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Factors That Improve RT-QuIC Detection of Prion Seeding Activity
Christina Doriana Orru (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Andrew G. Hughson (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Bradley R. Groveman (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Katrina J. Campbell (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Kelsie J. Anson (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Matteo Manca (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Allison Kraus (National Institutes of Health, National Institute of Allergy and Infectious Diseases), Byron W. Caughey (National Institutes of Health, National Institute of Allergy and Infectious Diseases)
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.