title: A Rapid Field-Deployable Reverse Transcription-Insulated Isothermal Polymerase Chain Reaction Assay for Sensitive and Specific Detection of Bluetongue Virus.
abstract: Bluetongue is a non-contagious, haemorrhagic, Culicoides-borne disease of ruminants. The causative agent, bluetongue virus (BTV), is a member of the Orbivirus genus of the Reoviridae family. So far, 26 BTV serotypes have been identified worldwide. The global distribution of bluetongue has been expanding, and rapid detection of BTV, preferably in the field, is critical for timely implementation of animal movement restrictions and vector control measures. To date, many laboratory-based, molecular assays for detection of BTV have been developed. These methods require the samples to be shipped to a central laboratory with sophisticated instruments and highly skilled technicians to perform the assays, conduct analyses and interpret the results. Here, we report the development and evaluation of a rapid, portable, user-friendly, pan-BTV reverse transcription-insulated isothermal polymerase chain reaction (RT-iiPCR) assay that can potentially be used in low-resource field conditions. The total length of the assay was <60 min, and at the end of the assay, the results were automatically displayed as '+' or '-' without the need for data interpretation. The RT-iiPCR assay detected 36 BTV isolates and two in vitro transcribed RNA samples representing all 26 BTV serotypes. The assay did not cross-react with other animal viruses tested, including two closely related orbiviruses. The analytical sensitivity of the assay was as low as nine copies of in vitro transcribed double-stranded BTV RNA. Analysis of BTV-infected whole blood samples showed that the BTV RT-iiPCR assay was as sensitive as real-time RT-PCR. The assay can potentially be used for rapid screening of animals for BTV in routine diagnostics and for monitoring bluetongue outbreaks both in ruminants and in Culicoides vectors in the field and in the laboratory.
authors.affiliation: National Centres for Animal Disease, Lethbridge Laboratory, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: A
authors.initials: A
authors.lastname: Ambagala
authors.affiliation: National Centres for Animal Disease, Lethbridge Laboratory, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Pahari
authors.affiliation: National Centres for Animal Disease, Lethbridge Laboratory, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: M
authors.initials: M
authors.lastname: Fisher
authors.affiliation: Department of Research and Development, GeneReach USA, Lexington, MA, USA.
authors.forename: P-Y A
authors.initials: PA
authors.lastname: Lee
authors.affiliation: National Centres for Animal Disease, Winnipeg Laboratory, Canadian Food Inspection Agency, Winnipeg, MB, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Pasick
authors.affiliation: Diagnostic Virology Laboratory, National Veterinary Services Laboratories, STAS, APHIS, VS, USDA, Ames, IA, USA.
authors.forename: E N
authors.initials: EN
authors.lastname: Ostlund
authors.affiliation: Diagnostic Virology Laboratory, National Veterinary Services Laboratories, STAS, APHIS, VS, USDA, Ames, IA, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Johnson
authors.affiliation: National Centres for Animal Disease, Lethbridge Laboratory, Canadian Food Inspection Agency, Lethbridge, AB, Canada.
authors.forename: O
authors.initials: O
authors.lastname: Lung
mesh_terms: D000818:Animals; D001820:Bluetongue virus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012418:Ruminants; D012680:Sensitivity and Specificity
chemical_list: D012367:RNA, Viral
keywords: bluetongue virus; detection; field-deployable; portable; reverse transcription-insulated isothermal polymerase chain reaction