| 两种抽提乙型肝炎病毒共价闭合环状DNA方法的效果比较 |
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| 引用本文： | 胡孔颖,金宇焘,叶建宇,宰文静,王洋,李雨檬,陈捷亮. 两种抽提乙型肝炎病毒共价闭合环状DNA方法的效果比较[J]. 微生物与感染, 2021, 16(5): 313-321. DOI: 10.3969/j.issn.1673-6184.2021.05.004 |
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| 作者姓名： | 胡孔颖 金宇焘 叶建宇 宰文静 王洋 李雨檬 陈捷亮 |
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| 作者单位： | 复旦大学上海医学院，基础医学院病原生物学系，医学分子病毒学教育部/卫健委重点实验室，上海 200032 |
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| 基金项目： | 国家自然科学基金;上海市青年科技启明星计划 |
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| 摘 要： | 乙型肝炎病毒(hepatitis B virus，HBV)作为一种嗜肝DNA病毒，在感染肝细胞后会在细胞核中形成病毒转录复制的模板和基因储存库--共价闭合环状DNA(covalently closed circular DNA, cccDNA)，其持续存在是乙型肝炎慢性化和难以治愈的核心，也是此研究领域内的重点。从细胞样品中稳定抽提获取cccDNA对于保证cccDNA检测的准确性至关重要。Hirt法是一种抽提真核细胞染色体外DNA的方法，被用于HBV cccDNA的抽提，但存在操作复杂和耗时长等问题。为简化操作，有研究对Hirt法进行改良，结合硅胶膜离心柱来抽提染色体外DNA，但尚不清楚该法用于HBV cccDNA抽提与传统Hirt法的效果差异。本研究基于HBV cccDNA细胞转染系统、HBV复制细胞系及感染系统，以DNA印迹(Southern blot)和定量聚合酶链式反应(quantitative polymerase chain reaction, qPCR)作为检测评价手段，平行比较了传统Hirt-酚/氯仿法与改良Hirt-过柱法抽提HBV cccDNA的效果。结果表明，两种方法具有相当的抽提效率和抽提特异性，而改良Hirt-过柱法耗时更短，提示在进行细胞HBV cccDNA抽提时可选择改良Hirt-过柱法以提高实验效率。
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| 关 键 词： | 乙型肝炎病毒 共价闭合环状DNA 抽提方法 Hirt法 硅胶膜离心柱 |
Comparison of two methods for extracting hepatitis B virus covalently closed circular DNA |
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| HU Kongying,JIN Yutao,YE Jianyu,ZAI Wenjing,WANG Yang,LI Yumeng,CHEN Jieliang. Comparison of two methods for extracting hepatitis B virus covalently closed circular DNA[J]. Journal of Microbes and Infection, 2021, 16(5): 313-321. DOI: 10.3969/j.issn.1673-6184.2021.05.004 |
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| Authors: | HU Kongying JIN Yutao YE Jianyu ZAI Wenjing WANG Yang LI Yumeng CHEN Jieliang |
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| Affiliation: | Department of Medical Microbiology and Parasitology, MOE & NHC Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China |
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| Abstract: | Hepatitis B virus (HBV) is a hepatotropic DNA virus that can deposit a covalently closed circular DNA form (cccDNA) in the nucleus of infected hepatocytes, which serves as a reservoir for HBV genome and the template for transcribing HBV RNAs. The persistence of cccDNA is the key of HBV chronicity and the main obstacle for HBV cure, and thus cccDNA has always been a focus of HBV research. The efficient extraction of cccDNA from cells is required for accurate detection and quantification. Hirt DNA extraction is a method for isolation of the extrachromosomal DNA in eukaryotes and has been applied for cccDNA extraction. However, the procedure of Hirt DNA extraction is laborious and time-consuming. To simplify the procedure, there has been reports of ”modified Hirt DNA extraction” combined with silicon-based resins column for rapid extraction of extrachromosomal DNA. However, the difference of extraction efficacy between the two methods remains unknown. Here, the two methods were parallelly compared by using Southern Blot and qPCR in various cell culture-based HBV transfection, replication and infection systems. The results suggested that “the modified Hirt-Silica Gel Membrane method” has the potential to enable a fast HBV cccDNA extraction with equally extraction efficacy and specificity compared with“the original Hirt-Phenol/Chloroform method”． |
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| Keywords: | Hepatitis B virus,cccDNA,Extraction method,Hirt ,Silica gel membrane, |
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