Sequence-validated trigger gating for false-positive control in point-of-care isothermal nucleic acid detection

Nonspecific by-products generated during isothermal amplification can be propagated by downstream signal amplifiers, causing false-positive visual readouts and limiting the reliability of point-of-care nucleic acid tests. Here, we report a sequence-validated trigger-gating strategy for catalytic hairpin assembly (CHA) that improves false-positive control in isothermal visual RNA detection. In this design, upstream amplification products must pass sequence-dependent validation before they can release the trigger strand required to activate entropy-driven CHA, thereby limiting the carry-through of nonspecific amplification products into downstream signal amplification. Using severe fever with thrombocytopenia syndrome virus (SFTSV) RNA as a model target, we implemented this concept in a regulated catalytic hairpin assembly platform (RecHA) coupled to lateral flow readout. The assay reduced background activation, improved test line discrimination, operated at 34 °C, and supported direct loading of lysis products. RecHA detected SFTSV RNA down to 10 fM. In clinical validation using 70 qRT-PCR-positive serum samples and 30 negative controls, it achieved an AUC of 0.994, a sensitivity of 1.000, and a specificity of 0.967 in this serum cohort, with high concordance with qRT-PCR. Sequence-validated trigger gating enhances the selectivity and reliability of CHA-based visual RNA detection and offers a practical strategy for portable molecular diagnostics in resource-limited settings.