Feline calicivirus (FCV) is a well-known causative pathogen for upper respiratory infection in cats. Its high genetic variability challenges existing molecular diagnostic methods in clinical settings. Thus, we developed two sensitive and visual assays for FCV nucleic acid detection based on RPA reaction and CRISPR-Cas13a trans-cleavage activity. Recombinant plasmid DNA, crRNAs, and RPA primers were designed and prepared, respectively, targeting to FCV ORF1 gene. Besides, purified LwCas13a protein was produced by E.coli prokaryotic expression system. To confirm the validity of FCV-Cas13a assays, seven reaction systems (RSs) with different components were tested, and visual readouts were displayed by lateral flow dipstick (FCV-Cas13a-LFD) and fluorescence detector (FCV-Cas13a-FLUOR), respectively. The established FCV-Cas13a assays were capable of detecting FCV nucleic acid in presetting RSs without cross-reaction with other feline-associated pathogens, and the detection limit was as low as 5.5 copies/μl for both visual methods. Moreover, the positive rate of 56 clinical specimens detected by FCV-Cas13a assays (67.9%, 38/56) was notably higher than that of RT-qPCR (44.6%, 25/56) (p < 0.001), including 13 presumptive positive specimens. Taken together, FCV-Cas13a assays provided reliable and visual diagnostic alternatives for FCV field detection.
Keywords: Crispr-cas13a; RPA; feline calicivirus; nucleic acid detection; visual approach.
Copyright © 2022 Huang, Liu, He, Yang and Li.