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Establishment of a loop-mediated isothermal amplification system for on-site diagnosis of viral disease in Allomyrina dichotoma
Korean horn beetles, Allomyrina dichotoma (Linnaeus, 1771) reared in many local farms are suffering from a fatal viral disease as reported in 2015, and recent sequencing analysis revealed that the virus is very closely related to Oryctes rhinoceros nudivirus. In a nationwide investigation, it was concluded that the virus can transmit vertically from an infected adult to the offspring by indirect manner, and 70 to 77% of young larvae dead in early stage were diagnosed as this virus positive. Here, we report for the establishment of on-site diagnosis method for the viral disease using loop-mediated isothermal amplification (LAMP) assay. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting OrNV_gp102 gene. To evaluate the LAMP specificity, two bacterial pathogens, Bacillus thuringiensis and Serratia marcescens, causing disease in A. dichotoma were tested along with OrNV. Only from the OrNV-infected larvae the reaction resulted positive. Also, to avoid DNA extraction process, the LAMP assay used diluted hemolymph directly and 50-fold dilution was set for diagnosis standardization and convenient on-site application for infected larvae from local farms. The LAMP diagnosis is fast and convenient for nontechnical user in the field and expected to stop this foreign virus spread all over the country.
