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

Zhang, Xiangying; Tian, Yuan; Xu, Ling; Fan, Zihao; Cao, Yongli; Ma, Yingmin; Li, Hao; Ren, Feng

doi:10.1007/s12072-022-10311-0

Cited by 23 publications

(22 citation statements)

References 19 publications

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“…We previous 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 [ 38 ]. The goal of this study is to drive the detection of HBV infection to effectively adaptable for any small to medium-sized laboratory or a field survey.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients

Tian

Fan

et al. 2023

Emerging Microbes & Infections

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Background & Aims: The WHO declared to eliminate hepatitis B virus (HBV) by 2030. However, an increasing number of patients are presenting with low-level viremia (LLV) with the widespread use of antiviral medications. The diagnostic efficiency and coverage area of HBV infection are low. Hence, this study intended to drive the HBV infection detection to effectively adaptable for any small to medium-sized laboratory or field survey. Methods: We established, optimized, and evaluated a colloidal gold test strip for detection of HBV DNA based on CRISPR/Cas13a combined with recombinase-aided amplification (RAA) technology. Furthermore, 180 HBV-infected patients (including patients with different viral loads, LLV patients and dynamic plasma samples of patients on antiviral therapy) were enrolled for clinical validation. Results: The strip detection of HBV DNA was established based on RAA-CRISPR-Cas13a technology with a sensitivity of 10 1 copies/μL and a specificity of 100%. HBV DNA gradient concentration plasmids and clinical samples were effectively identified by this approach. The positive coincidence rate for LLV patients was 87%, while the negative coincidence rate was 100%. The positive coincidence rate reached 100% in LLV patients (viral loading >100 IU/mL). The sensitivity, specificity, positive predictive agreement (PPA) and negative predictive agreement (NPA) values of dynamic plasma detection in patients on antiviral therapy were 100%, 92.15%, 93.75%, and 100%, respectively. Conclusions: We develop rapid and portable RAA-CRISPR/Cas13a-based strip of HBV DNA detection for LLV patients. This study provides a visual and faster alternative to current PCR-based diagnosis for HBV infection.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients

Tian

Fan

et al. 2023

Emerging Microbes & Infections

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“…This assay can detect 1 copy/µL HBV cccDNA by CRISPR/Cas13-assisted fluorescence readout. Furthermore, by performing ddPCR, qPCR, RCA-qPCR, PCR-CRISPR and RCA-PCR-CRISPR, this assay was validated for the detection of cccDNA in HBV-associated liver tissues, plasma, whole blood, and peripheral blood mononuclear cells (PBMCs) [ 93 ].…”

Section: Potential Of Crispr/cas System In Viral Hepatitis Diagnosismentioning

confidence: 99%

Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection

Bahrulolum

Tarrahimofrad

Rouzbahani

et al. 2023

Virol J

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Viral hepatitis, the most common cause of inflammatory liver disease, affects hundreds of millions of people worldwide. It is most commonly associated with one of the five nominal hepatitis viruses (hepatitis A–E viruses). HBV and HCV can cause acute infections and lifelong, persistent chronic infections, while HAV and HEV cause self-limiting acute infections. HAV and HEV are predominantly transmitted through the fecal-oral route, while diseases transmitted by the other forms are blood-borne diseases. Despite the success in the treatment of viral hepatitis and the development of HAV and HBV vaccines, there is still no accurate diagnosis at the genetic level for these diseases. Timely diagnosis of viral hepatitis is a prerequisite for efficient therapeutic intervention. Due to the specificity and sensitivity of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated sequences (Cas) technology, it has the potential to meet critical needs in the field of diagnosis of viral diseases and can be used in versatile point-of-care (POC) diagnostic applications to detect viruses with both DNA and RNA genomes. In this review, we discuss recent advances in CRISPR–Cas diagnostics tools and assess their potential and prospects in rapid and effective strategies for the diagnosis and control of viral hepatitis infection.

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“…Besides, Cui et al (2022) performed a picoinjection-aided digital reaction unlocking (PADLOCK) assay, which combines a droplet generator with the addition of the reaction initiator MgOAc through a droplet microfluidic device to avoid premature amplification, allowing detection of HPV16 at as low as 5 copies/μl. Recently, Zhang X. et al (2022) established an RCA-PCR-CRISPR assay that combines the Cas13a reaction with dual amplification—rolling circle amplification (RCA) and PCR—to detect covalently closed circular DNA (cccDNA) of the hepatitis B virus (HBV). Using this approach, they could detect 1 copy/μL HBV cccDNA, with a higher positive detection rate than those of real-time PCR (qPCR), droplet digital PCR (ddPCR), RCA-qPCR, and PCR-CRISPR.…”

Section: Cas13a-based Detection For Virusesmentioning

confidence: 99%

CRISPR-Cas13a system: A novel tool for molecular diagnostics

Zhao

Qiu

et al. 2022

Front. Microbiol.

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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.

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

Cited by 23 publications

References 19 publications

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients

CRISPR/Cas13a-assisted rapid and portable HBV DNA detection for low-level viremia patients

Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection

CRISPR-Cas13a system: A novel tool for molecular diagnostics

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