Rapid Detection of Feline Calicivirus Using Lateral Flow Dipsticks Based on CRISPR/Cas13a System

Feline calicivirus (FCV) is one of the most common viral pathogens in domestic cats worldwide, which mainly causes upper respiratory tract infections in felines and seriously threatens the health of felines. Consequently, it is crucial to establish a rapid detection method to efficiently take control and prevent the spread of FCV. To construct the Cas13a-RAA-LFD reaction system, this study specifically designed recombinase-aided amplification (RAA) primers added with a T7 promoter and CRISPR RNA (crRNA), which were both based on the FCV relatively conserved sequence. The Cas13a protein cleaved the reporting probes only when crRNA recognized the target sequence. The results could be directly observed by lateral flow dipsticks (LFDs). To evaluate this system, factors such as RAA amplification time, Cas13a protein concentration, crRNA concentration, and CRISPR reaction time were optimized. Then, a comparison of the coincidence rate for clinical samples between this method and the polymerase chain reaction (PCR) agarose electrophoresis method was performed to evaluate the reliability of the method. Eventually, the results indicated that the target gene could be effectively amplified by the Cas13a-RAA-LFD method, and the results could be visually observed by LFD. The method could detect FCV specifically, whilst having no cross-reaction with other common viruses which infect felines, such as feline parvovirus (FPV), feline coronavirus (FCoV) and feline herpesvirus (FHV). This method is extremely sensitive and has been validated to detect viral nucleic acids down to 10⁰ copies/μL. The good reproducibility and stability of the method were also verified by this study. Testing of clinical samples proved that the coincidence rate of clinical detection reached 96.39%. In summary, this study established a simplistic, efficient, accurate, and visualized FCV detection method, which can be utilized for early prevention and control of FCV.