Device-Free Nucleic Acid Detection Enabled by Body Temperature-Assisted DEMBA

We have developed a novel nucleic acid detection method, the body temperature endonuclease-dependent molecular beacon assay (DEMBA). In DEMBA, the fluorescence signal is significantly lower in the absence of the target sequence compared to its presence, demonstrating the assay's responsiveness. This study utilized two DEMBA probes incorporating restriction endonucleases (Bpu 10I and Bpu 1102I), whose secondary structures were predicted using NUPACK software. Experimental characterization included 4.5% agarose gel electrophoresis to analyze probe behavior, target interaction, and enzymatic cleavage products, while real-time PCR monitored fluorescence changes in response to varying ssDNA/ssRNA concentrations. To enable visual detection, we integrated the system with colloidal gold-based lateral flow assays (LFAs). Theoretically, any endonuclease with an optimal activity around 37 °C is suitable for DEMBA, enhancing its versatility. A key advantage of DEMBA is its ability to directly hybridize with target RNA sequences, eliminating the need for reverse transcription and thereby reducing assay time. Concurrently, RNA exhibits enhanced stability at this physiological temperature, improving detection reliability. Furthermore, DEMBA can be integrated with double-labeled test strips for DNA or RNA detection, enabling visual readout without specialized equipment. Notably, the 37 °C reaction condition can be met using the patient's body temperature, facilitating device-free nucleic acid testing. This study successfully achieved nucleic acid detection at 37 °C using DEMBA probes, establishing a foundation for the development of body temperature-based nucleic acid detection technologies.