title: Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion.
abstract: Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200  mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15  h. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.
authors.forename: Rohana P
authors.initials: RP
authors.lastname: Dassanayake
authors.affiliation: Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Orrú
authors.affiliation: Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Hughson
authors.affiliation: Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.
authors.forename: Byron
authors.initials: B
authors.lastname: Caughey
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.|The Paul G. Allen School for Global Animal Health, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7090, USA.
authors.forename: Telmo
authors.initials: T
authors.lastname: Graça
authors.affiliation: Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, WA 99164-6630, USA.
authors.forename: Dongyue
authors.initials: D
authors.lastname: Zhuang
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.
authors.forename: Sally A
authors.initials: SA
authors.lastname: Madsen-Bouterse
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.|Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, WA 99164-6630, USA.
authors.forename: Donald P
authors.initials: DP
authors.lastname: Knowles
authors.affiliation: Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.|Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, WA 99164-6630, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Schneider
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D001921:Brain / Q000378:metabolism*; D015511:Goat Diseases / Q000175:diagnosis / Q000235:genetics / Q000378:metabolism*; D006041:Goats; D018621:PrPC Proteins / Q000235:genetics / Q000378:metabolism*; D018620:PrPSc Proteins / Q000235:genetics / Q000378:metabolism*; D012608:Scrapie / Q000175:diagnosis* / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins