Development of a rapid RPA-CRISPR/Cas12b assay for dual detection of Hantaan virus and Seoul virus.

Hantaan virus (HTNV) and Seoul virus (SEOV) are the primary causative agents of hemorrhagic fever with renal syndrome (HFRS), presenting a persistent challenge to global public health. Prompt and precise diagnostic tools are essential for clinical investigation and the containment of HFRS outbreaks. Leveraging the programmable precision of CRISPR/Cas12b system, which has significantly advanced the field of molecular diagnostics, we developed a rapid, high-sensitivity assay for the dual detection of HTNV and SEOV. Each virus was detected in a separate, target-specific single-tube reaction. The assay combines recombinase polymerase amplification (RPA) with CRISPR/Cas12b-mediated fluorescence detection and uses virus-specific sgRNAs and RPA primers targeting conserved genomic regions. Using recombinant plasmid standards, both targets were detected at 1 × 101 copies/test in three technical replicates; this is a preliminary analytical detection threshold and does not incorporate extraction losses or matrix-associated inhibition. No cross-reactivity was observed within the evaluated four-organism non-target panel. In a preliminary feasibility assessment, all nine RT-qPCR-positive rodent samples (seven HTNV and two SEOV) were positive by the RPA-CRISPR/Cas12b assay, indicating complete positive agreement in this all-positive sample set. Because no negative samples were included, negative agreement, diagnostic specificity, false-positive rate, and overall clinical diagnostic accuracy could not be assessed. These results support further development of the assay as a potential screening tool; larger studies using well-characterized positive and negative clinical or field specimens are required.