Dual Detection of Hepatitis B and C Viruses Using CRISPR-Cas Systems and Lateral Flow Assay.

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.