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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

引用本文:
Citation:

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
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

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

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:   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。

     

  • 图  1  HBV cccDNA与rcDNA结构示意图

    图  2  ddPCR标准曲线

    图  3  梯度稀释阳性质粒ddPCR微滴荧光分布

    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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  • 收稿日期:  2021-01-19
  • 录用日期:  2021-03-19
  • 出版日期:  2021-08-20
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