Preliminary development of visual and real-time loop-mediated isothermal amplification assays for Megalocytivirus detection in Betta fish (Betta splendens)

This study developed rapid detection of Megalocytivirus using visual and real-time loop-mediated isothermal amplification (LAMP) assays. The primer was designed with the major capsid protein gene of Megalocytivirus, including red sea bream iridovirus (RSIV), infectious spleen and kidney necrosis virus (ISKNV), and turbot reddish body iridovirus (TRBIV). The visual and real-time LAMP assays were evaluated using DNA extracted from symptomatic betta fish that tested negative for Megalocytivirus by the broad-range PCR assay, as well as a chemically synthesized Megalocytivirus DNA template. Visual LAMP was found sensitive enough to detect up to 10 pg per reaction of the target DNA within 30 min. Real-time LAMP efficiently detected 10 ng per reaction of the target DNA in around 10 min. Compared with the broad-range PCR assay, the novel assays demonstrated 100% positive percent agreement when tested with the chemically synthesized Megalocytivirus DNA template and 100% negative percent agreement when evaluated using DNA extracted from symptomatic betta fish that tested negative for Megalocytivirus. The developed LAMP assays when combined with a fast DNA extraction method, could facilitate on-site Megalocytivirus screening.