Local genetic distance-guided qPCR method for genotyping canine/feline coronaviruses

Canine coronavirus (CCoV) and feline coronavirus (FCoV) are highly infectious in companion animals. Nevertheless, due to genomic recombination and mutagenesis of the two CoVs, these difficulties dampen their precise diagnosis especially by genotypes. Herein, we addressed these problems and developed a simple real-time quantitative polymerase chain reaction (qPCR) method capable of genotyping major CCoV and FCoV, and partially surveyed the current epidemiological prevalence in dog and cat populations. Exploiting both evolutionary analysis and genome-wide genetic distance analysis, we identified locally conserved genomic regions that are specific to each genotype, including representative CCoV (sub)genotypes (I, IIa, IIb and IIc) and FCoV genotypes (I and II). By targeting these conserved regions, genotype-specific primer pairs were designed and cross-validated by in silico BLAST analysis, PCR melt curve and sequencing analysis. These primer pairs were integrated into the development of a simple qPCR approach and applied for genotyping potential CCoVs and FCoVs in Chengdu, China. We found 62.71% positivity in symptomatic dogs (n = 59) and 66.00% positivity in symptomatic cats (n = 50), with CCoV-I and FCoV-I being major genotypes in their native hosts during 2024-2026. Interestingly, many dogs and cats respectively tested positive for multiple CCoV and FCoV genotypes, with cross-host detection of FCoV RNA in dogs and CCoV RNA in cats. Collectively, we established a genotyping qPCR approach that enables the detection of CCoV and FCoV (sub)genotypes within a single analytical workflow, using one reaction per genotype.