Establishment of hydrolysis probe system real-time PCR assay for rapid detection of canine circovirus

Abstract

In the study, we established a hydrolysis probe-based real-time polymerase chain reaction (PCR) assay to rapidly detect Canine circovirus (CanineCV) DNA in faecal samples. We designed a pair of specific primers and one probe targeting Rep in CanineCV, and sensitivity, specificity, and repeatability tests were performed to evaluate the efficacy of the assay. The assay showed high sensitivity and a minimum detection limit of 8.42\u2009×\u2009101 copies/μL, which is 1000-fold more sensitive compared to traditional PCR. The method was also highly specific, without cross-reaction with other common canine viruses. Moreover, the assay showed high repeatability, and the mean intra-assay and inter-assay coefficients of variation were 0.26 and 0.36%, respectively. The results of the detection of clinical samples showed that the positive detection rate of CanineCV was 14.04% (8/57). Notably, 8% of clinical samples were co-infected with other canine pathogens. In conclusion, the establishment of a hydrolysis probe-based real-time PCR method provides a fast, sensitive, specific, reliable, and repeatable method for CanineCV detection.