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Updated: Jun 7, 2026
DNA Virus Detection System Based on RPA-CRISPR/Cas12a-SPM and Deep Learning
Published on: May 10, 2024
CRISPR-Cas13a/Cas12a Assisted Dual Portable and Visualized HDV and HBV Detection
Yuan Tian1, Yaling Cao2, Zhenzhen Pan1
1Beijing Institute of Hepatology/Beijing Youan Hospital, Capital Medical University, Beijing, PR China.
Insights
A new CRISPR-Cas dual detection method accurately identifies hepatitis B virus (HBV) DNA and hepatitis D virus (HDV) RNA. This sensitive assay aids in early diagnosis and monitoring of co-infections.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Hepatitis B virus (HBV) and hepatitis D virus (HDV) co-infection exacerbates liver disease progression.
- Current diagnostics lack a combined method for detecting both HBV and HDV nucleic acids simultaneously.
Purpose of the Study:
- To develop a sensitive and specific dual detection assay for HDV RNA and HBV DNA.
- To establish a CRISPR-Cas-based system for simultaneous detection of both viruses.
Main Methods:
- Developed a dual detection method using CRISPR-Cas12a with recombinase polymerase amplification (RAA) for HBV DNA and CRISPR-Cas13a with RT-RAA for HDV RNA.
- Optimized RAA primers and crRNAs for dual fluorescence (DF) and lateral flow strip (DL) detection.
- Validated the assay using synthetic samples and plasma from 70 co-infected patients.
Main Results:
- The CRISPR-Cas system demonstrated 100% specificity.
- Both DF and DL methods achieved a sensitivity of 10 copies/μL for synthetic samples.
- Positive concordance rates in clinical samples were 85.7% for DF and 82.9% for DL.
Conclusions:
- A CRISPR-Cas13a/Cas12a-based dual assay provides sensitive, specific, and accurate detection of HDV RNA and HBV DNA.
- This assay is a valuable tool for early detection, treatment, and monitoring of HBV/HDV co-infections.
Abstract:
Co-infection with both hepatitis B virus (HBV) and hepatitis D virus (HDV) can aggravate the severity of the end-stage liver disease and accelerate its progression. However, no combined diagnostic method for HDV and HBV nucleic acids exists. In this study, we have developed a highly sensitive and specific dual detection method for HDV RNA and HBV DNA using the CRISPR-Cas system. We established a dual detection method combining CRISPR-Cas12a with recombinase polymerase amplification (RAA) for HBV, and CRISPR-Cas13a with RT-RAA for HDV. Validation was performed using specimens from 70 co-infected patients. RAA primers and crRNAs were designed and optimized to establish a dual fluorescence detection method (DF) and lateral flow strip-based dual detection (DL) within the same CRISPR-Cas13a/Cas12a system for HDV RNA and HBV DNA. The system demonstrated 100% specificity, and both DF and DL methods exhibited a sensitivity of 10 copies/μL for synthetic positive plasmids and samples. Peak fluorescence detection was achieved with T7 RNA polymerase, while the best detection efficiency was at ssRNA: ssDNA ratio of 1:1.5. In the validation of plasma samples from 70 co-infected clinical patients, the positive concordance rates for RT-RAA-CRISPR-Cas13a/Cas12a DF and DL were 85.7% (60/70) and 82.9% (58/70), respectively. We developed a CRISPR-Cas13a/Cas12a-based dual assay for sensitive, specific, and accurate detection of HDV RNA and HBV DNA, offering an effective tool for the early detection, treatment, and monitoring of HDV and HBV infections.
