RT-QuIC Assay for Prion Detection

The RT-QuIC was performed as reported before6 (link)8 (link). 85 μL of reaction buffer (consisting of 5xPBS (pH 6.9), 170 mM sodium chloride (SAFC Biosciences), 1 mM EDTA (Sigma-Aldrich), 10 μM Th-T (Sigma-Aldrich), and 0.1 mg/mL recPrP^C (Thermo Fisher Scientific/Prionics) was seeded with 15 μL CSF or 15 μL of brain homogenate at a dilution of 10⁻³ to a final volume of 100 μL in a 96-well black optical bottom plate (Fisher-Scientific). Each patient sample (brain and CSF) was analysed in triplicate. Prepared plates were sealed with sealing tape (Nunc/Sigma-Aldrich) and incubated in a FLUO Star OPTIMA plate reader (BMG Labtech) at 42 °C for 80 hours with intermittent quaking cycles, consisting of one-minute double-orbital quaking at 600 rpm followed by a one-minute incubation break. Beta-sheet formation kinetic was determined by measuring the Th-T fluorescence signal (450 nm excitation and 480 nm emission) every 30 minutes in relative fluorescence units (rfu).