Factors That Improve RT-QuIC Detection of Prion Seeding Activity

Viruses

Allison Kraus¹  Byron Caughey¹  Christina D. Orrú¹  Kelsie J. Anson¹  Bradley R. Groveman¹  Katrina J. Campbell¹  Matteo Manca¹  Andrew G. Hughson¹ 

[1] Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA;

关键词: prion;    CJD;    olfactory mucosa;    RT-QuIC;    scrapie;    CWD;    BSE;   

DOI  :  10.3390/v8050140

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown