The development of a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity offers great potential for SARS-CoV-2 diagnosis.

SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Compared to the conventional culture-based or immunoassay-based approaches, a nucleic acid-based approach relies primarily on a strand-displacing polymerase or T7 RNA polymerase at a constant temperature. In particular, the SplintR ligase can efficiently ligate two DNA probes using a target single-stranded RNA as a splint, enabling the sequence-specific detection of RNA molecules.

SENSR integrates all component reaction steps using the specially designed probes that contain all required functional parts: promoter, hybridization sequence to target, and an aptamer template. Even with the multifaceted features of the SENSR probes, the design process is systematic and straightforward. The probe design is unique in that two DNA probes are designed to expose single-stranded target recognition parts, enabling hybridization of the target RNA and the probes at 37°C.