Rapid and sensitivie detection of iridovirus by loop-mediated isothermal amplification (LAMP)

Abstract

A loop-mediated isothermal amplification (LAMP) assay is an established nucleic acid amplification method providing sensitive, rapid diagnosis of infectious diseases. Red seabream iridovirus (RSIV), which belongs to the iridoviridae family, have been responsible for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economical losses on a fish farm. The rapid molecular detection either through surveillance or diagnosis of RSIV has been considered mainly as a critical feature in responding and resolving the prevalence of RSIV infection. In the present study, with RSIV DNA sequence obtained from NCBI database, not samples of the infected fish, we developed a novel and highly specific LAMP assay for sensitive and rapid detection of RSIV infection. Using a set of the synthesized primers matching the specific region of the RSIV genome, the efficiency and specificity of LAMP assay on RSIV gene sequence were optimized in terms of the concentration of DNA polymerase and reaction temperature, showing that they act as main determinants in sensitivity and specificity in RAMP assay. In particular, we demonstrated that they were even applied to detect RSIV infection in red sea bream efficiently. These data suggest that the development of RAMP primers based on genetic information provided by public database, not the infected sample, can provide very simple and convenient method have been responsible for some of the most explosive epidemics of emerging viral diseases in many Asian countries, leading to huge economical losses on a fish farm. The rapid molecular detection either through surveillance or diagnosis of RSIV has been considered mainly as a critical feature in responding and resolving the prevalence of RSIV infection. In the present study, with RSIV DNA sequence obtained from NCBI database, not samples of the infected fish, we developed a novel and highly specific LAMP assay for sensitive and rapid detection of RSIV infection. Using a set of the synthesized primers matching the specific region of the RSIV genome, the efficiency and specificity of LAMP assay on RSIV gene sequence were optimized in terms of the concentration of DNA polymerase and reaction temperature, showing that they act as main determinants in sensitivity and specificity in RAMP assay. In particular, we demonstrated that they were even applied to detect RSIV infection in red sea bream efficiently. These data suggest that the development of RAMP primers based on genetic information provided by public database, not the infected sample, can provide very simple and convenient method