Rapid and visual detection of hepatitis B virus using the ERA/Cas12f1_ge4.1-based lateral flow assay system†

Hepatitis B virus (HBV) is a major pathogen posing significant challenges to global public health, making early diagnosis critical for preventing severe liver diseases. We previously developed a fluorescent biosensor named PAM-dependent dsDNA Target-activated Cas12f1 Trans Reporter (PDTCTR). However, its reliance on specialized fluorescence equipment and lack of visual readout limited its application in resource-limited settings. To address these limitations, we developed a lateral flow assay (LFA) that integrates enzymatic recombinase amplification (ERA) with the Cas12f1_ge4.1 system. This approach enables the specific amplification of the HBV target gene through ERA and leverages the precise cleavage activity of Cas12f1_ge4.1 for enhanced signal amplification. The entire detection process is completed within 50 minutes, with results readily interpretable through visual inspection. The assay achieves a minimum detection limit of 100 copies per μL and demonstrates high specificity, showing no cross-reactivity with related viruses. In a validation study involving 71 clinical samples, the system achieved a sensitivity of 94.23%, specificity of 100%, and a kappa value of 0.90 compared to quantitative PCR (qPCR), indicating high reliability. This method thus shows promise as an effective tool for early HBV diagnosis, particularly suited for rapid, on-site detection in resource-constrained environments, and holds broad potential for diverse applications.