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Viruses. 2016 May 23;8(5). pii: E140. doi: 10.3390/v8050140.

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

Author information

1
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. christina.orru@nih.gov.
2
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. ahughson@niaid.nih.gov.
3
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. bradley.groveman@nih.gov.
4
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. katrina.campbell@nih.gov.
5
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. kelsiejaness@gmail.com.
6
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. matteo.manca@nih.gov.
7
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. allison.kraus@nih.gov.
8
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute for Allergy and Infectious Diseases, National Institutes of Health, Hamilton, 59840 MT, USA. bcaughey@nih.gov.

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.

KEYWORDS:

BSE; CJD; CWD; RT-QuIC; olfactory mucosa; prion; scrapie

PMID:
27223300
PMCID:
PMC4885095
DOI:
10.3390/v8050140
[Indexed for MEDLINE]
Free PMC Article

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