Development And Application Of Loop Mediated Isothermal Amplification Assay For Aquatic Animal Diseases

Objective:Basing on the differences of six viruses, red sea bream iridovirus (RSFV), koi herpesvirus (KHV), epizootic haematopoietic necrosis virus (EHNV), spring viremia virus disease virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and hemorrhagic disease virus (VHSV), we’ve established the multiple Loop mediated isothermal amplification assay in this work. Multiple LAMP assay can detect different pathogenic synchronous in the same host, which can save detection time and cost, greatly.Method:In the light of the specificity of the six different viruses, six LAMP assays for detection of them were established, optimized the reaction conditions and verified sensitivity and specificity. Multiple LAMP assay was established by assembling unitary LAMP assays to detect mixed pathogens simultaneously, and therefore we could test specificity and sensitivity of this method. The specificity of the method was further verified by enzyme digestion..Result:The detection limit of LAMP of RSIV, KHV, EHNV, SVCV, IHNV and VHSV were 100 copies/μl, 10copies/μl, 1TCID50,10 copies/μl, 1TCID50,ITCID50. These methods could not crosswise react with other important aquatic animal disease pathogens crooswise, which illustrated that these LAMP detection methods possessed specificity. The detection limit of the study two established method is 100copies/μL. Such a method could not react with other important aquatic animal disease pathogens crosswise, which displays its specificity of the method. However, the multiple LAMP assay is lower than unitary LAMP method in sensitivity. The detection limit of the study three established method was 1TID50. Such a method could not react with other important aquatic animal disease pathogens crosswise, which displays its specificity of the method. The products of LAMP were digested by specific restriction enzyme, and the results showed that the specific detection could be achieved by the restriction of the fragment of the enzyme.Conclusion:Multiple LAMP assay was established by assembling unitary LAMP assays to detect mixed pathogens simultaneously, the pathogens were detected simultaneously from the same host.