Semi-automated diagnostic system and severe fever with thrombocytopenia syndrome (SFTS) viral RNA reference materials encapsulated in virus-like particles for on-site diagnosis of SFTS

Severe Fever with Thrombocytopenia Syndrome (SFTS), caused by the SFTS virus (SFTSV), is a tick-borne infectious disease prevalent in East Asia, characterized by acute fever and significant thrombocytopenia. Early and accurate diagnosis of SFTS is critical, particularly in rural areas where laboratory facilities are limited. Current diagnostic approaches, including RT-qPCR and immunological tests, face challenges due to their complexity, low sensitivity and the need for laboratory infrastructure. To address this, we developed a semi-automated diagnostic system for on-site testing of SFTS, integrating nucleic acid extraction with rapid, naked-eye detection using a reverse transcription loop-mediated isothermal amplification coupled with lateral flow assay. This system uses innovative SFTSV RNA reference materials encapsulated in virus-like particles, ensuring both stability and homogeneity, making them ideal for calibrating and validating diagnostic assays. Additionally, the semi-automated system, equipped with a microfluidic chip and optimized extraction processes, demonstrates a limit of detection (LOD) of 1.82 × 10² copies/µL with SFTSV RNA reference materials. Finally, this system allows for the rapid and accurate detection of SFTSV RNA from 12 clinical specimens within 100 minutes, with results visible to the naked eye in just 2 minutes post-amplification. This system offers a practical solution for on-site SFTS diagnosis, particularly in resource-limited settings, and represents a significant advancement in the field of molecular diagnostics.