in Cell-Free DNA［J］. Journal of Molecular Diagnostics, 2020, 22(7): 943-956.
［17］	Odogwu L, Mathieu L, Goldberg K B, et al. FDA Benefit-Risk Assessment of Osimertinib for the Treatment of Metastatic Non-Small Cell Lung Cancer Harboring Epidermal Growth Factor Receptor T790M Mutation［J］. Oncologist, 2018, 23(3): 353-359.