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通过数字PCR测量的乙型肝炎病毒cccDNA合成细胞周期蛋白依赖

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Ann Hepatol. 2019 Dec 28. pii: S1665-2681(19)32295-1. doi: 10.1016/j.aohep.2019.12.005. [Epub ahead of print]
Requirement of cyclin-dependent kinase function for hepatitis B virus cccDNA synthesis as measured by digital PCR.
Bao CY1, Hung HC1, Chen YW2, Fan CY2, Huang CJ3, Huang W4.
Author information

1
    Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
2
    Cold Spring Biotech Corp, New Taipei City, Taiwan.
3
    Department of Internal Medicine, Taipei City Hospital, Taipei, Taiwan.
4
    Department of Medical Laboratory Science and Biotechnology, College of Medicine, National Cheng Kung University, Tainan, Taiwan; Institute of Basic Medicine, National Cheng Kung University, Tainan, Taiwan; Department of Pathology, National Cheng Kung University Hospital, Tainan, Taiwan. Electronic address: [email protected].

Abstract
INTRODUCTION AND OBJECTIVES:

HBV covalently closed circular (ccc) DNA is the key player in viral persistence and an important predictive biomarker for hepatitis relapse. Precise quantification of intracellular cccDNA is challenging because cccDNA is present in very low levels in hepatocytes, where it also co-exists with a large excess amount of relaxed circular (rc) DNA. We aimed to develop a highly sensitive cccDNA detection method for cccDNA quantification by digital PCR (dPCR).
PATIENTS OR MATERIALS AND METHODS:

A standard plasmid containing the whole HBV genome in the closed circular conformation was employed to characterize the performance of dPCR. rcDNA in the growth medium of HBV-producing HepAD38 cells was used as a matrix for cccDNA detection. Intrahepatic cccDNA measurement by dPCR and qPCR was performed to determine the correlation of the analysis results for the two methods.
RESULTS:

The limit of detection (LOD) of the cccDNA dPCR was 1.05copy/μl, and the linear range of detection was 1.02×104copies/μl, achieving a dynamic detection range of 104-fold. cccDNA measurement using excess rcDNA as the matrix did not reveal false-positive detection, indicating that dPCR was highly specific. In the HepAD38 cells, the cccDNA levels measured by dPCR were highly correlated with those measured by qPCR but had a higher sensitivity. The CDK inhibitor AZD-5438 was found to block intracellular cccDNA synthesis.
CONCLUSIONS:

Dpcr greatly improved the sensitivity and specificity of cccDNA detection. Host CDK activities are likely required for cccDNA synthesis. dPCR can potentially be applied for drug screening for effective cccDNA inhibitors.

Copyright © 2019. Published by Elsevier España, S.L.U.
KEYWORDS:

Cyclin-dependent kinase; Digital PCR; Hepatitis B virus; Protein kinase C; cccDNA

PMID:
    31964596
DOI:
    10.1016/j.aohep.2019.12.005

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才高八斗

发表于 2020-1-23 17:34 |显示全部帖子
安·赫帕托尔。 2019年12月28日.pii:S1665-2681(19)32295-1。 Doi:10.1016 / j.aohep.2019.12.005。 [Epub提前发行]
通过数字PCR测量的乙型肝炎病毒cccDNA合成细胞周期蛋白依赖性激酶功能的要求。
包CY1,洪HC1,陈宇W2,范CY2,黄CJ3,黄W4。
作者信息

1个
国立成功大学医学院医学实验室科学与生物技术系,台湾台南。
2
台湾新北市冷泉生物科技股份有限公司。
3
台湾台北市台北市立医院内科。
4
国立成功大学医学院医学实验室科学与生物技术系,台湾台南;国立成功大学基础医学研究所,台湾台南;台湾台南国立成功大学医院病理科。电子地址:[email protected]

抽象
简介和目标:

HBV共价闭合环状(ccc)DNA是病毒持久性的关键因素,也是肝炎复发的重要预测生物标志物。精确定量细胞内cccDNA具有挑战性,因为cccDNA在肝细胞中的含量非常低,在肝细胞中cccDNA也与大量过量的松弛环状(rc)DNA共存。我们旨在开发一种高度敏感的cccDNA检测方法,用于通过数字PCR(dPCR)定量cccDNA。
患者或材料与方法:

使用以封闭的环状构象包含整个HBV基因组的标准质粒来表征dPCR的性能。产生HBV的HepAD38细胞生长培养基中的RcDNA被用作cccDNA检测的基质。通过dPCR和qPCR进行肝内cccDNA测量,以确定两种方法的分析结果之间的相关性。
结果:

cccDNA dPCR的检出限(LOD)为1.05复制/μl,线性检测范围为1.02×104拷贝/μl,动态检测范围为104倍。使用过量的rcDNA作为基质的CccDNA测量未显示假阳性检测结果,表明dPCR具有高度特异性。在HepAD38细胞中,通过dPCR测量的cccDNA水平与通过qPCR测量的cccDNA水平高度相关,但灵敏度更高。发现CDK抑制剂AZD-5438阻断细胞内cccDNA的合成。
结论:

Dpcr大大提高了cccDNA检测的灵敏度和特异性。 cccDNA合成可能需要宿主CDK活动。 DPCR可潜在地用于筛选有效cccDNA抑制剂的药物。

版权所有©2019.ElsevierEspaña,S.L.U.
关键字:

细胞周期蛋白依赖性激酶;数字PCR;乙型肝炎病毒;蛋白激酶C;基因

PMID:
31964596
DOI:
10.1016 / j.aohep.2019.12.005

Rank: 8Rank: 8

现金
62111 元 
精华
26 
帖子
30441 
注册时间
2009-10-5 
最后登录
2022-12-28 

才高八斗

发表于 2020-1-23 17:35 |显示全部帖子
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