Recombinase polymerase amplification-based rapid detection of Oryctes rhinoceros nudivirus (OrNV) infection in coconut rhinoceros beetles

The coconut rhinoceros beetle (CRB) is an important pest of palms in the Asia/Pacific regions. A natural pathogen of CRB, Oryctes rhinoceros nudivirus (OrNV) has successfully been introduced into virus-free, invasive CRB populations in many Pacific islands leading to pest suppression. While PCR is currently used for virus monitoring, a more portable, simplified in-field method of virus detection would be helpful. Here, we developed a Recombinase Polymerase Amplification (RPA)-based assay to detect the partial gp54 gene of OrNV (OrNV-RPA) using fluorescence-based chemistry. The assay has a high degree of specificity with no cross-reactivity to the genomic DNA of various insects, soil, and bacterial species commonly found in the environment. Additionally, the assay has a high level of sensitivity with the ability to detect approximately 1610 copies of the gp54 gene (15 min reaction). OrNV-RPA assay results showed high consistency with the current standard PCR-based assay, whether using purified DNA (95.4%) or crude lysate (93.5%) from CRB midgut tissue, validating the ability of the OrNV-RPA assay to detect virus-free and virus-infected CRB specimens. The newly developed RPA-based assay provides an option for rapid in-field detection of OrNV to help improve CRB management in the Pacific Islands and other regions.