Instrument-Free, Low-Cost and Dual-System Detection for Norovirus Based on Self-Assembling DNA Circuitry

Norovirus is a major cause of acute gastroenteritis, yet rapid diagnostics that combine scalability, high analytical sensitivity, and minimal instrumentation remain limited. Here, we report an enzyme-free nucleic acid detection platform that integrates catalytic hairpin assembly (CHA) with lateral flow readout in two formats: a visual colloidal-gold strip (CHA-GICA) and a fluorescence strip (CHA-FICA). A conserved 23-nt sequence shared across norovirus GI and GII triggers CHA at 35 °C to generate biotin- and digoxin-labeled duplex products, which are captured on the test line via antidigoxin binding, while excess reporters are retained at the control line. The workflow requires 30 min total (15 min reaction and 15 min strip development). Under optimized conditions, CHA-GICA achieved a limit of detection of 10 pM, and CHA-FICA reached 100 fM, with strong discrimination against single-base substitutions and small insertion/deletion variants. Hairpin probes remained stable for at least 4 weeks at −20 °C and were suitable for short-term storage at 4 °C. In 55 clinical fecal specimens benchmarked against RT-qPCR (15 positive, 40 negative), CHA-GICA showed 73.3% sensitivity and 100% specificity (AUC = 0.853), while CHA-FICA improved sensitivity to 86.7% with 100% specificity (AUC = 0.988). This dual-format, enzyme-free CHA lateral flow platform enables rapid norovirus screening with flexible deployment, supporting instrument-free field testing by CHA-GICA and higher-sensitivity detection by CHA-FICA.