Abstract
Spring viremia of carp (SVC), caused by spring viremia of carp virus (SVCV), is a highly contagious disease that poses a serious threat to cyprinid aquaculture and international trade, and it is listed as a notifiable disease by the World Organization for Animal Health (WOAH). Effective surveillance and control of SVCV rely on accurate and highly sensitive molecular diagnostic methods. However, several previously published RT-qPCR assays contain mismatches between primer/probe sequences and viral genomes, which may lead to false-negative results and reduced diagnostic reliability. In this study, a whole-genome comparison of 24 representative SVCV strains covering all four genotypes (SVCVa-d) was conducted, and a new primer-probe set (Cefas AR) targeting a highly conserved region of the L gene was designed. Reaction conditions were optimized, and the assay was rigorously validated in accordance with the WOAH Manual of Diagnostic Tests for Aquatic Animals. The developed RT-qPCR assay exhibited excellent analytical performance, with a limit of detection of 1.28 copies/μL, diagnostic sensitivities of 100% for cell-culture isolates and 96.6% for tissue samples, and a diagnostic specificity of 100%. In addition, the assay demonstrated strong reproducibility and consistency across nine independent laboratories. In conclusion, the WOAH-validated RT-qPCR assay developed in this study provides a highly sensitive, specific, and reliable tool for rapid screening, routine surveillance, and confirmatory diagnosis of SVCV, supporting sustainable aquaculture development and international aquatic animal health management.