The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV).
Bluetongue virus (BTV) is an infectious, non-contagious viral disease of domestic and wild ruminants that is transmitted by adult females of certain Culicoides species. Since 2006, several serotypes including BTV-1, 2, 4, 6, 8, 9 and 16, have spread from the Mediterranean basin into Northern Europe for the first time. BTV-8 in particular, caused a major epidemic in northern Europe. As a result, it is evident that most European countries are at risk of BTV infection. The objective of this study was to develop and validate a real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assay based on TaqMan technology for the detection of representative strains of all BTV serotypes. Primers and probes were based on genome segment 10 of the virus, the NS3 gene. The assay was tested for sensitivity, and specificity. The analytical sensitivity of the rRT-PCR assay was 200 copies of RNA per reaction. The assay did not amplify the closely related orbivirus epizootic hemorrhagic disease virus (EHDV) but successfully detected all BTV reference strains including clinical samples from animals experimentally infected with BTV-8. This real time RT-PCR assay offers a sensitive, specific and rapid alternative assay for the pan detection of BTV that could be used as part of a panel of diagnostic assays for the detection of all serotypes of BTV.
Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK; Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Ireland. Electronic address: catherine.mulholland@list.lu.
Catherine
C
Mulholland
Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
Michael J
MJ
McMenamy
Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
Bernd
B
Hoffmann
Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany Co. Meath, Ireland.
Bernadette
B
Earley
Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Ireland.
Bryan
B
Markey
Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Ireland.
Joseph
J
Cassidy
Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
Gordon
G
Allan
Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
Michael D
MD
Welsh
Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
John
J
McKillen
D000818:Animals; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers; D015342:DNA Probes; D057230:Limit of Detection; D025202:Molecular Diagnostic Techniques / Q000379:methods; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D012756:Sheep; D012757:Sheep Diseases / Q000175:diagnosis / Q000821:virology
D017931:DNA Primers; D015342:DNA Probes; D012367:RNA, Viral
BTV; Real-time RT-PCR; TaqMan