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Dual Detection of Hepatitis B and C Viruses Using CRISPR-Cas Systems and Lateral Flow Assay
Syeda Najidah Shahni1, Sarah Albogami2, Iqbal Azmi1
1Multidisciplinary Centre for Advanced Research and Studies, Jamia Millia Islamia, New Delhi, India.
Insights
This study introduces CRISPR-Cas12 and CRISPR-Cas13 for sensitive hepatitis B (HBV) and C (HCV) virus detection. A novel dual-enzyme lateral flow assay enables simultaneous, point-of-care diagnosis of both infections.
Area of Science:
- Molecular Biology
- Biotechnology
- Infectious Diseases
Background:
- Sensitive and specific diagnostic tools are crucial for managing hepatitis B virus (HBV) and hepatitis C virus (HCV).
- Current diagnostic methods may have limitations in sensitivity and multiplexing capabilities.
- CRISPR-Cas systems offer potential for highly specific nucleic acid detection.
Purpose of the Study:
- To develop and optimize CRISPR-Cas12 and CRISPR-Cas13 based assays for HBV and HCV detection.
- To enhance diagnostic sensitivity and specificity using a dual-enzyme approach.
- To create a multiplexed lateral flow assay (LFA) for simultaneous point-of-care (POC) detection of HBV and HCV.
Main Methods:
- CRISPR-Cas12 and CRISPR-Cas13 systems were employed for HBV (DNA) and HCV (RNA) detection, respectively.
- Guide RNAs (gRNAs) were designed and validated; cleavage was confirmed via gel electrophoresis and fluorescent reporter assays.
- Optimized gRNAs were integrated into a lateral flow assay (LFA), and a dual-enzyme strategy combining Cas12 and Cas13 was implemented.
Main Results:
- Optimized gRNAs enabled sensitive detection of HBV and HCV via LFA with concentration-dependent signal.
- The dual-enzyme approach significantly improved detection limits for both viruses.
- A dual antigen detection LFA strip successfully detected both HBV and HCV simultaneously without cross-reactivity.
Conclusions:
- CRISPR-Cas systems can be effectively utilized for highly sensitive and specific detection of HBV and HCV.
- The developed dual-enzyme LFA offers a promising platform for simultaneous, ultrasensitive POC diagnostics.
- This technology addresses limitations in current CRISPR-based diagnostics, enabling multiplexed detection in a single test strip.
Abstract:
The development of sensitive and specific diagnostic tools for hepatitis B virus (HBV) and hepatitis C virus (HCV) remains crucial for effective disease management and control. In this study, we utilized CRISPR-Cas12 and CRISPR-Cas13 systems for the detection of HBV (DNA virus) and HCV (RNA virus), respectively. We designed and tested multiple guide RNAs (gRNAs) targeting both viruses, confirming successful cleavage of target sequences through gel electrophoresis and a fluorescent reporter assay. Using optimized gRNAs, we developed a lateral flow assay (LFA) for sensitive detection of HBV and HCV, demonstrating a concentration-dependent signal increase. Importantly, no cross-reactivity was observed with other viral targets. To further enhance sensitivity, we employed a dual-enzyme approach, combining Cas12 and Cas13 in a single reaction, which significantly improved detection limits for both viruses. Finally, we developed a dual antigen detection LFA strip capable of simultaneously detecting both HBV and HCV in a single sample. This approach holds promise for point-of-care (POC) diagnostics where the specific viral infection is unknown. This study addresses the current limitations in CRISPR-Cas based diagnostics, namely, the need for ultrasensitive detection methods and the ability to detect multiple antigens using a single test strip. Our findings demonstrate the feasibility of using CRISPR-Cas systems for highly sensitive and specific detection of HBV and HCV, paving the way for potential POC application.
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