title: Exploring physical and chemical factors influencing the properties of recombinant prion protein and the real-time quaking-induced conversion (RT-QuIC) assay.
abstract: Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada ; Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Keding
authors.initials: K
authors.lastname: Cheng
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Angela
authors.initials: A
authors.lastname: Sloan
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Avery
authors.affiliation: Centre for Food-borne, Environmental and Zoonotic Infectious Diseases, Public Health Agency of Canada, Ottawa, Ontario, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Coulthart
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada ; Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: Michael
authors.initials: M
authors.lastname: Carpenter
authors.affiliation: National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada ; Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.
authors.forename: J David
authors.initials: JD
authors.lastname: Knox
mesh_terms: D001681:Biological Assay / Q000379:methods*; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis*; D006801:Humans; D011328:Prions / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011328:Prions; D011994:Recombinant Proteins