Establishment of a duplex SYBR green I-based real-time polymerase chain reaction assay for the rapid detection of canine circovirus and canine astrovirus

Abstract

The similar clinical characteristics of canine circovirus (CaCV) and canine astrovirus (CaAstV) infections and high frequency of co-infection make diagnosis difficult. In this study, a duplex SYBR Green I-based real-time polymerase chain reaction (PCR) assay was established for the rapid, simultaneous detection of CaCV and CaAstV. Two pairs of specific primers were designed based on the Rep gene of CaCV and the Cap gene of CaAstV. By using the real-time PCR assay method, the two viruses can be distinguished by the difference in melting temperatures, 79 °C and 86 °C for CaCV and CaAstV, respectively. This assay had high specificity, showing no cross-reaction with other common canine viruses, as well as high sensitivity, with minimum detection limits of 9.25 × 10 1 copies/μL and 6.15 × 10 1 copies/μL for CaCV and CaAstV, respectively. Based on the mean coefficient of variation, the method had good reproducibility and reliability. In a clinical test of 57 fecal samples, the rates of positive detection by real-time PCR were 14.04% (8/57) and 12.28% (7/57) for CaCV and CaAstV, respectively, and the rate of co-infection was 8.77% (5/57). In conclusion, the newly established duplex SYBR Green I-based real-time PCR assay is sensitive, specific, reliable, and rapid and is an effective tool for the detection of co-infections with CaCV and CaAstV.

Development of a real-time TaqMan PCR assay for the detection of porcine circovirus 4

“…Compared with loop-mediated isothermal amplification, the TaqMan-based real-time PCR assay has superior accuracy. Isothermal amplification requires specialised personnel for operation, and the samples are vulnerable to contamination; therefore, the potential for false positive results is high and it is difficult to guarantee the accuracy of detection ( 18 ). Compared with SYBR, the cost of TaqMan assays is higher, where the expense probe synthesis particularly is a consideration.…”

Reconstruction of the Evolutionary Origin, Phylodynamics, and Phylogeography of the Porcine Circovirus Type 3

“…Therefore, these results showed that circovirus can maintain a certain degree of virulence in feces and can infect other animals through the fecal-oral route. Phylogenetic analysis showed that PCV3 has a close relationship with birds and penguins ( Figure 3 ), which suggests that the transmission mode of circovirus might be similar to that of influenza, in which birds play a crucial role as the intermediate host ( Zhou et al, 2018 ; Karakus et al, 2019 ; Wang et al, 2020 ). However, in this cycle, whether the source of porcine circovirus is directly transmitted by birds and/or wild boars is worthy of further analysis.…”

A Duplex Sybr Green I-Based Real-Time Polymerase Chain Reaction Assay for Rapid Detection of Canine Kobuvirus and Canine Circovirus

“…Compared to these traditional detection methods, real-time uorescence quantitative PCR has great advantages in terms of sensitivity and speci city [33,35], and detects virus even at lower concentrations [36]. In addition, compared with TaqMan-based real-time PCR, the SYBR Green I-based real-time PCR method is cheaper and easier [12].…”

“…Canine Circovirus (CaCV) is a single-stranded, non-capsule DNA virus belonging to the circovirus family [12]. It has two ORFs that encode replicase proteins and capsid proteins, respectively [13].…”