Optimisation of RT-QuIC to detect PrP^Sc in BSE-infected sheep brain tissue.

Different substrates were tested by RT-QuIC, including (A) truncated ovine recPrP (amino acids 94–233. PNRP genotype ARQ), (B) full length ovine recPrP (resi 25–233. PNRP genotype ARQ), (C) hamster-sheep recPrP (resi 23–137 Syrian golden hamster PrP followed by resi 141–234 ovine PrP, PNRP genotype ARQ), and (D) full length bovine recPrP (resi 25–241). (E-H) Further experiments were performed to determine the optimal concentration of SDS required for RT-QuIC using truncated ovine recPrP. Based on previous literature, SDS concentrations within the 0.025–0.1% (w/v) range were tested. RT-QuIC reactions were seeded with a 10⁻⁴ dilution of BSE-infected sheep brain homogenate (purple) or brain homogenate from mock-infected negative control sheep (red). Unseeded reactions (“mock-seeded” with PBS buffer) are plotted in blue. In most experiments, a 10⁻⁴ dilution of 263K scrapie-infected hamster brain tissue was used as a positive control (green). Data points represent the mean ThT fluorescence from n = 4 replicates.

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