Abstract: This study is to establish a real-time fluorescence detection method for rapidly detecting the hepatitis B virus using enzyme recombinase amplification (ERA) technology. The primers and probes were designed according to the conserved sequence of the polymerase coding region (P region) of the hepatitis B virus, and the reaction conditions, such as primers, temperature, and fluorescent probe concentration, were optimized to establish the optimal reaction system of the ERA real-time fluorescence assay, which was further validated in terms of sensitivity, specificity, anti-jamming capacity, and clinical samples. The results showed that the ERA real-time fluorescence method could detect the presence of the hepatitis B virus within 20 minutes at 42 ℃, with a limit of detection of 102copies/μL. The other four viruses (hepatitis A virus, hepatitis C virus, Epstein-Barr virus, and Cytomegalovirus) did not show fluorescence amplification curves, indicating reasonable specificity and anti-interference ability. When compared to the real-time fluorescence PCR method, the ERA real-time fluorescence method for 58 hepatitis B virus clinical samples exhibited a sensitivity of 97.37%, a specificity of 100%, a negative predictive value of 95.24%, a positive predictive value of 100%, and Kappa value of 0.96. This study successfully established a simple, rapid, sensitive, specific, and low-cost method for early detection of hepatitis B virus to meet the need for rapid detection.

Key words: enzymatic recombinase amplification (ERA) technology, rapid detection, hepatitis B virus, the ERA real-time fluorescence method, early diagnosis