Factors That Improve RT-QuIC Detection of Prion Seeding Activity
AbstractRapid 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
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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.
Orrú CD, Hughson AG, Groveman BR, Campbell KJ, Anson KJ, Manca M, Kraus A, Caughey B. Factors That Improve RT-QuIC Detection of Prion Seeding Activity. Viruses. 2016; 8(5):140.Chicago/Turabian Style
Orrú, Christina D.; Hughson, Andrew G.; Groveman, Bradley R.; Campbell, Katrina J.; Anson, Kelsie J.; Manca, Matteo; Kraus, Allison; Caughey, Byron. 2016. "Factors That Improve RT-QuIC Detection of Prion Seeding Activity." Viruses 8, no. 5: 140.
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