A rapid and sensitive yellow fever virus detection method based on CRISPR/Cas13a and reverse transcription recombinase-aided amplification with special lateral-flow test strips.

Yellow fever (YF) is an epidemic disease caused by the yellow fever virus (YFV). Historically, it has caused several epidemics and continues to result in fatalities in South Sudan and other regions today. Due to its limited therapeutic options, high mortality rate, and high transmissibility, YFV is classified as a biosafety level-3 (BSL-3) pathogen. The development of a rapid and sensitive YFV detection method is therefore important for epidemic prevention and response. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) 13a system, reverse transcription recombinase-aided amplification (RT-RAA), and the easy-readout, sensitive enhanced lateral flow strip (ERASE LFS) to establish a new detection method for YFV. YFV ribonucleic acid and infectious YFV 17D particles were effectively detected using this approach. The RT-RAA-CRISPR-ERASE LFS (RCE) assay for YFV RNA demonstrated a detection sensitivity of 100 copies/μL with no cross-reactivity observed with five other common flaviviruses. The lyophilized RCE kit successfully detected YFV 17D in spiked human serum samples at a titer of 102 plaque-forming units (PFU)/mL. Furthermore, the optimized RCE reaction required only 35 min with a portable heat block. The RCE assay we established enabled rapid and sensitive on-site detection of YFV, holding substantial biosecurity significance for resource-limited regions with inadequate healthcare infrastructure.