Name: Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material (Video) | JoVE Journal
Uploaded: 2017-09-29T15:00:00.000+00:00
Duration: 9 min 50 s
Description: Learn the RT-QuIC assay for detecting chronic wasting disease prions in cervid fecal samples. This sensitive, high-throughput method enables researchers to study prion shedding timing and transmission dynamics in wildlife populations.

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5 minutes

We are grateful to Dr. Byron Caughey (NIH Rocky Mountain Laboratories) for providing training and the cervid PrP bacterial expression plasmid. SG is supported by the Canada Research Chair program. We acknowledge funding for this research to SG from Genome Canada, Alberta Prion Research Institute and Alberta Agriculture and Forestry through Genome Alberta, and the University of Calgary in support of this work. We acknowledge a research grant from the Margaret Gunn Foundation for Animal Research.

1. RT-QuIC Using Fecal Material
<ol>
	<li>Preparation of cervid feces extracts
	<ol>
		<li>Make fecal homogenate by adding 1 g of fecal material to 10 mL of feces extract buffer (20 mM sodium phosphate, pH 7.1, 130 mM NaCl, 0.05% Tween 20, 1 mM PMSF and 1x complete protease inhibitors, EDTA-free) to give a final concentration of 10% (w/v). The homogenization buffer can be prepared prior to the utilization and stored at -20 &#176;C.</li>
		<li>Homogenize fecal pellets (1 g) and buffer (10 mL) in tubes (e.g., gentleMACS M tubes) using a dissociator with a pre-set program from the manufacturer for proteins for 1 min at room temperature. Repeat this step two to three times until the fecal samples are completely homogenized in the buffer.</li>
		<li>Seal the tubes with parafilm and place them onto a rocking platform or rotary shaker for a 1 h incubation at room temperature.</li>
		<li>Centrifuge the tubes at 18,000 x g for 5 min at room temperature.</li>
		<li>Collect the supernatants which contain the protein extracts and aliquot them into 1.5 mL tubes. Aliquots can be stored at -80 &#176;C for further applications.</li>
	</ol>
	</li>
	<li>Concentration of PrPSc in feces extracts 
	NOTE: In order to concentrate CWD prions in the feces extract samples, thereby improving the sensitivity of detection by RT-QuIC, a protein precipitation step is added to the protocol.
	<ol>
		<li>NaPTA precipitation method</li>
		<li>Add 250 &#181;L of 10% (w/v) of N-lauryl sarcosinate (sarkosyl) to 1 mL of fecal protein extract to give a final concentration of sarkosyl of 2% (v/v).</li>
		<li>Seal the tubes which contain the samples with parafilm and incubate at 37 &#176;C for 30 min under constant shaking at 1,400 rpm in a thermomixer.</li>
		<li>Adjust the mix of fecal protein extract and sarkosyl with a stock solution containing 10% (w/v) of sodium phosphotungstic acid and 170 mM of magnesium chloride, pH 7.4 to obtain a final concentration of 0.3% (w/v) sodium phosphotungstic acid in the samples.</li>
		<li>Incubate the samples at 37 &#176;C for 2 h with constant shaking at 400 rpm.</li>
		<li>Centrifuge the samples at 14 &#176;C for 30 min at 15,800 x g. Remove the supernatants carefully.</li>
		<li>Wash the pellets by resuspending them in wash buffer (10 mM Tris-Cl, pH 7.5, 100 mM NaCl, 0.5% Triton-X 100, 10 mM EDTA, 0.5% sodium deoxycholate (w/v), and 0.1% sarkosyl (w/v)).</li>
		<li>Centrifuge the samples at 14 &#176;C for 15 min at 15,800 x g. Remove the supernatants carefully.</li>
		<li>Resuspend the pellets in 100 &#181;L (1/10 of the original volume of the fecal protein extract) of RT-QuIC dilution buffer. 
