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CRISPR/Cas13-assisted hepatitis B virus covalently closed circular DNA detection

BACKGROUND AND AIMS: 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. METHODS...

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Detalles Bibliográficos
Autores principales: Zhang, Xiangying, Tian, Yuan, Xu, Ling, Fan, Zihao, Cao, Yaling, Ma, Yingmin, Li, Hao, Ren, Feng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer India 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9013339/
https://www.ncbi.nlm.nih.gov/pubmed/35298777
http://dx.doi.org/10.1007/s12072-022-10311-0
Descripción
Sumario:BACKGROUND AND AIMS: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12072-022-10311-0.