title: Factors That Improve RT-QuIC Detection of Prion Seeding Activity.
abstract: Rapid and sensitive detection of prions is important in managing prion diseases. The real-time quaking-induced conversion (RT-QuIC) assay for prion seeding activity has been applied to many prion diseases and provides for specific antemortem diagnostic testing. We evaluated RT-QuIC's long-term consistency and varied multiple reaction parameters. Repeated assays of a single scrapie sample using multiple plate readers and recombinant prion protein (rPrP(Sen)) substrates gave comparable results. N-terminal truncated hamster rPrP(Sen) (residues 90-231) hastened both prion-seeded and prion-independent reactions but maintained a clear kinetic distinction between the two. Raising temperatures or shaking speeds accelerated RT-QuIC reactions without compromising specificity. When applied to nasal brushings from Creutzfeldt-Jakob disease patients, higher temperatures accelerated RT-QuIC kinetics, and the use of hamster rPrP(Sen) (90-231) strengthened RT-QuIC responses. Elongation of shaking periods reduced scrapie-seeded reaction times, but continuous shaking promoted false-positive reactions. Furthermore, pH 7.4 provided for more rapid RT-QuIC reactions than more acidic pHs. Additionally, we show that small variations in the amount of sodium dodecyl sulfate (SDS) significantly impacted the assay. Finally, RT-QuIC performed in multiplate thermoshakers followed by fluorescence readings in separate plate readers enhanced assay throughput economically. Collectively, these results demonstrate improved speed, efficacy and practicality of RT-QuIC assays and highlight variables to be optimized for future applications.
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. christina.orru@nih.gov.
authors.forename: Christina D
authors.initials: CD
authors.lastname: Orrú
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. ahughson@niaid.nih.gov.
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Hughson
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. bradley.groveman@nih.gov.
authors.forename: Bradley R
authors.initials: BR
authors.lastname: Groveman
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. katrina.campbell@nih.gov.
authors.forename: Katrina J
authors.initials: KJ
authors.lastname: Campbell
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. kelsiejaness@gmail.com.
authors.forename: Kelsie J
authors.initials: KJ
authors.lastname: Anson
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. matteo.manca@nih.gov.
authors.forename: Matteo
authors.initials: M
authors.lastname: Manca
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. allison.kraus@nih.gov.
authors.forename: Allison
authors.initials: A
authors.lastname: Kraus
authors.affiliation: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. bcaughey@nih.gov.
authors.forename: Byron
authors.initials: B
authors.lastname: Caughey
mesh_terms: D000818:Animals; D019411:Clinical Laboratory Techniques / Q000379:methods*; D007562:Creutzfeldt-Jakob Syndrome / Q000175:diagnosis*; D003955:Diagnostic Tests, Routine / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011328:Prions / Q000032:analysis*; D015203:Reproducibility of Results; D012608:Scrapie / Q000175:diagnosis*; D013048:Specimen Handling / Q000379:methods*; D013696:Temperature; D013997:Time Factors
chemical_list: D011328:Prions
keywords: BSE; CJD; CWD; RT-QuIC; olfactory mucosa; prion; scrapie