RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.