Development and application of quantitative detection method for nervous necrosis virus(NNV) isolate

来源 :Asian Pacific Journal of Tropical Medicine | 被引量 : 0次 | 上传用户:snailswuya
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Objective:To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from sevenband grouper Hyporhodus septemfasciatus.Methods:The 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(TCID_(50)) using in vitro and in vivo samples.Results:The phylogenetic analysis of viral genes demonstrated the relationship of SGYeosu08 with members of red-spotted grouper nervous necrosis virus(RGNNV).The qNNV_Rl primer set(R1_F and R1_R) and the qNNV_R2 primer set(R2_F and R2_R) revealed 93%primer efficiency(regression:y=-0.2861 x + 9.9401,R~2= 0.9976)and the revealed 108%primer efficiency(regression:y=-0.3172 x + 10.0611,R~2= 0.9982),respectively.Its comparison with viral infectivity calculated by TCID_(50) method showed similar kinetic pattern at in vitro and NNV challenged fish(in vivo) samples.Conclusions:Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method(TCID_(50)). Objective: To develop the rapid and efficient quantitative detection tool for nervous necrosis virus isolated from seven band grouper Hyporhodus septemfasciatus. Methods: The viral genes of the NNV (SGYeosu08) isolated from seven band grouper were phylogenetically analyzed. Addition, novel quantitative PCR primers based on the genomic sequence of SGYeosu08 isolate were designed and compared it with the conventional bio-assay method (TCID 50) using in vitro and in vivo samples. Results: The phylogenetic analysis of viral genes demonstrated the relationship of SG Yeosu 08 with members of red- The results showed that qNNV_Rl primer set (R1_F and R1_R) and the qNNV_R2 primer set (R2_F and R2_R) revealed 93% primer efficiency (regression: y = -0.2861 x + 9.9401, R ~ 2 = 0.9976) and the revealed 108% primer efficiency (regression: y = -0.3172 x + 10.0611, R ~ 2 = 0.9982), respectively.Its comparison with viral infectivity calculated by TCID_ (50) method showed similar kinetic pattern at in vitro a nd NNV challenged fish (in vivo) samples.Conclusions: Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method (TCID 50).
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