Factors That Improve RT-QuIC Detection of Prion Seeding Activity.
Viruses
; 8(5)2016 05 23.
Article
em En
| MEDLINE
| ID: mdl-27223300
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.
Palavras-chave
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Scrapie
/
Manejo de Espécimes
/
Príons
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Síndrome de Creutzfeldt-Jakob
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Técnicas de Laboratório Clínico
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Testes Diagnósticos de Rotina
Tipo de estudo:
Diagnostic_studies
/
Evaluation_studies
Limite:
Animals
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Humans
Idioma:
En
Ano de publicação:
2016
Tipo de documento:
Article