title: The development of an accelerated reverse-transcription loop mediated isothermal amplification for the serotype specific detection of bluetongue virus 8 in clinical samples.
abstract: In 2006 bluetongue virus serotype 8 (BTV 8) was identified for the first time in the Netherlands causing a major epidemic in sheep and cattle that quickly spread to neighbouring Belgium, Germany and beyond to France and the UK. This resulted in severe animal health and welfare problems as well as substantial economic losses to the agrifood industries of these countries. Given that the early diagnosis of BTV infection 'in-the-field' is extremely useful to its subsequent management and control, this study was established to design a novel, sensitive and rapid nucleic acid diagnostic test for the serotype-specific detection of BTV 8, which could be used without the use of advanced laboratory support and equipment. Primers for the detection of BTV 8 were based on genome segment 2 of the virus, the VP2 gene. The assay was assessed using a full panel of BTV reference strains and clinical samples. Positive amplification was observed using a fluorescent detection reagent. The sensitivity of the RT-LAMP assay was 102 copies of RNA. The assay did not amplify the closely related orbivirus EHDV. This novel RT-LAMP offers a sensitive, specific and rapid method of detecting BTV 8. The approach is inexpensive and easy to use and could potentially be used in a 'pen-side' setting 'in the field' or by smaller less well-equipped laboratories in developing countries.
authors.affiliation: Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK; Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland; School of Veterinary Medicine, Veterinary Sciences Centre, University College Dublin, Belfield, Ireland. Electronic address: catherine.mulholland@afbini.gov.uk.
authors.forename: Catherine
authors.initials: C
authors.lastname: Mulholland
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Hoffmann
authors.affiliation: Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: McMenamy
authors.affiliation: Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald-Insel Riems, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Korthase
authors.affiliation: Animal and Bioscience Research Department, Animal & Grassland Research and Innovation Centre, Teagasc, Grange, Dunsany, Co. Meath, Ireland.
authors.forename: Bernadette
authors.initials: B
authors.lastname: Earley
authors.affiliation: School of Veterinary Medicine, Veterinary Sciences Centre, University College Dublin, Belfield, Ireland.
authors.forename: Bryan
authors.initials: B
authors.lastname: Markey
authors.affiliation: School of Veterinary Medicine, Veterinary Sciences Centre, University College Dublin, Belfield, Ireland.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Cassidy
authors.affiliation: Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
authors.forename: John
authors.initials: J
authors.lastname: McKillen
authors.affiliation: Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
authors.forename: Gordon
authors.initials: G
authors.lastname: Allan
authors.affiliation: Agri Food Biosciences Institute, Veterinary Sciences Division, Stoney Road, Stormont, Belfast BT4 3SD, UK.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Welsh
mesh_terms: D000818:Animals; D001819:Bluetongue / Q000175:diagnosis* / Q000821:virology; D001820:Bluetongue virus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D060005:Genotyping Techniques / Q000379:methods*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012680:Sensitivity and Specificity; D012756:Sheep; D014730:Veterinary Medicine / Q000379:methods*; D014773:Virology / Q000379:methods
chemical_list: D017931:DNA Primers
keywords: BTV; BTV 8; Detection; Isothermal amplification; LAMP