Development and application of a real-time VPCR method for ultra-fast quantitative analysis and Visual Detection of...

Hepatitis B virus (HBV) infection remains a major global health challenge, necessitating rapid and accurate diagnostic methods for effective disease management. Although quantitative real-time PCR (qPCR) is widely used, its detection time is relatively long. We developed an ultra-fast HBV-DNA quantification system based on rapid VPCR technology, which, combined with a 10-min nucleic acid extraction process, reduces the entire workflow to approximately 35 min. Crucially, despite this significant increase in speed, the system maintains analytical sensitivity, specificity, and reproducibility comparable to those of conventional qPCR. The system's performance was then evaluated using 143 clinical samples and compared to that of standard qPCR, demonstrating that the developed real-time VPCR system significantly shortened the amplification time while maintaining excellent performance, with 100% sensitivity and 97.09% specificity. Additionally, a cost-effective qualitative endpoint visual detection method based on probe cleavage was established, offering an alternative readout strategy suitable for HBV screening in resource-limited settings without requiring expensive instruments. Collectively, this study presents a rapid quantification system and a qualitative visual detection method that significantly enhance diagnostic efficiency, providing strong technical support for clinical HBV management and screening.IMPORTANCEThe rapid hepatitis B virus (HBV) quantification system established in this study completes the entire workflow within 35 min and delivers excellent performance, meeting the demand for rapid decision-making in time-sensitive scenarios such as emergency care and acute liver failure. Its "test-and-treat" workflow is particularly suitable for patients in remote areas, allowing diagnosis and treatment in a single visit. Meanwhile, the accompanying visual qualitative method provides a low-cost screening strategy for resource-limited settings. This system greatly improves detection efficiency and clinical accessibility, showing important application value for the rapid diagnosis of hepatitis B and other pathogens.