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.

In coping with prion diseases, it is important to have tests that are practical enough for routine applications in medicine, agriculture, wildlife biology, and research, yet sensitive enough to detect minimal amounts of infectivity. Real-time quaking-induced conversion (RT-QuIC) assays have evolved to the point where they fulfill these criteria in applications to various human and animal prion diseases. For example, RT-QuIC assays of cerebrospinal fluid and nasal brushings allow for highly sensitive (77-97%) and specific (99-100%) identification of human sCJD patients. Recent improvements have markedly enhanced sensitivity and reduced the assay time required for many samples to a matter of hours rather than days. By combining analyses of cerebrospinal fluid and nasal brushings, diagnostic sensitivities and specificities of nearly 100% can be achieved. RT-QuIC assays are based on prion-seeded amyloid fibril formation by recombinant prion protein (rPrP) in multiwell plates using a Thioflavin T fluorescence readout. Here we describe our current RT-QuIC methodologies as well as technical considerations in executing, troubleshooting, and adapting the assay to new strains of prions and sample types.

Background: Key factors modulating conversion of prion protein into prions remain unclear. Results: Neutralization of a cluster of lysines within residues 101-110 promoted formation of an N-terminally extended recombinant prion protein amyloid core. Conclusion: A central lysine cluster strongly modulates folding of prion protein amyloids. Significance: These findings highlight a key structural factor in the PrP Sc -like folding of prion protein.

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