Abstract
BackgroundCanine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Early detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Recombinase polymerase amplification (RPA), a novel isothermal gene amplification technique, has been developed for the molecular detection of diverse pathogens. In this study, a real-time RPA assay was developed for the detection of CPV-2 using primers and an exo probe targeting the CPV-2 nucleocapsid protein gene.ResultsThe real-time RPA assay was performed successfully at 38 °C, and the results were obtained within 4–12 min for 105–101 molecules of template DNA. The assay only detected CPV-2, and did not show cross-detection of other viral pathogens, demonstrating a high level of specificity. The analytical sensitivity of the real-time RPA was 101 copies/reaction of a standard DNA template, which was 10 times more sensitive than the common RPA method. The clinical sensitivity of the real-time RPA assay matched 100% (n = 91) to the real-time PCR results.ConclusionThe real-time RPA assay is a simple, rapid, reliable and affordable method that can potentially be applied for the detection of CPV-2 in the research laboratory and point-of-care diagnosis.
Highlights
Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species
Using 10 ng of viral DNA, cDNA or canine genome as template, the results showed that only CPV-2a and CPV-2b were detected by the real-time Recombinase polymerase amplification (RPA) assay, while the other four viruses, including Canine distemper virus (CDV), Canine coronavirus (CCoV), Canine parainfluenza virus (CPIV) and Pseudorabies virus (PRV), and canine genome, were not (Fig. 1, n = 5)
To evaluate the sensitivity of the real-time RPA method, a dilution range of 105–100 copies/μL of standard DNA was used as templates, and the real-time RPA and realtime polymerase chain reaction (PCR) were performed simultaneously
Summary
Canine parvovirus 2, a linear single-stranded DNA virus belonging to the genus Parvovirus within the family Parvoviridae, is a highly contagious pathogen of domestic dogs and several wild canidae species. Detection of canine parvovirus (CPV-2) is crucial to initiating appropriate outbreak control strategies. Canine parvovirus diseases, caused by canine parvovirus type 2 (CPV-2), is highly contagious and prevalent worldwide in domestic and wild canids. CPV-2 emerged as a novel pathogen in 1978 and spread rapidly worldwide [1, 2]. CPV-2 is a small non-enveloped, linear single-stranded DNA virus belonging to the family. Early, rapid and accurate diagnosis of CPV-2 infection would help veterinarians to implement appropriate strategies in time to improve disease management and prevent outbreaks, within a shelter environment
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