title: The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases.
abstract: The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPScseed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays-such as protein misfolding cyclic amplification (PMCA)-which are based on sonication, the RT-QuIC technique is based on prion seed-induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes ∼87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Schmitz
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Cramm
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Franc
authors.initials: F
authors.lastname: Llorens
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Müller-Cramm
authors.affiliation: Department of Medicine, The University of Melbourne, Parkville, Victoria, Australia.
authors.forename: Steven
authors.initials: S
authors.lastname: Collins
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Atarashi
authors.affiliation: Department of Molecular Microbiology and Immunology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Satoh
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, USA.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Orrù
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, USA.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Groveman
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Saima
authors.initials: S
authors.lastname: Zafar
authors.affiliation: Department of Neuropathology, University Medical Center Göttingen, Georg-August University, Göttingen, Germany.
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Schulz-Schaeffer
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Hamilton, Montana, USA.
authors.forename: Byron
authors.identifier: http://orcid.org/0000-0002-3928-6101
authors.initials: B
authors.lastname: Caughey
authors.affiliation: Department of Neurology, University Medical Center Göttingen and German Center for Neurodegenerative Diseases (DZNE), Göttingen, Germany.
authors.forename: Inga
authors.initials: I
authors.lastname: Zerr
mesh_terms: D000818:Animals; D019963:Clinical Chemistry Tests / Q000379:methods*; D006224:Cricetinae; D006801:Humans; D057230:Limit of Detection; D018620:PrPSc Proteins / Q000097:blood / Q000134:cerebrospinal fluid*; D017096:Prion Diseases / Q000097:blood / Q000134:cerebrospinal fluid* / Q000175:diagnosis*; D057165:Proteostasis Deficiencies / Q000097:blood / Q000134:cerebrospinal fluid* / Q000175:diagnosis*; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D018620:PrPSc Proteins