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χ^(2)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1×10^(3)、1×10^(2)、1×10^(1)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(χ^(2)=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。
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(χ^(2)=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.
作者
田原
徐玲
范子豪
曹亚玲
张向颖
陈煜
段钟平
任锋
TIAN Yuan;XU Ling;FAN Zihao;CAO Yaling;ZHANG Xiangying;CHEN Yu;DUAN Zhongping;REN Feng(Beijing YouAn Hospital,Capital Medical University,Beijing Institute of Hepatology,Beijing 100069,China;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)
出处
《临床肝胆病杂志》
CAS
北大核心
2021年第8期1806-1810,共5页
Journal of Clinical Hepatology
基金
国家自然科学基金(81770611,82002243)
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(首发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,Z191100006619097)
科技部传染病重大专项项目(2018ZX10301407-005-002,2018ZX10302205-004-004)
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)。
