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

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 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 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.

Large‐scale prion protein genotyping in Canadian caribou populations and potential impact on chronic wasting disease susceptibility

“…Prions are highly resistant to many major forms of inactivation (Giles, Woerman, Berry, & Prusiner, 2017). It is exceptionally challenging and not possible to contain and eradicate CWD to date, as prions are shed in excrement and bodily fluids of infected free‐ranging cervids (Cheng, Hannaoui, et al, 2017; Haley et al., 2011; Henderson et al., 2015; Safar et al., 2008). This results in contamination of the environment, particularly soil, with prions that remain infectious for years (Bartelt‐Hunt & Bartz, 2013; Kuznetsova, McKenzie, Cullingham, & Aiken, 2020; Pritzkow et al., 2018; Somerville et al., 2019).…”

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry

“…We and others have used RT-QuIC to detect and estimate levels of prion seeding activity in multiple tissues, excreta, and body fluids with the goal of increasing sensitivity and speed in CWD detection pre-and post-mortem [19][20][21][22][23][24][25][26]. Here we provide a dynamic longitudinal profile of progressing CWD infection in white-tailed deer after low dose oral exposure to CWD prions as assessed by serial lymphoid tissue biopsies examined by both the RT-QuIC and IHC methods.…”

Comparison of conventional, amplification and bio-assay detection methods for a chronic wasting disease inoculum pool

“…The major challenge to early diagnosis of prion diseases has been the very low prion concentrations and/or inhibitors that are present in accessible biological samples [19–21]. In addition, prion infectivity is comprised of variably protease sensitive particles [22], yet conventional prion detection methods require the use of protease digestion, which ablates sensitive forms of prion infectivity (PrP Sen ) and reduces sensitivity.…”