An isothermal amplification-bridged ratiometric DNA switch for sensitive detection of viral gene fragments

The detection of viral gene fragments is essential for diagnosing and screening viral infections, managing virus transmission, and controlling epidemics. In this study, we developed an isothermal amplification-bridged DNA switch designed for the sensitive detection of viral gene fragments. Specifically, the binding of the target viral gene fragment to the recognition sequences triggers isothermal amplification, resulting in the production of multiple DNA products. Consequently, a single target gene fragment is transformed into approximately 1250 DNA products, which can subsequently activate the DNA switches. This activation leads to the opening of the DNA switch and the occurrence of Förster resonance energy transfer (FRET), generating a ratiometric fluorescent signal. The effective conversion of the target facilitated by isothermal amplification enables the generation of amplified ratiometric fluorescent signals, thereby achieving sensitive detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gene fragment with a limit of detection of 0.04 fM. An extensive linear range spanning approximately eight orders of magnitude, characterized by two distinct linear relationships, is obtained, making it particularly effective for detecting viral gene fragments in various biological samples with significantly varying concentrations of target gene fragments. The analysis of actual samples from patients presenting with upper respiratory symptoms underscores the method's potential for clinical diagnostic applications. Additionally, by modifying the recognition strands, the method allows for the sensitive and selective detection of the Rabies virus (RABV) gene fragment, highlighting its versatility and adaptability. Therefore, this research presents a novel and sensitive approach for detecting viral gene fragments, offering considerable promise for the clinical diagnosis of infectious diseases.