Optimization of a thermal shaker-aided quaking-induced conversion platform for the rapid detection of prion seeding activity in human samples

Creutzfeldt-Jakob disease (CJD) is a rapidly progressive, fatal neurodegenerative disorder caused by the misfolding of the normal prion protein (PrPC) into its pathogenic form (PrPSc). Therefore, effective clinical management and infection control require diagnostic assays that combine sub-attomole sensitivity with a turnaround time compatible with routine practice. While real-time quaking-induced conversion (RT-QuIC) offers excellent analytical performance, its protracted reaction time and dependence on plate readers that integrate high-speed shaking with continuous fluorescence detection restrict its widespread implementation. Endpoint QuIC (EP-QuIC) is an established alternative approach that utilizes vigorous shaking in a ThermoMixer(R) C and relies on a single endpoint fluorescence readout. In this study, we introduced minor modifications to assay conditions to enhance the speed, robustness, and reproducibility of EP-QuIC. The refined assay detected prion seeding activity in sporadic CJD brain samples within 6 h--and, in some cases, as early as 2 h. Unlike RT-QuIC, problematic recombinant PrP batches that impaired assay reliability in RT-QuIC--exhibiting delayed kinetics and spontaneous ThT fluorescence in unseeded reactions--did not produce such effects in EP-QuIC. Instead, they yielded strong, specific fluorescence signals that clearly identified sCJD samples, demonstrating improved batch tolerance and diagnostic efficiency of this approach. When applied to CSF, the EP-QuIC assay showed 100% sensitivity and specificity, highlighting its potential as a rapid, reliable diagnostic tool for prion diseases.