# Practical Real-Time Quaking-Induced Conversion for Detecting Classical Bovine Spongiform Encephalopathy and Classical and Atypical Scrapie Prions

## TL;DR

This study improves a prion detection method by optimizing protein substrates and removing inhibitors to detect bovine spongiform encephalopathy and scrapie prions in brain tissue.

## Contribution

The study introduces optimized recombinant prion proteins and a pretreatment method to enhance RT-QuIC for detecting classical and atypical prion diseases.

## Key findings

- A single-step lipid extraction effectively removes inhibitors from brain homogenates.
- Recombinant sheep PrP is optimal for classical BSE detection, while specific recombinant cervid PrP variants excel for classical and atypical scrapie.
- Optimized rPrP substrates combined with inhibitor removal significantly improve RT-QuIC performance.

## Abstract

Real-time quaking-induced conversion (RT-QuIC) is highly sensitive for prion detection; however, inhibitory factors present in tissue homogenates readily interfere with the assay. We previously reported that recombinant cervid prion protein (rCerPrP) enabled the establishment of practical RT-QuIC for detecting chronic wasting disease and atypical bovine spongiform encephalopathy (BSE) prions, i.e., detecting low levels of prions in high concentration of brain tissue homogenates. Accordingly, the present study aimed to establish RT-QuIC for detecting classical BSE (C-BSE) and classical and atypical scrapie (C- and A-scrapie, respectively). A single-step lipid extraction using a 3:1 mixture of 2-butanol and methanol was effective as a pretreatment to remove inhibitors from brain homogenates. Among three rPrPs extensively evaluated, recombinant sheep PrP (rShPrP) was the most suitable substrate for practical detection of C-BSE prions. rCerPrP-173S/177N and rCerPrP-98S/173S/177N, which carry sheep-type amino acid substations at codons 173 and 177 and at codons 98, 173, and 177, showed excellent performance for detecting C-scrapie prions. Moreover, rCerPrP-98S/173S/177N, but not rCerPrP-173S/177N, was identified as an optimal substrate for detecting A-scrapie prions. These results suggested that combining inhibitor-removal pretreatment with the optimization of rPrP substrate for each animal prions further enhanced of RT-QuIC performance.