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OBJECTIVE@#To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection.@*METHODS@#A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus.@*RESULTS@#The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus.@*CONCLUSIONS@#The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.
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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 (TCID50) 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_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, R(2) = 0.9976) and the revealed 108% primer efficiency (regression: y = -0.3172x + 10.0611, R(2) = 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.@*CONCLUSIONS@#Result show that this method is rapid and efficient to diagnose NNV infection compare to traditional bioassay method (TCID50).
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Objective: To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. Methods: A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. Results: The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. Conclusions: The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.
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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
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The present study was performed to trace the decisive evidence for mixed infection of 2 Myxobolus species, M. episquamalis and Myxobolus sp., in the gray mullet, Mugil cephalus, from Korean waters. Mullets with whitish cyst-like plasmodia on their scales were collected near a sewage plant in Yeosu, southern part of Korea, in 2009. The cysts were mainly located on scales and also found in the intestine. The spores from scales were oval in a frontal view, tapering anteriorly to a blunt apex, and measured 7.2 microm (5.8-8.0) in length and 5.3 microm (4.7-6.1) in width. Two polar capsules were pyriform and extended over the anterior half of the spore, measuring 3.5 microm (2.3-4.8) in length and 2.0 microm (1.5-2.2) in width. In contrast, the spores from the intestine were ellipsoidal, 10.4 microm (9.0-11.9) in length and 8.4 microm (7.3-10.1) in width. The polar capsules were pyriform but did not extend over the anterior half of the spore, 3.7 microm (2.5-4.5) in length and 2.2 microm (1.8-2.9) in width. The nucleotide sequences of the 18S rDNA gene of the 2 myxosporean spores from scales and intestine showed 88.1% identity to each other and 100% identity with M. episquamalis and 94.5% identity with M. spinacurvatura from mullet, respectively. By the above findings, it is first confirmed that mullets from the Korean water are infected with 2 myxosporean species, M. episquamalis and Myxobolus sp.