Development of a Novel Loop Mediated Isothermal Amplification Assay (LAMP) for the Rapid Detection of Epizootic Haemorrhagic Disease Virus

• Published in: Viruses 2021-10, Vol.13 (11), p.2187
• Main Authors: Rajko-Nenow, Paulina, Howson, Emma L A, Clark, Duncan, Hilton, Natasha, Ambagala, Aruna, Svitek, Nicholas, Flannery, John, Batten, Carrie
• Format: Article
• Language: English
• Subjects: AB 7500 fast instrument; Animals; Automation; Bluetongue; Bluetongue - virology; Cattle; Cell culture; Deer; Design; Disease; DNA Primers - genetics; EHDV; Epidemics; Gene amplification; Genomes; Hemorrhagic disease; Hemorrhagic Disease Virus, Epizootic - isolation & purification; High-throughput screening; Laboratories; Molecular Diagnostic Techniques - methods; multiplex LAMP; Nucleic Acid Amplification Techniques - methods; rapid diagnostics; Reagents; Real-Time Polymerase Chain Reaction; Reoviridae Infections - diagnosis; Reoviridae Infections - virology; Reverse Transcription; RNA, Viral - genetics; RT-LAMP; Sensitivity and Specificity; Severe acute respiratory syndrome coronavirus 2; Viruses
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v13112187

Epizootic haemorragic disease (EHD) is an important disease of white-tailed deer and can cause a bluetongue-like illness in cattle. A definitive diagnosis of EHD relies on molecular assays such as real-time RT-qPCR or conventional PCR. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a cost-effective, specific, and sensitive technique that provides an alternative to RT-qPCR. We designed two sets of specific primers targeting segment-9 of the EHD virus genome to enable the detection of western and eastern topotypes, and evaluated their performance in singleplex and multiplex formats using cell culture isolates (n = 43), field specimens (n = 20), and a proficiency panel (n = 10). The limit of detection of the eastern and western RT-LAMP assays was estimated as ~24.36 C and as ~29.37 C in relation to real-time RT-qPCR, respectively, indicating a greater sensitivity of the western topotype singleplex RT-LAMP. The sensitivity of the western topotype RT-LAMP assay, relative to the RT-qPCR assay, was 72.2%, indicating that it could be theoretically used to detect viraemic cervines and bovines. For the first time, an RT-LAMP assay was developed for the rapid detection of the EHD virus that could be used as either a field test or high throughput screening tool in established laboratories to control the spread of EHD.