Abstract
Bluetongue is an economically important disease of ruminants caused by the bluetongue virus (BTV). BTV is serologically diverse, which complicates vaccination strategies. Rapid identification of the causative BTV serotypes is critical, however, real-time PCR (RT-qPCR) can be costly and time consuming to perform when the circulating serotypes are unknown. The Luminex xMAP technology is a high-throughput platform that uses fluorescent beads to detect multiple targets simultaneously. We utilized existing BTV serotyping RT-qPCR assays for BTV-1 to BTV-24 and adapted them for use with the xMAP platform. The xMAP assay specifically detected all 24 BTV serotypes when testing reference strains. In all BTV-positive samples, the sensitivity of the BTV xMAP was 87.55% whereas the sensitivity of the serotype-specific RT-qPCR was 79.85%. The BTV xMAP assay allowed for the specific detection of BTV serotypes 1–24 at a lower cost than current RT-qPCR assays. Overall, the assay provides a useful novel diagnostic tool, particularly when analyzing large sample sets. The use of the BTV xMAP assay will allow for the rapid assessment of BTV epidemiology and may inform decision-making related to control and prevention measures.
Highlights
Bluetongue (BT) is a world Organization for Animal Health (OIE) notifiable disease of ruminants
Published primers [34] were used in the RT-PCR step of the BT virus (BTV) xMAP assay, one exception was the BTV-24 forward primer, which was lengthened by 3 nucleotides to increase the primer melting temperature (Table S1)
The development of a high-throughput, multiplex assay for serotyping BTV would provide a cost-effective means of identifying the true picture of BTV epidemiology and may go towards the deployment of appropriate control strategies
Summary
Bluetongue (BT) is a world Organization for Animal Health (OIE) notifiable disease of ruminants. Acute clinical disease is mainly seen in sheep, with cattle thought to be the reservoir host; goats and some deer species can be infected [1,2]. The BTV genome consists of ten segments of linear double-stranded RNA that encode for a number of structural (VP1-VP7) and non-structural (NS1-NS5 and NS3a) viral proteins [4,5]. The structural protein VP2, encoded by segment 2, shows the highest degree of variation and is the main determinant of BTV serotype [6]. Until 2008, 24 BTV serotypes had been detected based on interactions between VP2 and neutralizing antibodies in the ruminant host. Since that time, a number of additional BTV serotypes (of reduced virulence) have been detected, which include serotypes 25 to 29 [7,8,9,10], in addition to the as-yet unclassified BTV strains TUN2017, BTV-X ITL2015, V196/XJ/2014, and SPvvvv/02 [11,12,13,14]
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