Nucleic acid-based differential diagnostic assays for feline coronavirus

Feline coronavirus (FCoV) is a pleomorphic, enveloped, positive-sense single-stranded RNA virus. Owing to the differences in its genotype, FCoV belongs to a separate clade along with other viruses, such as transmissible gastroenteritis virus (TGEV) and canine coronavirus (CCoV), which can be isolated from cats. In this study, a PCR assay was developed to differentiate these coronaviruses concurrently. Multiplex differential RT-PCR was performed with primers based on the highly conserved coronavirus membrane protein. Three primer sets were designed: a primer pair (S1 and S2) that can bind to conserved sequences in all target coronaviruses, a CCoV-specific primer (S3), and a TGEV-specific primer (S4). Because of the high sequence homology among FCoV, CCoV, and TGEV, a nucleotide preceding the last pair of dissimilar nucleotides in S3 and S4 was substituted with an inosine to allow primer binding. This assay could detect and differentiate FCoV (n = 7), CCoV (n = 4), and TGEV (n = 8) precisely and did not show any cross-reactivity with other pathogens. These results suggest that this molecular approach provides a rapid and reliable way to detect FCoV, especially in feline clinical specimens.