Simultaneous detection of canine astrovirus and canine kobuvirus by duplex qPCR: validation and evidence of extraintestinal viral RNA in naturally infected dogs

Canine astrovirus (mamastrovirus 5 [MAstV5], formerly CaAstV; family <i>Astroviridae</i>, taxon species <i>Mamastrovirus canis</i>) and canine kobuvirus (aichivirus A2 [AiV-A2], formerly CaKoV; <i>Picornaviridae</i>, <i>Kobuvirus aichi</i>) are emerging enteric viruses increasingly detected in both diarrheic and subclinical dogs. Although primarily associated with gastrointestinal illness, recent evidence suggests a potential for systemic dissemination, which remains insufficiently explored. To improve detection, we developed and validated a duplex quantitative real-time PCR (dqPCR) assay for simultaneous identification of MAstV5 and AiV-A2. Primers and TaqMan probes were designed based on conserved regions of the MAstV5 <i>ORF1b</i> and AiV-A2 <i>3D</i> polymerase genes. Assay optimization included primer-probe concentration titration and thermal gradient analysis. Analytical performance was assessed using synthetic plasmid standards and RNA from non-target viruses, including canine parvovirus (CPV), canine morbillivirus (CDV), and canine enteric coronavirus (CCoV). Our dqPCR assay had high linearity (<i>R</i>² > 0.99) and sensitivity, with limits of detection of 10 copies/μL for MAstV5 and 100 copies/μL for AiV-A2. No cross-reactivity or interference was observed in coinfection simulations across various target ratios. Intra- and inter-assay CV values were <2.2%, indicating excellent reproducibility. Validation using 50 clinical rectal swabs and tissue samples from 25 autopsied dogs revealed 1 additional AiV-A2-positive case undetected by RT-PCR but confirmed by sequencing. Importantly, viral RNA was also found in extraintestinal tissues-spleen, liver, trachea, and mesenteric lymph nodes-suggesting systemic distribution in naturally infected dogs. Our dqPCR assay provides a sensitive, specific, and efficient tool for the detection of MAstV5 and AiV-A2, supporting both clinical testing and epidemiologic studies.