Jun 13, 2024

Public workspaceRT-QuIC Assay for the Detection of Chronic Wasting Disease in Rectal Mucosa of White-Tailed Deer

DOI
dx.doi.org/10.17504/protocols.io.yxmvmn2y6g3p/v1
RT-QuIC Assay for the Detection of Chronic Wasting Disease in Rectal Mucosa of White-Tailed Deer
  • Robert B. Piel1,
  • David A. Schneider1,
  • Aaron Lomax2,
  • Daniel Walsh3,
  • Eric M. Nicholson4,
  • Tracy A. Nichols5,
  • Susan E. Veneziano4
  • 1USDA-ARS-Animal Disease Research Unit, Pullman, WA;
  • 2University of Wisconsin, Madison, WI;
  • 3USGS-National Wildlife Health Center, Madison, WI;
  • 4USDA-ARS-National Animal Disease Center, Ames, IA;
  • 5USDA-APHIS-Veterinary Services Cervid Health Program, Fort Collins, CO
David A. Schneider
Open access
DOI: dx.doi.org/10.17504/protocols.io.yxmvmn2y6g3p/v1
Protocol Citation: Robert B. Piel, David A. Schneider, Aaron Lomax, Daniel Walsh, Eric M. Nicholson, Tracy A. Nichols, Susan E. Veneziano 2024. RT-QuIC Assay for the Detection of Chronic Wasting Disease in Rectal Mucosa of White-Tailed Deer. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmn2y6g3p/v1
Manuscript citation:
Piel, R. B., Veneziano, S. E., Nicholson, E. M., Walsh, D. P., Lomax, A. D., Nichols, T. A., Seabury, C. M., & Schneider, D. A. (2024). 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). PLoS ONE. https://doi.org/10.1371/journal.pone.0303037

Piel, Robert B.; Veneziano, Susan; M. Nicholson, Eric; Walsh, Daniel P.; Lomax, Aaron; A. Nichols, Tracy; et al. (2023). 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). Ag Data Commons. Dataset. https://doi.org/10.15482/USDA.ADC/24727866.v2
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
See the data produced by this protocol: Piel, Robert B.; Veneziano, Susan; M. Nicholson, Eric; Walsh, Daniel P.; Lomax, Aaron; A. Nichols, Tracy; et al. (2023). 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). Ag Data Commons. Dataset. https://doi.org/10.15482/USDA.ADC/24727866.v2
Created: February 18, 2022
Last Modified: June 13, 2024
Protocol Integer ID: 58412
Keywords: RT-QuIC, Real-Time Quaking-Induced Conversion, Misfolding, Amplification, Prion, PrP, CWD, Chronic Wasting Disease, Deer, WTD, ThT, Rectoanal Mucosa-Associated Lymphoid Tissue, RAMALT, rPrP, Thioflavin T, NP103, prion protein, rectal mucosa
Funders Acknowledgement:
USDA-ARS CRIS
Grant ID: 2090-32000-035-000D
USDA-ARS Non-Assistance Cooperative Agreement
Grant ID: 2090-32000-042-005-S
USDA-ARS CRIS
Grant ID: 5030-32000-228-000-D
Disclaimer
Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.
Abstract
This protocol details a Real-Time Quaking-Induced Conversion (RT-QuIC) assay to detect the prion-seeding activity associated with Chronic Wasting Disease prions in rectal mucosa samples from white-tailed deer. The authors tested this protocol as part of an agreement between USDA-APHIS, USDA-ARS, and USGS.


