Field-Deployable Nucleic Acid Testing via Functional Double-Strand to Single-Strand Conversion

Abstract Efficient double-stranded DNA (dsDNA) to single-stranded DNA (ssDNA) conversion is critical for nucleic acid testing. We present a functional dsDNA-to-ssDNA conversion (FDSC) strategy that integrates auxiliary hybridization probes with a multicomponent DNAzyme, enabling simultaneous strand separation and catalytic signal amplification. FDSC is compatible with recombinase polymerase amplification (RPA) and was engineered into a lyophilized single-tube assay with extraction-free urine processing and a portable fluorescence reader. Clinically validated for BK polyomavirus detection in kidney transplant recipients, the RPA-FDSC platform achieved 100% specificity and 93.3% sensitivity in unprocessed urine, offering a robust, cost-effective route for field-deployable molecular diagnostics.