Rapid detection of Akabane virus by a novel reverse transcription loop-mediated isothermal amplification assay (RT-LAMP)

Jun Qiao,Guochao Wang,Haibo Yang,Yucheng Liu,Chuangfu Chen,Qingling Meng,Xuepeng Cai,Zhihao He,Zaichao Zhang,Junwei Wang

doi:10.1186/1743-422x-10-288

Abstract

BackgroundAkabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage.Methods and resultsIn this study, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid detection of Akabane virus was successfully developed. The primers were designed to target the highly conserved fragment of nucleoprotein from the Akabane virus. The results indicate that the assay is highly specific and sensitive with a detection limit of 5.0 TCID50 /mL within a 60-min incubation time. A total of 126 abortive samples collected from Xinjiang province were detected by the established RT-LAMP. The results of RT-LAMP assay showed 96.8% agreement with the semi-nested RT-PCR.ConclusionThis study is to first to develop a rapid, sensitive, and accurate method for the detection of Akabane virus, which may be used to screen clinical samples in developing countries or regions.

Highlights

Akabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage
The results demonstrated that only Akabane virus was detectable by reverse transcription loop-mediated isothermal amplification (RT-loop-mediated isothermal amplification (LAMP)) assay (Figure 1)
The results showed that the RT-LAMP assay is highly sensitive as it can detect a template at 10 –5 dilution with a detection limit of 5.0 Median tissue culture infective dose (TCID50)/mL (Figure 3)

Summary

Introduction

Akabane disease, caused by Akabane virus, is an insect-transmitted disease of ruminants that is primarily characterized by fetal damage. The causative agent of enzootic bovine arthrogryposis and hydranencephaly, is an arbovirus in the genus Orthobunyavirus and the Simbu sero-group of the family Bunyaviridae. This particular virus is transmitted among animals by hematophagous arthropod vectors such as mosquitoes and culicoides biting midges [1]. Laboratory diagnostic techniques for Akabane disease such as viral neutralization test (VNT), enzymelinked immunosorbent assays (ELISAs) [9-12], immunoperoxidase staining [13,14], dot immunobinding assay [15], nested reverse transcription polymerase chain reaction (RT-PCR) [16], multiplex RT-PCR [17] and real-time RT-PCR [18] have been established, most of the current serological tests have cross-reactions with other related viruses, those in the simbu serogroup [9,15,19]. It is necessary to develop new assays for the detection of Akabane virus in the clinic

Objectives

Methods

Results