Design of a Universal Backbone Improving the Endonuclease Dependence Molecular Beacon Assay

The endonuclease dependence molecular beacon assay (DEMBA) is an isothermal detection method celebrated for its high sensitivity, operational ease, and time-saving capabilities. However, the intricate design of the probes limits the extensive application of DEMBA. The main challenge is the requirement for the probe sequence to include restriction endonuclease sites, two target sequence binding sites, and self-binding sites, which frequently overlap. To overcome this issue, we introduce a strategy involving a universal probe backbone. This design permits the modification of only the target sequence binding sites for the detection of various nucleic acids. In this research, we successfully created universal backbone probes and implemented them in DEMBA assays. Our results demonstrate that these probes facilitate the sensitive and specific detection of nucleic acids, with an enhanced capability for targeting single-stranded RNA (ssRNA). Considering the increasing incidence of ssRNA virus outbreaks, the DEMBA with a universal backbone is expected to gain prominence in the prevention of infectious diseases.