Development of a point-of-care testing method for Feline Coronavirus using CRISPR techniques

Feline Coronavirus (FCoV) is a recognized etiological agent for intestinal infection in cats and its mutants can cause lethal Feline Infectious Peritonitis (FIP). Its high genetic variability challenges existing molecular diagnostic techniques in clinical environments. Thus, we developed visual assays for the detection of FCoV nucleic acid based on CRISPR-Cas13a trans-cleavage activity. Recombinant plasmid DNA and CRISPR RNAs (crRNAs) were synthesized, respectively, targeting to FCoV RdRp, S, E and N genes of FCoV. Additionally, the Cas13a protein was purified through an Escherichia coli prokaryotic expression system. To validate the efficacy of the FCoV-CRISPR-Cas13a strip, five distinct reaction systems comprising various components were evaluated, with visual outputs assessed via fluorescence detection and colloidal gold immunochromatographic test strips. The developed FCoV-CRISPR-Cas13a strip demonstrated the ability to detect FCoV nucleic acids without cross-reactivity with other feline-associated pathogens, and the detection limit was as low as 102 TCID50 for the visual method. Furthermore, the positive detection rate of 200 clinical specimens using the FCoV-CRISPR-Cas13a strip (30.5%, 61/200) was notably higher than that of RT-qPCR (29.0%, 58/200), and the overall coincidence rate was 98.50%. In conclusion, FCoV-CRISPR-Cas13a strip provide a reliable, sensitive, and expeditious point-of-care testing (POCT) method for the detection of FCoV, thereby facilitating etiological surveillance and the prevention of FIPV.