Development of a One-Step RT-LAMP-CRISPR/Cas12b Assay for Rapid On-Site Detection of Dabie Bandavirus

Abstract

Severe fever with thrombocytopenia syndrome (SFTS), caused by Dabie bandavirus (DBV), has rapidly spread to multiple countries worldwide since its discovery in 2009, posing a serious threat to global public health security. SFTS is a tick-borne disease, and people living in mountainous and hilly areas are at increased risk of infection, which highlights the urgent need for rapid, accurate, and field-applicable diagnostic strategies for DBV. In this study, LAMP primers and CRISPR sgRNA were designed based on the conserved region of the DBV L segment. A one-pot, one-step molecular assay integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12b technology was established after systematic optimization of key parameters. The assay operates under isothermal conditions at 60 °C, delivering results within 40 min. Analytical evaluation demonstrated a limit of detection of 25.6 RNA copies per reaction. No cross-reactivity was observed with Hantaan virus, Seoul virus, Chikungunya virus, Japanese encephalitis virus, Dengue virus, and Zika virus. Clinical validation using 42 specimens showed complete concordance with gold-standard qRT-PCR, achieving 100% sensitivity (30/30) and 100% specificity (12/12). For point-of-care application, fluorescence visualization was incorporated into the CRISPR/Cas-based detection workflow, enabling straightforward result interpretation. This integrated RT-LAMP-CRISPR/Cas12b platform provides rapid and specific detection of DBV through a simplified workflow that does not require complex instrumentation, supporting its potential application in resource-limited environments.