Development and application of quantitative detection method for nervous necrosis virus (NNV) isolated from sevenband grouper Hyporthodus septemfasciatus

ObjectiveTo develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus.MethodsThe viral genes of the NNV (SGYeosu08) isolated from sevenband grouper were phylogenetically analyzed. In addition, novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method (TCID50) using in vitro and in vivo samples.ResultsThe phylogenetic analysis of viral genes demonstrated the relationship of SGYeosu08 with members of red-spotted grouper nervous necrosis virus (RGNNV). The qNNV_R1 primer set (R1_F and R1_R) and the qNNV_R2 primer set (R2_F and R2_R) revealed 93% primer efficiency (regression: y = −0.2861x + 9.9401, R2 = 0.9976) and the revealed 108% primer efficiency (regression: y = −0.3172x + 10.0611, R2 = 0.9982), respectively. Its comparison with viral infectivity calculated by TCID50 method showed similar kinetic pattern at in vitro and NNV challenged fish (in vivo) samples.ConclusionsResult show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method (TCID50).