High Throughput Detection of Bluetongue Virus by a New Real-Time Fluorogenic Reverse Transcription—Polymerase Chain Reaction: Application on Clinical Samples from Current Mediterranean Outbreaks

Abstract

Abstract.A real-time reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of bluetongue virus (BTV) in blood samples. A combination of primers specific for a highly conserved region in RNA segment 5 (based on Mediterranean BTV sequences) and a DNA probe bound to 5Ј-Taq nuclease-3Ј minor groove binder (TaqMan᭧ MGB) was used to detect a range of isolates. This real-time RT-PCR assay could detect 5.4 ϫ 10 Ϫ3 tissue culture infectious doses (TCID 50 ) of virus per milliliter of sample, which was comparable to our current BTV diagnostic nested RT-PCR assay. The assay detected all recent Mediterranean isolates (including serotypes 2, 4, and 16), BTV vaccine strains for serotypes 2 and 4, and 15 out of the 24 BTV reference strains available (all serotypes), but did not detect the related orbiviruses epizootic hemorrhagic disease and African horse sickness viruses. Following assay evaluation, the ability of this assay to identify BTV in recent isolates (2003, 2004) from ovine and bovine samples from an epizootic outbreak in Spain was also tested. Minor nucleotide changes (detected by sequencing viral genomes) within the probebinding region were found to have a profound effect on virus detection. This assay has the benefits of being fast and simple, and the 96-well format enables large-scale epidemiological screening for BTV, especially when combined with a high-throughput nucleic acid extraction method.

A Multiplex Real-Time Reverse Transcription Polymerase Chain Reaction Assay for Detection and Differentiation ofBluetongue VirusandEpizootic Hemorrhagic Disease VirusSerogroups

“…12 A number of real-time RT-PCR assays were recently developed for BTV. 7,14,29,32,36 The genome segments are commonly denoted as L1-3, M4-6, and S7-10, based on sequence size, similar to the Reoviridae nomenclature. The coding assignments are based on sequence size and protein produced, with structural proteins denoted as VP1-7 and nonstructural protein denoted as NS1-3.…”

Serotype-specific detection of African horsesickness virus by real-time PCR and the influence of genetic variations

“…This is in the same order of magnitude as that of the reverse blot serotype-specific hybridization (Koekemoer and Van Dijk, 2004) and the RT-PCR test that uses serotype-specific primers (Sailleau et al, 2000). Sensitivity is less than what is reported for the group-specific real-time tests for AHSV (Agüero et al, 2008) and BTV (Jiménez-Clavero et al, 2006). These methods, however, make use of more sensitive 5 -Taq nuclease-3 -minor groove binder-DNA probes that hybridize to smaller amplification products.…”

“…From previous sequence analyses of genome segment 2 of AHSV 7 (Koekemoer et al, 2003) and AHSV 2 (unpublished data) field isolates, these variations were known to be a possibility but their numerous appearance in the short (42-51 bp) hybridization probe targets was not predicted. Other workers have reported similar problems with BTV identification using real-time PCR (Jiménez-Clavero et al, 2006) and this phenomenon is something that is warned against when using melting curve analysis as a diagnostic tool (Whiley and Sloots, 2005). Sequence variation in the probe target site occurred in 10 out of 48 bases when comparing the reference and one of the field isolates of AHSV 9 (Fig.…”

Diagnostic evaluation of a multiplexed RT-PCR microsphere array assay for the detection of foot-and-mouth and look-alike disease viruses

“…rRT-PCR assays have also been reported for the detection of other viruses which cause vesicular disease of livestock including swine vesicular disease (SVD) (29), vesicular stomatitis (VS) (11,28) and vesicular exanthema of swine (VES) (31) or symptomatic look-alike diseases including bluetongue (13,27,33), bovine viral diarrhea…”