One-step multiplex TaqMan probe-based method for real-time PCR detection of four canine diarrhea viruses

Abstract

Viral canine diarrhea has high morbidity and mortality and is prevalent worldwide, resulting in severe economic and spiritual losses to pet owners. However, diarrhea pathogens have similar clinical symptoms and are difficult to diagnose clinically. Thus, fast and accurate diagnostic methods are of great significance for prevention and accurate treatment. In this study, we developed a one-step multiplex TaqMan probe-based real-time PCR for the differential diagnosis of four viruses causing canine diarrhea including, CPV (Canine Parvovirus), CCoV (Canine Coronavirus), CAstV (Canine Astrovirus), and CaKoV (Canine Kobuviruses). The limit of detection was up to 10² copies/μL and performed well with high sensitivity and specificity. This assay was optimized and used to identify possible antagonistic relationships between viruses. From this, artificial pre-experiments were performed for mixed infections, and a total of 82 canine diarrhea field samples were collected from different animal hospitals in Zhejiang, China to assess the method. The virus prevalence was significantly higher than what previously reported based on RT-PCR (Reverse Transcription-Polymerase Chain Reaction). Taken together, these results suggest that the method can be used as a preferred tool for monitoring laboratory epidemics, timely prevention, and effective monitoring of disease progression.

Keywords: Canine diarrhea; Multiplex real-time PCR; TaqMan probe.

Fig. 1

Optimization of primers and probes. The blue, red, yellow and orange amplification curves represent CPV(A), CCoV(B), CaKoV(C) and CAstV(D), respectively. The pale blue curve represents primer and probe concentrations of 0.2 μM and 0.05 μM. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Amplification and standard curves of (A) CPV, (B) CCoV, (C) CaKoV, and (D) CAstV. The standard curve was evaluated using standards containing 10⁷ to 10¹ copies/μL. (E) Equation, correlation coefficient (R²) and amplification efficiency.

Fig. 3

Multiplex real-time PCR specificity. The X axis and Y axis represent the number of cycles and the fluorescence intensity, respectively.

Fig. 4

Simulation of virus co-infection. (A) 6 cases of double virus infection that was verified using 10³ and 10² copies/μl respectively. (B) 4 cases of three virus infections verified using 10³ and 10² copies/μL. The color was the same as Fig. 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)