Unknown

Dataset Information

0

Factors That Improve RT-QuIC Detection of Prion Seeding Activity.


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.

SUBMITTER: Orru CD 

PROVIDER: S-EPMC4885095 | BioStudies | 2016-01-01

REPOSITORIES: biostudies

Similar Datasets

2012-01-01 | S-EPMC3489776 | BioStudies
2017-01-01 | S-EPMC5614645 | BioStudies
2014-01-01 | S-EPMC3880330 | BioStudies
2018-01-01 | S-EPMC6301698 | BioStudies
2010-01-01 | S-EPMC2996325 | BioStudies
1000-01-01 | S-EPMC4922112 | BioStudies
2016-01-01 | S-EPMC5972304 | BioStudies
2015-01-01 | S-EPMC4472236 | BioStudies
2017-01-01 | S-EPMC5780402 | BioStudies
2017-01-01 | S-EPMC5240994 | BioStudies