BACKGROUND: Canine respiratory coronavirus (CRCoV) is a major contributor to the canine infectious respiratory disease complex (CIRDC). Despite its widespread prevalence, molecular assays for CRCoV detection remain limited. Additionally, the efficiency and accuracy of detection can vary depending on the type of clinical sample used, such as nasal swabs (NS), oropharyngeal swabs (OS), and rectal swabs (RS). To address these challenges, we developed a nanoplate-based reverse transcription digital polymerase chain reaction (RT-dPCR) method for detecting the spike gene of CRCoV in various clinical samples. RESULTS: The RT-dPCR assay demonstrated consistent repeatability and reproducibility, ensuring reliable results. With a detection limit of 1.83 copies/µL, the RT-dPCR assay exhibited 100-fold greater sensitivity than probe-based reverse transcription quantitative polymerase chain reaction (RT-qPCR). It showed no cross-reactivity with other common CIRDC-associated viruses or coronaviruses, confirming its high specificity for CRCoV. The assay was further validated using 162 clinical swab samples (NS, OS, and RS) collected from both healthy dogs and those with respiratory distress. The RT-dPCR assay showed a higher overall positivity rate for CRCoV compared to RT-qPCR, with the most notable difference observed in rectal swabs (P <
0.05), where RT-dPCR detected CRCoV in 53.7% of samples compared to 22.22% by RT-qPCR. CONCLUSIONS: This study demonstrated that the RT-dPCR assay provided high sensitivity for detecting low viral loads across various sample types, making it a valuable tool for precise CRCoV detection. In contrast, RT-qPCR remains valuable for its broader detection range and suitability in initial screening. Both techniques proved to be versatile tools that can contribute to advancing CRCoV research and improving clinical diagnostics.