Dual-gene detection in a single-tube system based on CRISPR-Cas12a/Cas13a for severe fever thrombocytopenia syndrome virus

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease, which is caused by severe fever with thrombocytopenia syndrome virus (SFTSV). The disease results in high mortality and increased morbidity and threatens global public health. Rapid detection of SFTSV is crucial for epidemic prevention in low-resource settings. Here we developed deployable, sensitive and rapid detection methods based on CRISPR/Cas12a or Cas13a technologies. The CRISPR/Cas12a-based detection assay could stably detect the SFTSV L or M genes at 10 cp/mu l. The Cas13a-based method could detect the L gene as low as 0.75 cp/mu l. For point-of-care testing, we combined fluorescence visualization and lateral flow detection with CRISPR/Cas-based assays. Furthermore, using the orthogonal DNA/RNA collateral activity of the Cas12a/Cas13a system, we present the dual-gene detection platform for SFTSV, which can simultaneously detect the L and M genes in a single tube. Based on the dual-gene detection, we designed multiplexed test strips to detect SFTSV. All our methods were initially validated using 52 clinical samples, showing 100% sensitivity and specificity. These new CRISPR/Cas-based detection methods are promising candidates for on-site detection of SFTSV.

Automated summary

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that threatens global public health. Rapid and sensitive detection methods based on CRISPR/Cas12a or Cas13a technologies have been developed for on-site detection of SFTS, with promising results shown in initial clinical validation.