Note
*The authors appreciate and acknowledge the initial critiques and refinements made to this protocol as were provided by Christina Orru, Andrew Hughson, Natália do Carmo, and Byron Caughey at the NIH/NIAID Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, Hamilton, MT.
**The protocol described herein is a unification of the general method described by Orru, et al. (2017) with additional details and modifications supported in studies reporting sensitivity for CWD in cervid species. Notable publications included:
Henderson, et al. (2015). Quantitative assessment of prion infectivity in tissues and body fluids by real-time quaking-induced conversion. J Gen Virol, 96(Pt 1), 210-219. doi:10.1099/vir.0.069906-0

Haley, Siepker, Walter, et al. (2016). Antemortem Detection of Chronic Wasting Disease Prions in Nasal Brush Collections and Rectal Biopsy Specimens from White-Tailed Deer by Real-Time Quaking-Induced Conversion. J Clin Microbiol, 54(4), 1108-1116. doi:10.1128/JCM.02699-15

Haley, Siepker, Hoon-Hanks, et al. (2016). Seeded Amplification of Chronic Wasting Disease Prions in Nasal Brushings and Recto-anal Mucosa-Associated Lymphoid Tissues from Elk by Real-Time Quaking-Induced Conversion. J Clin Microbiol, 54(4), 1117-1126. doi:10.1128/JCM.02700-15

Orru, et al.. (2017). RT-QuIC Assays for Prion Disease Detection and Diagnostics. Methods Mol Biol, 1658, 185-203. doi:10.1007/978-1-4939-7244-9_14

Haley, Donner, et al. (2020). Cross-validation of the RT-QuIC assay for the antemortem detection of chronic wasting disease in elk. Prion, 14(1), 47-55. doi:10.1080/19336896.2020.1716657

Haley, Henderson, et al. (2020). Management of chronic wasting disease in ranched elk: conclusions from a longitudinal three-year study. Prion, 14(1), 76-87. doi:10.1080/19336896.2020.1724754

Henderson, et al. (2020). Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry. PLoS One, 15(2), e0228327. doi:10.1371/journal.pone.0228327

Guidelines
Protocol Notes
  1. SDS is included only in the Resuspension Buffer due to reports that long-term storage in the presence of SDS diminishes the seeding activity of homogenate samples (Caughey Lab, unpublished).
  2. N-2 media supplement is included to buffer adsorption of seeding material to the walls of dilution vessels in diluted homogenate samples. N-2 is not included in initial homogenization buffers as the quantity of total protein in these samples far exceeds the binding capacity of the vessels.


Materials
Required Laboratory Equipment:
  • Balance capable of mg accuracy
  • Bead beating grinder*
  • Pipettes 2 μL - 10 mL
  • Centrifuge capable of 1.5 mL tubes at 21,000 x g
  • Centrifuge capable of 0.2 mL tubes (single speed benchtop for PCR tubes)
  • Water-bath sonicator with fittings for 0.2 mL tubes*
  • Vortex mixer
  • Rocking/rotating incubator compatible with 1.5 mL tubes
  • RT-QuIC capable microplate reader*

Note
*Equipment models used to develop this protocol
Equipment
MP Biomedicals™ FastPrep -24™ Classic Instrument
NAME
Benchtop homogenizer
TYPE
Fisher Scientific
BRAND
12079310
SKU
https://www.fishersci.co.uk/shop/products/mp-biomedicals-fastprep-24-instrument/12079310
LINK

Equipment
Q700
NAME
Sonicator
TYPE
Qsonica
BRAND
Q700-110
SKU
https://www.sonicator.com/
LINK

Equipment
Q700 Microplate Horn
NAME
Sonicator Accessory
TYPE
Qsonica
BRAND
431MPXH
SKU

Equipment
FLUOstar Omega
NAME
Microplate Reader
TYPE
BMG Labtech
BRAND
FLUOstar Omega
SKU



Note
Optional Backup Power Supply:

This assay includes multi-day run-times with computer-controlled instruments. Consider using an uninterruptible power supply (UPS) system to avoid premature assay termination due to power outages/irregularities.

It is recommended to use a UPS that supplies continuous power through a battery to avoid interruptions due to power switching.

