A light-activated one-pot ERA/CRISPR-Cas12a assay for cost-effective dual-mode detection of HBV DNA.

Light-activatable CRISPR-Cas systems offer an effective strategy to overcome the kinetic incompatibility in one-pot nucleic acid assays; however, their practical application remains limited by high reagent cost, the lack of compatible reaction buffers, and reliance on instrument-dependent readout. In this work, we developed a cost-effective light-controlled one-pot ERA/CRISPR-Cas12a platform for rapid hepatitis B virus (HBV) DNA detection. NPOM-caged crRNA was employed to temporarily suppress Cas12a activity during amplification and to trigger target-dependent trans-cleavage by a brief 30 s UV irradiation. By replacing RPA with ERA, the per-reaction reagent cost of the one-pot assay was reduced from approximately US$6.0 to US$2.0, corresponding to a reduction of approximately 66.7%, while maintaining comparable analytical performance. In addition, a PEG-free unified buffer was established through systematic optimization, enabling efficient integration of ERA and Cas12a reactions in a sealed single-tube format. The proposed platform provided dual-mode readout through fluorescence and lateral flow assay (LFA), with a limit of detection of 1 copy μL-1 and a total assay time of 30 min. Clinical evaluation using 79 serum samples showed a sensitivity of 100% for fluorescence readout and 96.3% for LFA, with excellent agreement with qPCR (Cohen's κ = 0.94 - 1.00). Owing to its low cost, high sensitivity, and operational simplicity, this platform represents a promising tool for rapid HBV molecular diagnosis in both centralized laboratories and resource-limited settings.