基于微滴数字PCR技术建立HBV共价闭合环状DNA的检测方法

田原; 徐玲; 范子豪; 曹亚玲; 张向颖; 陈煜; 段钟平; 任锋

doi:10.3969/j.issn.1001-5256.2021.08.013

基于微滴数字PCR技术建立HBV共价闭合环状DNA的检测方法

DOI: 10.3969/j.issn.1001-5256.2021.08.013

田原¹,
徐玲¹,
范子豪¹,
曹亚玲¹,
张向颖¹,
陈煜²,
段钟平²,
任锋^1, ,

1.
首都医科大学附属北京佑安医院，北京市肝病研究所，北京 100069
2.
首都医科大学附属北京佑安医院肝病中心四科，肝衰竭与人工肝治疗研究北京市重点实验室，北京 100069

基金项目:

国家自然科学基金 (81770611);

国家自然科学基金 (82002243);

北京自然科学基金和北京市教委联合资助重点项目 (KZ202010025035);

首都卫生发展科研专项重点攻关项目 (2020-1-1151);

北京市科技计划“首都临床诊疗技术研究及示范应用”专项课题 (Z191100006619096);

北京市科技计划“首都临床诊疗技术研究及示范应用”专项课题 (Z191100006619097);

科技部传染病重大专项项目 (2018ZX10301407-005-002);

科技部传染病重大专项项目 (2018ZX10302205-004-004);

北京市优秀人才培养项目 (2018000021469G289);

北京市医院管理中心“青苗”计划专项 (QML20201702)

利益冲突说明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。

作者贡献声明：田原、徐玲负责资料分析，论文撰写；范子豪、曹亚玲、张向颖负责收集和分析数据；陈煜、段钟平、任锋负责拟定写作思路，指导撰写并最后定稿。

详细信息

通信作者:
任锋，renfeng7512@ccmu.edu.com

中图分类号: R512.62
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出版历程
- 收稿日期: 2021-01-19
- 录用日期: 2021-03-19
- 出版日期: 2021-08-20

Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA

1.
Beijing YouAn Hospital, Capital Medical University, Beijing Institute of Hepatology, Beijing 100069, China
2.
Fourth Department of Liver Disease, Beijing YouAn Hospital, Capital Medical University, Beijing Municipal Key Laboratory of Liver Failure and Artificial Liver Treatment Research, Beijing 100069, China

Research funding:

National Natural Science Foundation of China (81770611);

National Natural Science Foundation of China (82002243);

Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035);

Key Public Relations Project of Capital Health Development Scientific Research Project (2020-1-1151);

Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619096);

Demonstrating Application and Research of Clinical Diagnosis and Treatment Technology in Beijing (Z191100006619097);

National Science and Technology Key Project on Infectious Diseases (2018ZX10301407-005-002);

National Science and Technology Key Project on Infectious Diseases (2018ZX10302205-004-004);

Beijing Talents Foundation (2018000021469G289);

Beijing Hospitals Authority Youth Programme (QML20201702)

摘要

摘要: 目的建立一种用于检测HBV共价闭合环状DNA(cccDNA)的微滴数字PCR(ddPCR)方法。方法构建HBV cccDNA标准品，利用HBV cccDNA和松弛环状DNA(rcDNA)在结构上存在的差异，设计HBV cccDNA引物和探针，通过扩增HBV质粒得到HBV cccDNA标准品，把梯度稀释后的标准品作为HBV cccDNA检测的模板，建立ddPCR检测方法，并分析此方法的检出限和重复性；收集2017年6月—2020年10月在首都医科大学附属北京佑安医院就诊的20例临床患者的肝组织样本，均诊断为HBV感染，提取样本的DNA，利用质粒安全性ATP依赖的DNA酶(PSAD)进行酶切，得到HBV cccDNA模板，对ddPCR检测方法进行临床样本的评价，并与实时荧光定量PCR(qPCR)检测方法作对比。计数资料两组间比较采用χ²检验。结果建立了基于ddPCR的HBV cccDNA检测方法，梯度稀释的HBV cccDNA标准品均能准确检出，检出限为1拷贝/μl，其中1×10³、1×10²、1×10¹拷贝/μl标准品的变异系数分别为4.41%、3.98%、5.09%；检测20例临床HBV患者样本的HBV cccDNA，ddPCR检测方法能检出17例，阳性率为85%，qPCR检测方法能检出11例，阳性率为55%，两组比较差异有统计学意义(χ²=4.286，P=0.038)。结论建立的ddPCR检测HBV cccDNA方法具有较低的检出限和较好的重复性，为进一步的临床检测提供了有效的工具。
- 乙型肝炎病毒 /
- DNA, 环状 /
- 微滴数字PCR
Abstract: Objective To establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). Methods HBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. Results The HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×10³, 1×10², and 1×10¹ copies/μl standard substances were 4.41%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ²=4.286, P=0.038). Conclusion The established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.
- Hepatitis B Virus /
- DNA, Circular /
- Droplet Digital PCR

HTML全文

图 1 HBV cccDNA与rcDNA结构示意图

下载: 全尺寸图片幻灯片

图 2 ddPCR标准曲线

下载: 全尺寸图片幻灯片

图 3 梯度稀释阳性质粒ddPCR微滴荧光分布

下载: 全尺寸图片幻灯片

表 1 梯度稀释阳性质粒ddPCR检测结果

样本名称(拷贝/μl)	检测结果(拷贝/μl)
阳性对照质粒1×10⁴	2.03×10⁴
阳性对照质粒1×10³	1.06×10³
阳性对照质粒1×10²	2.66×10²
阳性对照质粒1×10¹	1.47×10¹
阳性对照质粒1×10⁰	2.4

下载: 导出CSV

表 2 重复性试验结果分析

质粒浓度 (拷贝/μl)	第1次重复 (拷贝/μl)	第2次重复 (拷贝/μl)	第3次重复 (拷贝/μl)	变异系数 (%)
1×10³	1.53×10³	1.67×10³	1.62×10³	4.41
1×10²	1.45×10²	1.57×10²	1.52×10²	3.98
1×10¹	1.25×10¹	1.38×10¹	1.29×10¹	5.09

下载: 导出CSV

表 3 荧光定量PCR和ddPCR检测结果比较

样本编号	荧光定量PCR检测 (拷贝/μl)	ddPCR检测 (拷贝/μl)
样本1	3.09×10⁵	3.32×10⁵
样本2	Undetermined	7.09×10
样本3	9.34×10²	4.28×10²
样本4	Undetermined	5.22×10
样本5	1.03×10⁵	2.19×10⁵
样本6	Undetermined	Undetermined
样本7	Undetermined	1.3×10
样本8	6.8×10⁴	5.9×10⁴
样本9	Undetermined	Undetermined
样本10	2.1×10⁴	10.6×10⁴
样本11	5.73×10²	1.9×10²
样本12	Undetermined	5.6
样本13	Undetermined	4.3×10²
样本14	3.8×10⁴	16.2×10⁴
样本15	9.5×10⁴	6.3×10³
样本16	Undetermined	Undetermined
样本17	1.17×10³	2.1×10³
样本18	Undetermined	3.8×10
样本19	4.16×10⁴	7.9×10⁴
样本20	2.36×10⁴	4.7×10³

下载: 导出CSV

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