Rowden, G. | Search bioRxiv

bioRxiv2022

Standardization of Data Analysis for RT-QuIC-based Detection of Chronic Wasting Disease

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

molecular biology↗

bioRxiv2021

A Field-Deployable Diagnostic Assay for the Visual Detection of Misfolded Prions

Chronic Wasting Disease (CWD), a prion disease of cervids, has been identified across North America, Northern Europe and Korea. Diagnostic tools for the rapid and reliable detection of prion diseases are limited. Here, we combine gold nanoparticles (AuNPs) and quaking induced conversion (QuIC) technologies for the visual detection of amplified misfolded prion proteins from tissues of wild white-tailed deer infected with Chronic Wasting Disease (CWD). Our newly developed diagnostic test, MN-QuIC, enables both naked-eye and light-absorbance measurements for the detection of misfolded prions. The MN-QuIC assay leverages basic laboratory equipment that is cost-effective and portable, thus facilitating real-time prion diagnostics across a variety of settings. To test the portability of our assay, we deployed to a rural field station in southeastern Minnesota and tested for CWD on site. We successfully demonstrated that MN-QuIC is functional in a non-traditional laboratory setting by performing a blinded analysis in the field and correctly identifying all CWD positive and CWD not detected (independently confirmed with ELISA and/or IHC tests) animals at the field site, thus documenting the portability of the assay. Additionally, we show that electrostatic forces and concentration effects help govern the AuNP/prion interactions and contribute to the differentiation of CWD-prion positive and negative samples. We examined 17 CWD-positive and 24 CWD-not-detected white-tailed deer tissues that were independently tested using ELISA, IHC, and RT-QuIC technologies, and results secured with MN-QuIC were 100% consistent with these tests. We conclude that hybrid AuNP and QuIC assays, such as MN-QuIC, have great potential for sensitive, field-deployable diagnostics for a variety of protein misfolding diseases.

molecular biology↗