HBV cccDNA检测方法的研究进展  

Quantatitative assays for covalently closed circular DNA of hepatitis B virus

作　　者：施恬树 曹佳莉 杨艺楠 袁权[1] Tianshu Shi;Jiali Cao;Yinan Yang;Quan Yuan(School of Public Health,State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics,Xiamen University,Xiamen 361100,China;School of Life Sciences,National Institute of Diagnostics and Vaccine Development in Infectious Diseases,Xiamen University,Xiamen 361100,China)

机构地区：[1]厦门大学公共卫生学院,分子疫苗学和分子诊断学国家重点实验室,厦门361100 [2]厦门大学生命科学学院,国家传染病诊断试剂与疫苗工程技术研究中心,厦门361100

出　　处：《科学通报》2020年第16期1529-1545,共17页Chinese Science Bulletin

摘　　要：目前全球约有2.57亿慢性乙型肝炎病毒(hepatitis B virus,HBV)感染者,是当前最为突出的公共卫生问题之一.HBV感染的肝细胞内稳定存在的共价闭合环状DNA(cccDNA)是造成HBV感染持续、抗病毒治疗停药后病毒反弹的重要原因.现有Anti-HBV药物难以有效清除或直接抑制肝内cccDNA是慢性乙肝难以治愈的关键瓶颈.缺乏简单易行的cccDNA定量检测方法,影响了靶向cccDNA的Anti-HBV新药研发进展,也阻碍了这一指标在慢性乙肝患者临床管理实践中的应用.Southern blot被认为是目前cccDNA实验室检测的"金标准",但其操作复杂且灵敏度低,不适合于高通量药物筛选及临床应用.基于PCR的cccDNA检测方法相对操作简单、检测灵敏度更高,但其可靠性仍有待考验.本文系统整理分析了cccDNA样品提取和测定方法的研究现状与进展,希望通过综合比较不同cccDNA检测技术的性能和特点,为cccDNA相关基础研究、药物发展与临床研究提供方法学参考.Chronic infection of hepatitis B virus(HBV)is still one of the public health concerns affecting human health.Although the prophylactic HBV vaccines was introduced over three decades ago,the total number of chronic hepatitis B(CHB)infection still accounts for 3.5%(257 million)of the world’s population,with approximately 800000 annual deaths related to the viral infection.Currently,drugs including PEG-IFN and NAs are widely used in the treatment of HBV infection with certain degree of success.It is still difficult to achieve a high proportion of"clinical cure"with existing treatment methods.The presence of covalently closed circular DNA(cccDNA)in the infected hepatocytes is the underlying mechanism for the long-term HBV persistence and is also essential for off-therapy viral relapse.The current treatments have no or limited effects on the suppression of intrahepatic cccDNA.The availability for a reliable cccDNA quantification assay would facilitate the developmentof anti-viral drugs targeting cccDNA-targeting.In this review,we summarized the advantages and disadvantages of the existing methods for cccDNA quantitation.Specifically,the extraction method of Hirt DNA combined with Exonuclease V can effectively remove other forms of HBV DNA,and suitable for cccDNA extraction,but it needs to be further optimized.Southern blot is considered as the"gold-standard"method for cccDNA detection,but it is less sensitive and requires cumbersome procedures,making it an unlikely method for high-throughput screening used for drug development or for clinical applications.The PCR-based methods are more convenient and sensitive for cccDNA detection.However,the reliability of PCR-based method requires further optimization and validation.Different PCR-based methods were developed for quantitation of HBV cccDNA.They include selective qPCR,droplet-digital PCR,rolling circle amplification PCR,in situ PCR,invader assay,magnetic capture hybridization qPCR,etc.These PCR-based methods are widely used in clinical and laboratory research for qu

关 键 词：乙型肝炎病毒 共价闭合环状DNA 抗乙肝药物 cccDNA检测方法 

分 类 号：R512.62[医药卫生—内科学]