Fluorogenic RNA aptamer output sensors via transcription activated by recombinase polymerase amplification for nucleic acid testing

[Display omitted] •A novel fluorogenic RNA aptamer output sensor via in vitro transcription (IVT) activated by RPA (ROSAR) was developed.•The proposed ROSAR for pathogens nucleic acid (DNA or RNA) testing displayed outstanding performance.•A paper-based ROSAR was developed for improving the stability of reagents and facilitate POC testing.•An integrated, low-cost, and user-friendly device was designed for paper-based ROSAR readout using mobile phone.•The proposed ROSAR and paper-based ROSAR were validated using clinical samples, showed the potential of clinical application. Sensitive, simple, and rapid nucleic acid testing used for pathogen detection are crucial for preventing outbreaks and transmission of infectious diseases. In this study, we present a fluorogenic RNA aptamer Output Sensors via transcription Activated by Recombinase polymerase amplification (RPA), termed ROSAR, for highly sensitive detection of DNA and RNA from pathogenic bacteria or viruses in clinical samples. The promoter primer and reporter primer facilitate the integration of the transcriptional promoter with the fluorogenic RNA aptamer transcription templates through the RPA of target DNA. Subsequently, the amplification products are transcribed by T7 RNA polymerase, producing RNA aptamer that binds to a fluorogenic dye. The assay, which can be completed within 50 min and can allow a limit of detection of level aM nucleic acid concentration, owning to the dual amplification effects of RPA and in vitro transcription. Furthermore, we develop a paper-based ROSAR by freeze-drying the reaction components, which is compatible with visual detection based on mobile phone fluorescent reader and suitable for point-of-care (POC) applications. The assay can detect various viral and bacterial nucleic acids, making it a highly promising tool for large-scale epidemic screening and rapid POC testing in home healthcare settings.