Development and characterization of a digital CRISPR/Cas13a based assay for rapid and sensitive diagnosis of severe fever with thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a re-emerging pathogen causing sporadic outbreaks in China, Japan, and Korea with mortality rates of up to 30 %. There are currently no licensed vaccines or therapeutics, and early diagnosis of infected cases is critical for outbreak control and prevention. Assays based on isothermal nucleic acid amplification and CRISPR/Cas have recently been developed against several pathogens without the need for specialized laboratory equipment. However, these assays are qualitative in nature and cannot provide quantitative results. Here, we developed three candidate diagnostic assays for field or clinical detection of SFTSV infections. The two-step CRISPR/Cas13a based assay was found to be comparable in sensitivity to previously published work (100 cp/mu L), whereas optimization of reaction conditions for the one-pot CRISPR/Cas13a based assay improved the sensitivity by 40-fold. The optimized one-pot assay has high sensitivity (5 cp/mu L), and the assays were found to be non-cross reactive against other (re)-emerging pathogens. To provide quantitative results for SFTSV detection, the digital CRISPR/Cas13a-based assay was developed, and found to be consistent with qRT-PCR results (100 %) using SFTSV clinical samples. This work will be important for the future development of diagnostics for SFTSV, with the potential for point-of-care testing.