Abstract:[Background] Hematopoietic necrosis caused by cyprinid herpesvirus-2 (CyHV-2) has led to serious economic losses in aquaculture due to its spread in crucian carp, goldfish and other species. [Objective] To develop an on-site colloidal gold test strip for detecting and monitoring the level of IgM antibodies against CyHV-2 in crucian carp. [Methods] CyHV-2 was combined with colloidal gold as the gold-labeled antigen. Protein A was employed as the detection line, and rabbit-derived rORF66 polyclonal antibody as the quality control line. The optimal preparation and assembly conditions of colloidal gold test strips were analyzed, and the optimal pH and concentration of colloidal gold-labeled antigen, as well as the optimal marking concentrations of test line (T line) and quality control line (C line), were determined. [Results] The colloidal gold test strips prepared in this study did not cross-react with other common fish antibody-positive sera, such as grass carp reovirus antibody-positive serum, mandarin fish iridovirus antibody-positive serum, largemouth black bass iridovirus antibody-positive serum, and carp spring viremia virus antibody-positive serum. The test strips can detect serum antibodies at a minimum dilution of 1:100, and the density of the detection line can help roughly judge the antibody level. The test strips were used to detect 10 crucian carp serum samples and compared with the detection method of goldfish haematopoietic necrosis virus (GB/T 36194—2018). The Kappa value was 0.80, indicating a high coincidence rate between the two methods. [Conclusion] The test strips prepared in this study have high sensitivity, high specificity, simple operation, and low cost, demonstrating the potential for fish quarantine and evaluation of fish antibody levels against CyHV-2.
