2.3. Detection and typing of feline coronavirus
The 3’UTR of FCoV was amplified using RT-nested PCR as described by Herrewegh et al. (Herrewegh et al., 1995), and a positive 3’UTR produced a 177 bp amplicon on electrophoresis. Furthermore, for samples in which the 3’UTR gene was detected, genotyping was performed with RT-nPCR as reported by Addie et al. (DD Addie, Schaap, Nicolson, & Jarrett, 2003), providing 360 and 218 bp amplicons of FCoV types I and II, respectively. The PCR detection was performed in a 25 μL volume, including 1 μL of template, 1 μL of each primer (10 pmol), 8 μL of ddH2O, and 12.5 μL of Quick Taq HS DyeMix at 2 × concentration (Toyobo, Japan). The PCR conditions were set at 94 °C for 2 min, 35 repeats of denaturing at 94 °C for 30 s, annealing at 51 °C for 30 s, extension at 68 °C for 1 min, and a final extension at 72 °C for 8 min. The RT-PCR products were analyzed using 1.5% agarose gel electrophoresis and visualized using ultraviolet illumination. Finally, the PCR products consistent with the expected size were sent to Sangon Biotech (Shanghai, China) for sequencing.