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

ABSTRACT 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.