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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

田原, 徐玲, 范子豪, 等. 基于微滴数字PCR技术建立HBV共价闭合环状DNA的检测方法[J]. 临床肝胆病杂志, 2021, 37(8): 1806-1810. DOI: 10.3969/j.issn.1001-5256.2021.08.013
引用本文: 田原, 徐玲, 范子豪, 等. 基于微滴数字PCR技术建立HBV共价闭合环状DNA的检测方法[J]. 临床肝胆病杂志, 2021, 37(8): 1806-1810. DOI: 10.3969/j.issn.1001-5256.2021.08.013
TIAN Y, XU L, FAN ZH, et al. Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA [J]. J Clin Hepatol, 2021, 37(8): 1806-1810. DOI: 10.3969/j.issn.1001-5256.2021.08.013
Citation: TIAN Y, XU L, FAN ZH, et al. Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA [J]. J Clin Hepatol, 2021, 37(8): 1806-1810. 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
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

详细信息
    通讯作者:

    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

Funds: 

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
  • 修回日期:  2021-03-19
  • 刊出日期:  2021-08-16
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