CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection.
CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection.
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DOI：
10.1007/s12072-022-10311-0
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发表时间：
2022-04
影响因子：
6.6
通讯作者：
Ren, Feng
中科院分区：
文献类型：
--
作者：
Zhang, Xiangying;Tian, Yuan;Xu, Ling;Fan, Zihao;Cao, Yaling;Ma, Yingmin;Li, Hao;Ren, Feng
关键词：
肝内共价闭合环状DNA（cccDNA）核内池的形成是持续性B型肝炎病毒（HBV）感染的主要原因。在这里，我们建立了基于CRISPR-Cas 13 a技术的高度敏感和特异性的方法来检测cccDNA。我们使用质粒安全的ATP依赖性DNA酶（PSAD）和HindIII消化松散环状rcDNA和双链线性DNA，通过滚环扩增（RCA）和PCR扩增特异性HBV cccDNA片段，并使用CRISPR-Cas 13 a技术检测目的基因。使用HBV相关肝组织、血浆、全血和外周血单核细胞（PBMC）进一步临床验证了用于检测cccDNA的基于CRISPR-Cas 13 a的测定。基于样品预处理步骤、扩增步骤和检测步骤，我们建立了一种新的基于CRISPR-Cas 13 a的检测cccDNA的方法。经RCA和PCR扩增后，CRISPR/Cas 13辅助荧光读出可检测到1拷贝/μl HBV cccDNA。我们采用ddPCR、qPCR、RCA-qPCR、PCR-CRISPR和RCA-PCR-CRISPR方法分别检测了40例HBV相关患者肝组织标本中的20、4、18、14和29例阳性样本。上述5种方法在20份HBV患者血液样本（包括血浆、全血和PBMC）中几乎完全未检出HBV cccDNA。我们开发了一种新型的基于CRISPR的检测方法，用于高度敏感和特异性地检测HBV cccDNA，为准确检测HBV感染、抗病毒治疗评估和治疗指导提供了一种有前途的替代方案。在线版本包含补充材料，可通过10.1007/s12072-022-10311-0获得。
The formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver is the main cause of persistent hepatitis B virus (HBV) infection. Here, we established highly sensitive and specific methods to detect cccDNA based on CRISPR-Cas13a technology. We used plasmid-safe ATP-dependent DNase (PSAD) enzymes and HindIII to digest loose circle rcDNA and double-stranded linear DNA, amplify specific HBV cccDNA fragments by rolling circle amplification (RCA) and PCR, and detect the target gene using CRISPR-Cas13a technology. The CRISPR-Cas13a-based assay for the detection of cccDNA was further clinically validated using HBV-related liver tissues, plasma, whole blood and peripheral blood mononuclear cells (PBMCs). Based on the sample pretreatment step, the amplification step and the detection step, we established a new CRISPR-Cas13a-based assay for the detection of cccDNA. After the amplification of RCA and PCR, 1 copy/μl HBV cccDNA could be detected by CRISPR/Cas13-assisted fluorescence readout. We used ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR methods to detect 20, 4, 18, 14 and 29 positive samples in liver tissue samples from 40 HBV-related patients, respectively. HBV cccDNA was almost completely undetected in the 20 blood samples of HBV patients (including plasma, whole blood and PBMCs) by the above 5 methods. We developed a novel CRISPR-based assay for the highly sensitive and specific detection of HBV cccDNA, presenting a promising alternative for accurate detection of HBV infection, antiviral therapy evaluation and treatment guidance. The online version contains supplementary material available at 10.1007/s12072-022-10311-0.
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影响因子：
3.7
作者：
Zhong Y;Hu S;Xu C;Zhao Y;Xu D;Zhao Y;Zhao J;Li Z;Zhang X;Zhang H;Li J
通讯作者：
Li J
影响因子：
29.4
作者：
Volz, Tassilo;Lutgehetmann, Marc;Petersen, Joerg
通讯作者：
Petersen, Joerg
DOI：
10.1016/j.jmii.2014.08.008
发表时间：
2016-08-01
影响因子：
7.4
作者：
Sai, Lin-Tao;Yao, Yong-Yuan;Ma, Li-Xian
通讯作者：
Ma, Li-Xian
DOI：
10.1007/978-1-61779-937-2_7
发表时间：
2012-01-01
期刊：
DIAGNOSIS OF SEXUALLY TRANSMITTED DISEASES: METHODS AND PROTOCOLS
影响因子：
--
作者：
Takkenberg, R. B.;Menting, S.;Beld, M. G. H. M.
通讯作者：
Beld, M. G. H. M.
影响因子：
5.4
作者：
Qu, Bingqian;Ni, Yi;Urban, Stephan
通讯作者：
Urban, Stephan