Establishment and application of ERA-LFD method for rapid detection of feline calicivirus

Feline calicivirus (FCV) has a single-stranded, positive-sense RNA genome, and it is responsible for many infectious respiratory diseases in cats. In addition, more worryingly, highly virulent strains of FCV can cause high mortality in felines. Therefore, a rapid and reliable diagnosis tool plays an important role in controlling the outbreak of FCV. In this study, enzymatic recombinase amplification (ERA) assay combined with lateral flow dipstick (LFD) was developed for the detection of FCV, targeting a relatively conversed position of FCV-ORF1. The results showed that the optimal reaction condition was at 40 °C for 30 min. ERA-LFD method was highly sensitive with the detection limit as low as 3.2 TCID₅₀ of FCV RNA per reaction. The specificity analysis demonstrated no cross-reactivity with feline parvovirus (FPV), feline herpesvirus (FHV) and feline infectious peritonitis virus (FIPV). ERA-LFD was highly repeatable and reproducible, with the intra-assay and inter-assay coefficients of variation for this method both less than 7%. The general test showed that all the recombinant plasmids with known mutant sites and FCV strains with different mutant sites stored in our laboratory were all detected by this method. Of the 23 samples, 14 samples were tested positive for FCV by ERA-LFD and RT-qPCR, respectively. In summary, ERA-LFD assay was a fast, accurate and convenient diagnosis tool for the detection of FCV. KEY POINTS: • The detection principle of ERA-LFD was introduced. • Almost all the currently known FCV strains can be detected. • ERA-LFD is easy to operate and can be used for field detection.