Coupling Exponential Rolling Circle Amplification to Lateral Flow Devices for Point-of-Care Detection of Viral RNA

Sensitive detection of viral RNA in biological samples remains challenging because of low viral loads, complex matrices, and RNA secondary structures. Here, we present a heat-free, reverse-transcription-free lateral flow platform that converts structured viral RNA into visual signals through RNase I-mediated primer generation, exponential rolling circle amplification (E-RCA), and toehold-mediated strand displacement (TMSD). The newly developed E-RCA circuit uses a single additional primer, P2, which is extended on primary RCA products and then processed by Φ29 DNA polymerase to generate a feedback primer, enabling rapid exponential amplification from a circular DNA template. RCA products and P2-extended products subsequently trigger a newly designed TMSD module to release bridging DNA strands that generate visible lateral flow signals. Using SARS-CoV-2 as a model, the assay achieved detection limits down to 500 aM, sample-to-result times of approximately 45 min, and 95%-100% accuracy across four viral RNA regions in clinical saliva samples, providing a platform for rapid point-of-care RNA diagnostics.