		NOTE: RT-QuIC dilution buffer (20 mM sodium phosphate, pH 6.9, 130 mM NaCl, 0.1% SDS (w/v) and 1 X N2 supplement) is prepared prior to the utilization. A total volume of 10 mL is filtered through a 0.2 &#181;m acrodisc syringe filter using a syringe and then stored at -20 &#176;C until use.</li>
		<li>Store the NaPTA treated samples at -20 &#176;C until utilizing them in RT-QuIC assay.</li>
	</ol>
	</li>
	<li>RT-QuIC assay
	<ol>
		<li>Prepare a fresh 10 mM Th-T stock solution (0.032 g of Th-T in 10 mL of double distilled H2O).</li>
		<li>Make a dilution of 1:10 of the Th-T stock solution in double distilled H2O to make a final concentration of Th-T of 1 mM the filter it through a 0.2 &#181;m acrodisc syringe filter using a syringe.</li>
		<li>Thaw mouse recombinant PrP (rPrP, aa 23-231) substrate (10 &#181;g/mL per RT-QuIC reaction) stored at -80 &#176;C on ice.</li>
		<li>Load 500 &#181;L of rPrP substrate at a time into 100 kDa size exclusion filter microtubes and centrifuge at 14,000 x g for 5 min at room temperature (one tube/filter can be used up to two times).</li>
		<li>Transfer the eluted substrate into a sterile 1.5 mL tube and keep it at 4 &#176;C until use.</li>
		<li>Thaw RT-QuIC dilution buffer stored at -20 &#176;C.</li>
		<li>Make dilutions of the seed (fecal protein extract) in RT-QuIC dilution buffer: 
		Undiluted sample: use the fecal protein extract undiluted 
		Diluted 2 x 10-1 
		Diluted 2 x 10-2 
		Diluted 2 x 10-3</li>
		<li>Prepare stock solutions for RT-QuIC reaction mixture: 
		Phosphate-buffered saline (PBS): 5X 
		NaCl: 2 M stock solution of NaCl prepared in double distilled H2O. 
		EDTA: 100 mM stock solution of EDTA prepared in double distilled H2O.</li>
		<li>Prior to utilization of all the solutions (step 1.3.8), filter each solution separately through a 0.2 &#181;m acrodisc syringe filter using a syringe and keep them sterile at room temperature.</li>
		<li>Prepare a total volume of RT-QuIC reaction mixture according to the number of samples (98 &#181;L volume of reaction per sample and four replicates per sample) to be used plus 10% of this volume to make sure to have enough mixture for all the reactions.
		<ol>
			<li>Use a 15 mL tube to prepare the RT-QuIC reaction mixture (20 mM sodium phosphate, pH 6.9, 300 mM NaCl, 1 mM EDTA, 10 &#181;M Th-T, 10 &#181;g/mL rPrP substrate, and double distilled water to adjust the rPrP final concentration in the reaction) using the stock solutions.</li>
			<li>Mix all the solutions well by pipetting up and down using a pipette with a filter tip. Avoid as much as possible the use of vortex.</li>
			<li>Pour the mixture into a 50 mL disposable pipetting reservoir.</li>
			<li>Use a multichannel pipette to load 98 &#181;L of RT-QuIC reaction mixture into each well of a 96-well optical bottom plate.</li>
		</ol>
		</li>
		<li>Add to each RT-QuIC reaction 2 &#181;L of undiluted or 10-fold serially diluted fecal protein extract. Test each sample in four replicates. 
		NOTE: In each experiment, serial dilutions of fecal samples (ranging from undiluted to 2 x 10-3) from CWD-negative and verified CWD-positive animals are used as negative and positive controls, respectively.</li>
		<li>Seal the plate with a sealing tape before incubating in a plate reader at 42 &#176;C for 25 h with repeated cycles of 1 min double orbital shaking (700 rpm) and 1 min resting throughout the incubation.</li>
		<li>Define fluorescence measurement settings: 
		Excitation: 450 nm 
		Emission: 480 nm 
		Bottom read, number of flashes: 20 
		Manual gain: 1000 (depends on the machine) 
		Integration time: 20 &#181;s</li>
		<li>Remove the plate from the plate reader at the end of the 25 h.</li>
		<li>Spin down the plate at 3,000 x g for 3 min to avoid potential contamination between the wells.</li>
		<li>Remove the seal from the plate.</li>
		<li>Prepare a new 96-well plate by adding 90 &#181;L of RT-QuIC reaction mixture containing fresh substrate and fresh fluorescence dye Th-T as described in section 1.3.1 to 1.3.6.</li>
		<li>Transfer 10 &#181;L from each well of the first plate to the newly prepared 96 well plate.</li>
		<li>Seal the new plate and continue the RT-QuIC assay for additional 50 h following the steps described in step 1.3.13. This additional step of 50 h will make the total reaction time of 75 h. 
