Development of a Sensitive Real-Time PCR Method for Detection of Carp Oedema Virus

Carp oedema virus disease (CEVD), caused by the carp oedema virus (CEV), results in significant economic losses to the common carp and koi aquaculture industry. The CEVD symptoms resemble those of koi herpesvirus (KHV) and include lethargy, swollen gills, sunken eyes, and skin haemorrhages. Consequently, the development of efficient detection methods is imperative for the prevention and diagnosis of CEVD. In this study, we developed a quantitative real-time PCR assay for the sensitive detection of CEV infection. Specific primers were designed based on a highly conserved region within the CEV p4a gene. The assay exhibited a sensitivity that was 100-fold greater than that of the conventional PCR technique, with high specificity, and no observed cross-reactivity with infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), spring viremia of carp virus (SVCV), salmonid alphavirus (SAV), or KHV. Inter- and intra-assay experiments also confirmed the method's high repeatability. In a clinical setting, it showed higher sensitivity compared to the diagnostic methods for CEVD developed by the Center for Environment, Fisheries and Aquaculture Science (CEFAS), while also significantly reducing detection costs. Consequently, the established assay could provide robust technical support for the clinical diagnosis and epidemiological investigation of CEVD.