Example UPS:
Equipment
SMART UPS SRT 1500VA 120V
NAME
Uninterruptible Power Supply
TYPE
APC
BRAND
SRT1500XLA
SKU


Consumables:
  • Single-use scalpel and cutting surface (e.g., weigh boat) for each sample
  • 2 mL tubes compatible with bead beating grinder
  • Reagent0.7mm Zirconia beads BioSpec ProductsCatalog #11079107ZX
  • 0.2 and 1.5 mL tubes for tissue processing and aliquoting
  • 15 and 50 mL conical tubes for buffer preparation
  • 0.5 mL 100 kDa molecular weight cut-off (MWCO) centrifugal filters
  • Reagent96-well optical bottom black plate Thermo Fisher ScientificCatalog #265301
  • ReagentNunc™ Sealing TapesThermo Fisher ScientificCatalog #232702
  • Serological pipettes 5-10 mL
  • Micro-pipette tips 2-1000 μL


Chemical Reagents:

  • 10X PBS (Phosphate-buffered Saline) pH 7.4
  • NaPTA (Sodium phosphotungstic acid)
  • MgCl2 (anhydrous*; m.w. = 95.21)
  • SDS (Sodium dodecyl sulfate)
  • ReagentN-2 Media Supplement Gibco - Thermo FisherCatalog #17502048
  • Na2HPO4 (anhydrous*, dibasic; m.w. = 142)
  • NaH2PO4 . H2O (monohydrate*, monobasic; m.w. = 138)
  • NaCl
  • EDTA (Ethylenediaminetetraacetic acid) solution 0.5 M, pH 8
  • ThT (Thioflavin T)

*specific hydration states of the listed reagents are not required; however, differences will impact the molecular weights of the reagents and change the buffer preparation calculations provided below.

Prion Protein Substrate:

  • recombinant truncated Syrian hamster prion protein: Ha(90-231) rPrPC
  • Substrate were shipped on dry ice and stored at -80°C without being allowed to thaw


_____________________________________________________________________________________

Solutions and Buffers:


4% NaPTA Solution

ABCD
Stock concentrationamountFinal concentration
NaPTA powder2 g4% (w/v)
MgCl2 (m.w. = 95.21)powder809 mg170 mM
watern/ato 50 mL


Resuspension Buffer

ABCD
Stock concentrationamountFinal concentration
PBS (pH 7.4)10X5 mL1X
SDSpowder50 mg0.1 % (w/v)
N-2 media supplement (Gibco-Fisher)100X0.5 mL1X
watern/ato 50 mL


RT-QuIC Assay Buffer

  • prepare fresh for each assay run
  • see below for stock solution recipes
  • see Step 15 for rPrPC substrate preparation instructions

To prepare 10 mL RT-QuIC Assay Buffer, combine:
ABCD
Stock concentrationVolume (µL)Final concentration
watern/ato 10 mL totaln/a
NaPO4 buffer pH 7.4100 mM100010 mM
NaCl2 M1500300 mM
EDTA0.5 M 201 mM
ThT1 mM10010 µM
Filtered rPrPC substrateX mg/mL1000 (µL/mL) / X (mg/mL)0.1 mg/mL

Stock solution recipes for RT-QuIC Assay buffer:

100 mM NaPO4 buffer pH 7.4
  • 3.1 g NaH2PO4•H2O (monobasic; m.w. = 138)
  • 10.9 g Na2HPO4 (dibasic; m.w. = 142)
  • water to 1 L

2 M NaCl
  • 5.8 g NaCl (m.w. = 58.44)
  • water to 50 mL

1 mM ThT
  • 16 mg ThT (m.w. = 318.86)
  • water to 50 mL

0.5 M EDTA
  • purchased as stock solution (see above)

Filtered rPrPC substrate
  • see Step 15












Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
Sample Preparation: Rectal Mucosa Homogenization – 10% w/v
6m 15s
For each sample, prepare a 2-mL screw cap tube containing Amount1 g of 0.7 mm Zirconia beads (BioSpec 11079107zx) and label with sample/animal ID.

Weigh and add biopsy sample (up to Amount150 mg *) to each tube.
Note
*Biopsy samples larger than Amount150 mg may be processed in multiple pieces/tubes and homogenates pooled prior to freezing.

Note
This protocol is designed for mucosa-only samples. Skin, muscle layers, or excess connective tissue should be removed if evident.

