Abstract
Prion diseases are neurodegenerative diseases affecting both humans and animal species. The phenotypic spectrum is broad and includes Creutzfeldt-Jakob disease (CJD) and its variant zoonotic form (vCJD) in humans, while in animals, scrapie of sheep and goats, bovine spongiform encephalopathy and chronic wasting disease of deer, elk and moose are naturally occurring forms. Transmission and pathogenesis appear causally linked to the misfolding of the normal form of the prion protein (PrP(C)) into disease associated conformers (PrP(D)), the latter enriched in β-strand secondary structure. Over the past 10 years two protein amplification techniques, the protein misfolding cyclic amplification (PMCA) assay and real-time quaking induced conversion (RT-QuIC) assay have been developed and successfully deployed in prion biology across a range of scientific and clinical applications, including generation of de novo prions, quantitation of prion infectivity and ultra-sensitive detection of PrP(D). While PMCA utilises sonication to facilitate protein amplification, RT-QuIC employs vigorous shaking to achieve this outcome, with both techniques sharing the ability to amplify miniscule quantities of PrP(D) seed present in various tissues and body fluids to levels detectable using routine biochemical methods. The enhanced specificity of the RT-QuIC for detection of PrP(D) in cerebrospinal fluid (CSF) has spawned international collaborations to rigorously assess and validate the assay for clinical diagnostic purposes. In parallel with collaborative CSF validation studies have been successful efforts to refine the RT-QuIC allowing its use for more accessible body fluids or tissues such as urine and nasal brushings, as well as promote higher sample throughput, shorten assay times and offer accurate quantification of PrP(D) even at levels below those detectable by animal bioassays. Animal studies support the generic capacity of the RT-QuIC for PrP(D) detection, underpinning the utility of this assay for studying prion disease and the high likelihood of inter-convertibility of technical refinements for human and animal use.