Development and Evaluation of a Loop-mediated Isothermal Amplification(LAMP) Assay for Rapid Detection of Chinese Giant Salamander Ranavirus

Loop-mediated isothermal amplification(LAMP) is a novel nucleic acid diagnostic method that can amplify rapidly a target template under isothermal conditions. In this study, a LAMP assay for rapid detection of Chinese giant salamander ranavirus(CGSRV) was developed from culture isolates and clinical samples. The LAMP assay was developed by designing one set of four specific primers, targeting the major capsid protein(MCP) gene of CGSRV. Reaction time and temperature were optimal for 40 min at 62°C. The developed LAMP assay is specific and highly sensitive for CGSRV detection, the detection limit could reach about 5 copies of cloned viral genomic fragments. The sensitivity of the LAMP assay was about 1000 and 10-fold higher than that of both conventional and nested PCR, respectively. The LAMP amplification produces a typical ladder-like pattern of products on an agarose gel that can be visually inspected after addition of ethidium bromide. The LAMP assay was evaluated further with clinical samples, and the results indicated the suitability and simplicity of the test as a rapid diagnostic tool for the detection of CGSRV.