Add 9 volumes 1X PBS Ph7.4 .

Note
Example: Amount100 mg biopsy sample + Amount900 µL 1X PBS

Homogenize using bead beating grinder.




3 cycles of Duration00:00:45 at speed 5.5 with Duration00:05:00 rest TemperatureOn ice between cycles.

5m 45s
Centrifuge at Centrifigation3000 x g, 00:00:30 .

30s
Collect supernatant*, pooling tubes from larger biopsy samples if necessary.

Note
*Un-disrupted connective tissue will likely remain following homogenization; do not collect.

Prepare Amount50 µL aliquots in 0.2-mL snap cap tubes and reserve remainder in one 2-mL tube. Store at Temperature-80 °C .
Note
We suggest preparing enough Amount50 µL aliquots for any planned experiments plus a few extra before storing the remainder. Though not fully characterized, it is likely that excessive freeze/thaw cycling may degrade the seeding activity present in sample homogenates.


Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
Sample Preparation: NaPTA Precipitation and Resuspension of Rectal Mucosa Homogenate
1h 31m 30s
Thaw a Amount50 µL aliquot of 10% homogenate from each sample to be tested.
Sonicate for Duration00:00:30 in a water bath sonicator at a power output of 180-200 W.


Note
Sonication is performed in a 0.2 mL tube using a sonicator equipped with a microplate horn. Other sonication methods have not been evaluated.



30s
Centrifuge ~Duration00:01:00 in a benchtop centrifuge at ~2,000 x g (or in a single speed 0.2-mL tube centrifuge) to pellet tissue debris.

1m
Supernatant will be added to the NaPTA reaction in the next step* .

Note
*Pipette carefully to avoid disturbing the pellet.
*Ensure the supernatant is free of insoluble material before adding it to the NaPTA reaction.

In a 1.5-mL centrifuge tube, combine:
AB
15 μLHomogenate supernatant from Step 9
1380 μL1X PBS pH 7.4
105 μL4% NaPTA solution
Incubate with gentle rocking or rotation Duration01:00:00 at TemperatureRoom temperature .

Note
*Tubes should be rocked/rotated in a horizontal position; adequate agitation can be visualized as the movement of the air bubble within the tube.


1h
Pellet by centrifugation Centrifigation21000 x g, 00:30:00 .

30m
Discard supernatant.*
Note
*It is important to remove the supernatant that contains NaPTA so that it does not interfere with resuspension in the subsequent step.
*We recommend using a vacuum flask with fresh 200-µL pipette tips on the suction line for each sample.
*The vacuum line should be protected from aerosol contamination following applicable regulations but minimally including an in-line High-Efficiency Particulate Air (HEPA) filter.

Critical
Resuspend the pellet* in Amount150 µL Resuspension Buffer**.
Note
*The pellet can be difficult to resuspend. As needed to resolubilize fully, transfer initial resuspension with all undissolved material to 0.2-mL tube and alternate vortexing and sonication (Duration00:00:30 in water bath sonicator at 180-200 W).


Note
**Resuspension Buffer is prepared fresh. N2 media supplement contains protein components that may degrade if stored for extended periods.



Critical
rPrPC Substrate Preparation
rPrPC Substrate Preparation
15m
Substrate Preparation Instructions:
  • prepare fresh for each assay run
  • rPrPC substrate was shipped as frozen aliquots and filtered before use.

To filter substrate:
  1. Thaw aliquot(s) completely at room temperature.
  2. Add substrate to 100 kDa centrifugal filter(s).
  3. Centrifuge at 3,000 x g for 10 min or until all liquid has passed through the filter
  4. Collect filtrate
  5. Determine protein concentration*


Note
*A loss of approximately 10-15% of protein concentration is expected following filtration.

rPrP concentration of the substrate may be measured by absorbance at 280 nm. A mass extinction coefficient (Abs 0.1% (=1 g/L)) of 1.4 is commonly used for truncated hamster (Ha90) substrate.

