A one-pot detection method based on restriction endonuclease recognition for rapid detection of canine parvovirus and precise genotyping of the 2c variant.

Canine parvovirus (CPV) is a highly contagious virus that primarily infects dogs, posing a significant threat to canine health and wildlife conservation. Among its variants, the 2c type is the predominant circulating strain with important clinical significance. Therefore, rapid detection and precise genotyping of CPV, particularly the 2c variant, are of great importance for disease prevention and control.In this study, we report a nucleic acid detection method termed CEPD, which integrates recombinase-aided amplification with restriction endonuclease recognition by Pyrococcus furiosus argonaute (PfAgo). A one-pot CEPD assay was developed to enable simultaneous detection of CPV and synchronous genotyping of the CPV-2c variant. The analytical limit of detection of the one-pot CEPD assay, determined using serially diluted recombinant plasmids, was 1 copy/μL for both CPV and the CPV-2c variant, without cross-reactivity with other pathogens. The results from clinical sample testing were fully consistent with those obtained by PCR sequencing.These findings demonstrate that the one-pot CEPD assay is convenient, sensitive, and specific, making it suitable for the detection of CPV and the 2c variant, with promising potential for practical applications.