Establishment of a loop‐mediated isothermal amplification system for on‐site diagnosis of Oryctes rhinoceros nudivirus in Allomyrina dichotoma (Coleoptera: Scarabaeidae)

Abstract

The Korean insect industry is rapidly developing, with the Korean horn beetle Allomyrina dichotoma as one of the most popular insects kept as pets. Korean horn beetles reared in local farms are suffering from Oryctes rhinoceros nudivirus (OrNV). In a nationwide investigation on the early death of young larvae due to this virus, 70 to 77% mortality was found. It was also confirmed that several larvae collected from some wild horn beetle habitats were infected with the virus. Thus, it was concluded that the virus could be transmitted vertically from an infected adult to the offspring and that the OrNV disease is a very serious threat to the wild horn beetle in Korea. With local farmers expecting that an early detection technique would be helpful for the quick removal of infected larvae, an on‐site diagnosis method for the viral disease using loop‐mediated isothermal amplification (LAMP) assay was thus tested. To avoid the DNA extraction process, the LAMP assay used a 50‐fold diluted hemolymph was set for diagnosis standardization and convenient on‐site application to infected larvae from local farms. A. dichotoma larvae were assayed for the OrNV infectivity with LAMP primers targeting the 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. The positive results were detected only from the OrNV‐infected larvae.

A role of mulberry leaves in improving resistance to virus‐mediated disease in Allomyrina dichotoma

“…Viral infection was confirmed through AdNV gene amplification by PCR using following primers; AV‐F1; 5′ ‐ TCC GGA AAT TAC ACG AGC CAC ‐ 3′, and AV‐R1; 5′ ‐ ATG CCG TAC GAG AGT ATA GGT CG ‐ 3′, following the protocol described by Lee et al . ().…”

“…In addition, gDNA was isolated from the midgut and feces or from virus-contaminated sawdust using the DNeasy Blood and Tissue kit (QIAGEN Co., Valencia, CA, USA). Viral infection was confirmed through AdNV gene amplification by PCR using following primers; AV-F1; 5′ -TCC GGA AAT TAC ACG AGC CAC -3′, and AV-R1; 5′ -ATG CCG TAC GAG AGT ATA GGT CG -3′, following the protocol described by Lee et al (2018).…”

“…Research has shown that AdNV (GenBank accession no: KM_233708.1, KM_233709.1, KM_233710.1) isolated from the ROK is 95-99% homologous to Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 from Malaysia (GenBank accession no: EU_747721.1) (Lee et al 2018;Wang et al 2011). However, there has been little research on methods for prevention and control of AdNV infection in the ROK.…”

Exploration of proteomic biomarkers and digital imaging analysis for Oryctes rhinoceros nudivirus infection

“…Nudiviruses are rod‐shaped, enveloped, double‐stranded DNA insect viruses, which infect the larval stages of various insect orders, including Coleoptera, Diptera, Lepidoptera, Orthoptera, as well as marine crustaceans; however, the viruses did not replicate in several insect cells (Petersen et al 2022). Among nudiviruses, Oryctes rhinoceros nudivirus (OrNV), a coleopteran nudivirus, can infect the Korean rhinoceros beetle ( Allomyrina dichotoma ) and pose a threat to the Korean beetle industry, though the source of this virus has not been identified in the area (Lee et al 2019; Lee et al 2015).…”