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Factors That Improve RT-QuIC Detection of Prion Seeding Activity

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA
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Author to whom correspondence should be addressed.
These authors contributed equally to this study.
Academic Editor: Eric Freed
Viruses 2016, 8(5), 140; https://doi.org/10.3390/v8050140
Received: 15 March 2016 / Revised: 9 May 2016 / Accepted: 12 May 2016 / Published: 23 May 2016
(This article belongs to the Section Prions)
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 (rPrPSen) substrates gave comparable results. N-terminal truncated hamster rPrPSen (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 rPrPSen (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. View Full-Text
Keywords: prion; CJD; olfactory mucosa; RT-QuIC; scrapie; CWD; BSE prion; CJD; olfactory mucosa; RT-QuIC; scrapie; CWD; BSE
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Orrú, C.D.; Hughson, A.G.; Groveman, B.R.; Campbell, K.J.; Anson, K.J.; Manca, M.; Kraus, A.; Caughey, B. Factors That Improve RT-QuIC Detection of Prion Seeding Activity. Viruses 2016, 8, 140.

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