Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus

Abstract

SummaryAfrican horse sickness (AHS) is a disease of equids caused by African Horse Sickness Virus (AHSV) and is transmitted by Culicoides midges. AHS is endemic in sub‐Saharan Africa, but during the past century, outbreaks of significant economic importance and elevated mortality have been recorded in Northern African countries, the Iberian and Arabian Peninsula, the Middle East and the Indian subcontinent. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Conventional reverse‐transcriptase (RT) PCR (RT‐PCR) and real‐time RT‐PCR (rRT‐PCR) assays have improved the sensitivity and rapidity of diagnosing AHS, resulting in the adoption of these methods as recommended tests by the World Organisation for Animal Health (OIE). However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for AHS would improve the fast implementation of control policies.Loop‐mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand‐displacement nucleic acid amplification technique which can be performed in the field. LAMP assays are attractive molecular assays because they are simple to use, rapid, portable and have sensitivity and specificity within the range of rRT‐PCR. This study describes the development of a novel RT‐LAMP assay for the detection of AHSV. The AHSV RT‐LAMP assay has an analytical sensitivity of 96.1% when considering an rRT‐PCR cut‐off value of C T > 36, or 91.3% when no rRT‐PCR cut‐off is applied. Diagnostic sensitivity and specificity were 100%. This assay provides for a rapid and low cost AHS diagnostic for use in the field.

A rapid RT-LAMP assay for the detection of all four lineages of Peste des Petits Ruminants Virus

“…Furthermore, LAMP chemistry is more resistant to inhibitors than PCR, enabling simplification of extraction procedures (Blomstrom et al, 2008; Waters et al, 2014; Howson et al, 2017). As such, RT-LAMP assays for a number of RNA viruses including bluetongue virus (Maan et al, 2016; Mohandas et al, 2015) and African horse sickness virus (Fowler et al, 2017) have been developed. An RT-LAMP assay for PPRV detection was first developed in 2010 (Li et al, 2010), but it was validated on a small sample set and the assay requires one hour incubation under isothermal conditions.…”

Awareness of African Horse Sickness as an Emerging Arbovirus Disease

“…Metode diagnosis cepat untuk AHS telah dikembangkan dengan menggunakan uji reverse transcription loop-mediated isothermal amplification assay (RT-LAMP). Metode ini lebih sederhana, mudah digunakan dan dapat diaplikasikan di lapang, cepat, sensitif dan spesifik (Fowler et al 2016). Hasil positif pada PCR dapat dilanjutkan dengan sekuensing dengan menggunakan gen AHS lengkap untuk mengetahui originalitas strain isolat yang diperoleh dan serotipe virus AHS (Bachanek-Bankowska et al 2014;Potgieter et al 2015).…”

Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme

“…Loop-mediated isothermal amplification (LAMP) is an isothermal, autocyling, strand-displacement DNA amplification technique [10] that can be performed at a constant temperature. Numerous LAMP assays have been developed for human, plant and veterinary pathogens including paramyxoviruses [11,12,13,14,15,16,17]. In case of PPRV, an RT-LAMP assay based on the M-protein gene [16] and another based on the N-protein gene [11] have been developed; however, they are not known to be extensively used in the field.…”