Molecular detection of genotype II grass carp reovirus based on nucleic acid sequence-based amplification combined with enzyme-linked immunosorbent assay (NASBA-ELISA)

Grass carp reovirus (GCRV) is the causative agent of the grass carp hemorrhagic disease that has resulted in severe economic losses in the grass carp (Ctenopharyngodon idella) farming industry in China. Early diagnosis and vaccine administration are important priorities for GCRV control. In this study, a nucleic acid sequence-based amplification with enzyme-linked immunosorbent assay (NASBA-ELISA) was developed for to detect genotype II GCRV (GCRV- II). Primers specifically targeting viral RNA genome segment 6 were utilized for amplification in an isothermal digoxigenin-labeling NASBA process, resulting in DIG-labeled RNA amplicons. The amplicons were hybridized to specific biotinylated DNA probes and the products were detected colorimetrically using horseradish peroxidase and a microplate reader. The new method is able to detect GCRV at 14 copies/μL within 5 h and had a diagnostic sensitivity and a specificity of 100% when GCRV-II and non-target virus were tested. This NASBA-ELISA was evaluated using a panel of clinical samples (n = 103) to demonstrate that it is a rapid, effective and sensitive method for GCRV detection in grass carp aquaculture.