Author:
Date:
2021-04-20
[Abstract] Hepatitis B virus (HBV) is the major cause of liver diseases and liver cancer worldwide. After infecting hepatocytes, the virus establishes a stable episome (covalently closed circular DNA, or cccDNA) that serves as the template for all viral transcripts. Specific and accurate quantification of cccDNA is difficult because infected cells contain abundant replicative intermediates of HBV DNA that share overlapping sequences but arranged in slightly different forms. HBV cccDNA can be detected by Southern blot or qPCR methods which involve enzymatic digestion. These assays are laborious, have limited sensitivity, or require degradation of cellular DNA (which precludes simple normalization). The method described in this protocol, cccDNA inversion quantitative (cinq)PCR, instead uses a series ...
[摘要] [摘要]乙型肝炎病毒(HBV)是全球范围内肝脏疾病和肝癌的主要原因。感染肝细胞后,病毒建立起稳定的附加体(共价闭合的环状DNA或cccDNA),作为所有病毒转录本的模板。cccDNA的特异性和准确定量非常困难,因为被感染的细胞含有丰富的HBV DNA复制中间体,这些中间体共享重叠序列,但排列形式略有不同。HBV cccDNA可以通过涉及酶消化的Southern印迹或qPCR方法进行检测。这些测定费力,灵敏度有限或需要细胞DNA降解(无法进行简单的标准化)。该协议中描述的方法cccDNA反向定量(cinq)PCR,而是使用一系列限制性酶介导的水解和连接反应,将cccDNA转化为反向线性扩增子,该扩增子无法从其他形式的HBV DNA扩增或检测到。重要的是,细胞DNA在样品制备过程中仍可定量,从而可以进行标准化并显着提高精确度。另外,第二线性片段(源自酶消化HBV DNA基因组的单独区域,并以所有形式的HBV DNA存在)可用于同时定量总HBV水平。
图形摘要:
HBV的cccDNA和总HBV DNA的选择性检测使用cinqPCR (转载自涂等人,2020一)。
[背景]乙型肝炎病毒(HBV)是一种小的有包膜病毒,其encapsidates一个部分双链环状DNA基因组,所谓松弛环状(RC)的DNA。感染人肝细胞后,核衣壳被转运至细胞核,其中rcDNA基因组被转化为共价闭合的环状(ccc)DNA。这种游离形式是高度稳定的,并保持慢性HBV感染(Tu等人,2020b ...
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