Data from: Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus virginianus)

Purpose of the study:

The diagnostic performance of a standardized RT-QuIC protocol using two sources of assay substrate was studied for detecting chronic wasting disease (CWD) in mock rectal mucosa biopsy samples from farmed white-tailed deer (Odocoileus virginianus).

The repository files

RTQuIC_reactions.xlsx

The entire collection of reaction data used to determine the diagnostic performance of a standardized RT-QuIC protocol (DOI: dx.doi.org/10.17504/protocols.io.yxmvmn2y6g3p/v1). The variables (columns) in the dataset include the name of the substrate source, the plate reader ID, plate well position, assay time in fractional hours, the ThT signal measured in relative fluorescence units, and also the following animal information for the sample tested: an anonymized white-tailed deer ID, sex, age in years, PRNP genotype at codons 95 and 96, and stage of infection. The animal's stage of infection was determined by immunohistochemistry for CWD-associated prion protein accumulation in the medial retropharyngeal lymph nodes (MRPLN) and the obex. All deer were from farms on which infected deer had been previously detected; thus, all deer were considered exposed. Based on the results of CWD-IHC, deer were classified for CWD as infection not detected in these tissues (ND), as early preclinical when CWD-IHC positive in only the MRPLN, and as late preclinical when CWD-IHC positive in both the MRPLN and obex. None of the deer were reported to be clinical at the time of sample collection.

S1 Fig ###.png

A series of 26 PNG files representing the reaction data from RTQuIC_reactions.xlsx.

Each PNG file plots the ThT data acquired from a 96-well plate over time. Each file includes a header identifying the substrate source, plate ID (1-13 for each substrate source), and plate reader ID. Each plot indicates the white-tailed deer ID being tested and shows the quadruplicate reactions recorded. Also included are the quadruplicate reaction data for the plate negative control (a sample from an ND deer) and positive control (a late preclinical deer).

S2 Fig 1.tif

Gross purity of recombinant prion protein substrates. After the initial performance testing of the standardized RT-QuIC assay protocol, the gross purities of the independently produced recombinant truncated hamster prion protein (rPrP Ha90) substrates were compared by denaturing gel electrophoresis and Coomassie staining. A subsequent production run of the commercial source (MNPROtein) and laboratory source rPrPs were obtained and routinely prepared in RT-QuIC reaction buffer at a working concentration of 0.1 mg/mL before processing for electrophoresis (NuPAGE^TM; Invitrogen). An image of the resultant gel is shown in which Precision Plus Protein WesternC (BioRad) markers were loaded into lanes 1 and 4, and the processed RT-QuIC reaction buffers containing MNPROtein or laboratory produced rPrP Ha90 substrates were respectively loaded into lanes 2 and 3. The molecular mass and amount of each production source of rPrP substrate in RT-QuIC reaction buffer appear equivalent and without gross evidence of impurities.