Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

Yo Ching Cheng; Samia Hannaoui; Theodore Ralph John; Sandor Dudas; Stefanie Czub; Sabine Gilch

doi:10.3791/56373

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Method Article

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

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DOI:

10.3791/56373

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September 29th, 2017

Yo Ching Cheng^*¹ , Samia Hannaoui^*¹ , Theodore Ralph John² , Sandor Dudas³ , Stefanie Czub³ , Sabine Gilch¹

¹Dept. of Ecosystem and Public Health, Calgary Prion Research Units, Faculty of Veterinary Medicine, University of Calgary, ²Dept. of Molecular Biology, University of Wyoming, ³Canadian Food Inspection Agency, Lethbridge Laboratories

^* These authors contributed equally

Summary

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

Abstract

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with ᵦ-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrP^Sc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrP^Sc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

Introduction

Prion diseases or transmissible spongiform encephalopathies (TSE) are neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids ¹^,². TSEs are characterized by distinctive spongiform appearance and loss of neurons in the brain. According to the "protein only" hypothesis, prions are mainly composed of PrP^Sc ('Sc' for scrapie) ³, a misfolded isoform of the host-encoded cellular prion protein, PrP^C. PrP^Sc....