Development of a novel droplet digital PCR assay for the sensitive detection of carp edema virus

Abstract

Carp edema virus (CEV) is a poxvirus associated with outbreaks of clinical disease that may lead to high mortality in both koi and common carp populations, potentially causing severe economic losses for carp aquaculture and the global koi trade. Therefore, the establishment of a sensitive and specific assay for detection of CEV is an essential requirement for the prevention of further spread of this emerging viral disease. In this study, a novel droplet-digital PCR (ddPCR) assay, targeting the p4a gene of CEV was developed. The assay exhibits good linearity, repeatability and reproducibility. Linearity was maintained at extremely low concentrations of CEV nucleic acid templates. The detection limit of ddPCR was 2.2 ± 0.26 copies of CEV DNA per reaction (n = 8), which represents a greater sensitivity than the quantitative PCR (qPCR) assay. Specificity analysis showed that the ddPCR assay for CEV had no cross-reactivity with other important aquatic pathogens. In clinical diagnosis of 151 koi and common-carp samples, 58 and 45 tested positively by ddPCR and qPCR assays, respectively. The overall agreement between the two assays was 91.39% (138/151). Linear regression analysis demonstrated a significant correlation between ddPCR and qPCR results with an R2 value of 0.9627. Our findings indicate that the ddPCR assay could provide a robust diagnostic tool for the sensitive detection of CEV, even in samples with a low viral load.