Note
Do not re-freeze rPrPC substrate

rPrPC loses sensitivity and shows increased propensity for spontaneous misfolding over time once thawed or when subject to repeated freeze/thaw cycles.

15m
RT-QuIC Assay
RT-QuIC Assay
3d 13h 16m
Prepare RT-QuIC Assay Buffer

  • must be prepared fresh for each assay run
  • see materials for buffer recipe and Step 15 for substrate handling instructions

Note
Prepare enough RT-QuIC Assay Buffer to test samples in quadruplicate.

(For full 96-well plate, prepare Amount10 mL RT-QuIC Assay Buffer.)



Critical
Add Amount98 µL RT-QuIC Assay Buffer to each well of a 96-well optical bottom black plate (Thermo Scientific Nunc 265301).
Add Amount2 µL prepared sample from Step 14 to each reaction well.



The following microplate layout is suggested for testing samples in quadruplicate and allows use of a 12-well multichannel pipette for reaction seeding:
AB
WellsSample ID
Column 1 Rows A-DSample 1
Column 2 Rows A-DSample 2
Column 3 Rows A-DSample 3
Column 4 Rows A-DSample 4
Column 5 Rows A-DSample 5
Column 6 Rows A-DSample 6
Column 7 Rows A-DSample 7
Column 8 Rows A-DSample 8
Column 9 Rows A-DSample 9
Column 10 Rows A-DSample 10
Column 11 Rows A-DSample 11
Column 12 Rows A-DSample 12
Column 1 Rows E-HSample 13
Column 2 Rows E-HSample 14
Column 3 Rows E-HSample 15
Column 4 Rows E-HSample 16
Column 5 Rows E-HSample 17
Column 6 Rows E-HSample 18
Column 7 Rows E-HSample 19
Column 8 Rows E-HSample 20
Column 9 Rows E-HSample 21
Column 10 Rows E-HSample 22
Column 11 Rows E-HSample 23
Column 12 Rows E-HSample 24
Seal the plate with film (Thermo Scientific Nunc 232702).
Insert the sealed plate into the microplate reader.


Incubation and Fluorescence Measurement Conditions:
Temperature: Temperature42 °C
Shaking: cycles of Shaker700 rpm, 00:01:00 double orbital followed by Duration00:01:00 rest.
Measure: at Duration00:43:00 (or Duration00:15:00 )* intervals: Bottom Read, 20 flashes/well.
Fluorescence: excitation: 450 ± 10 nm, emission: 480 ± 10 nm.
Gain: (manual) 1800.**
Assay length: Duration85:00:00


Note
*This protocol was optimized using a 43-minute measurement interval, which was the setting of an older plate reader program/script. We currently use and suggest 15-minute measurement intervals when running the protocol. This improves the estimation of baseline ThT fluorescence, which is generally used to calculate reaction threshold values. This also provides a more precise estimation of the time-to-threshold, an informative measure of reaction kinetics.

**Ideal gain settings may vary between individual plate readers and may be adjusted to allow better visualization of fluorescence curves with no impact on the assay reaction itself. A gain set too high will result in excessive baseline signal noise and/or saturated readings for positive reactions. A gain set too low may make it difficult to distinguish a positive signal from the baseline.

3d 13h 59m
Export to datafile
Export to datafile
For data analysis, we suggest exporting the data in a table format consisting of ThT relative fluorescence units corresponding to each Well/Sample ID at each Measurement Time.

Instructions for data export using BMG MARS software (version ####) are provided below:
Times can be recorded in hour decimal time to facilitate compatibility with downstream calculations:

  1. In the MARS software, open the Formats and Settings tab
  2. Select the Number Format Settings button
  3. Open the Number Formats tab
  4. Under Global Time Format Options, select the middle bubble and choose In hours from the drop down menu
To generate an Excel export table:

  1. In the MARS software, open the assay file
  2. Open the Table View tab
  3. Open the <Select a Cycle group> drop-down menu
  4. Select All Cycles
In the top left of the test run window, select the Excel Export button
image.png

  1. Save the exported file in Excel