		NOTE: All the procedures of RT-QuIC assay are done under biosafety cabinet.</li>
		<li>Collect the data.
		<ol>
			<li>Read and document the Th-T fluorescence signal of each well every 15 min.</li>
			<li>Collect data of total cycles using Data Analysis software and transfer into a spreadsheet with the average of quadruplicate reactions.</li>
			<li>Plot the averages of data. The x-axis corresponds to the reaction time of RT-QuIC (h) and the y-axis corresponds to the relative fluorescence units (RFU). Each curve corresponds to a dilution of a known sample.</li>
			<li>In each experiment, demarcate a threshold by calculating the highest mean values of the negative control plus 5 standard deviations as described in current published literature 41. 
			NOTE: All replicates which crossed the defined threshold are considered positive. If at least two out of four replicates cross the threshold, the sample is then considered positive.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Purification of Recombinant Prion Protein (rPrP)
<ol>
	<li>Growth of bacterial stock and expression of rPrP 
	NOTE: The Escherichia coli (E. coli) strain Rosetta (DE3) transformed with vector pET-24 encoding the mouse PrP (residues 23-231) was used to prepare the rPrP.
	<ol>
		<li>Thaw glycerol stock of Rosetta (DE3) transformed with pEt-24 mPrP(23-231) on ice.</li>
		<li>Streak LB (Luria Bertani) plates with a final concentration of kanamycin of 50 &#181;g/mL and incubate overnight in a 37 &#176;C incubator. Make sure that the plates are upside down to avoid humidity condensation in the solid media containing the bacteria. Prepare two plates to make sure to have growth in at least one of the two plates.</li>
		<li>Prepare 1 L of LB and autoclave it to make it sterile.</li>
		<li>Supplement 1 L of sterile LB media with 1 mL of kanamycin (50 mg/mL) and 1 mL of chloramphenicol (34 mg/mL).</li>
		<li>Pick one colony of each plate by using a disposable inoculation loop to inoculate 3 mL of LB medium containing kanamycin and chloramphenicol.</li>
		<li>Place the mini-cultures in an incubator at 37 &#176;C with constant shaking at 225 rpm for 5-6 h.</li>
		<li>Add the mini-cultures to the rest of the original 1 l of LB media together with Express Autoinduction System 1 for induction of protein expression.</li>
		<li>Place the culture in 2 L flask, or larger, in an incubator at 37 &#176;C with constant shaking at 200 rpm for 20-24 h.</li>
		<li>Split the 1 L culture into 4 x 250 mL centrifuge tubes.</li>
		<li>Spin down the tubes containing the culture at 3,750 x g for 20 min at room temperature.</li>
		<li>Discard the supernatant and collect the bacterial pellets in 50 mL centrifuge tubes, then freeze them at -80 &#176;C until further processing. The resulting pellets have weighed 3 to 4 g for a good protein yield.</li>
	</ol>
	</li>
	<li>Preparation of inclusion body
	<ol>
		<li>Thaw the frozen pellet (3 to 4 g) at 37 &#176;C for few minutes.</li>
		<li>Freeze the pellet one more time for 10 min at -80 &#176;C and thaw again. Repeat this step of freezing and thawing two more times.</li>
		<li>Resuspend the bacterial pellet in 1X&#160;lysis reagent (e.g. BugBuster Master Mix) by pipetting thoroughly. Use 5 mL of the reagent for 1 g of bacterial pellet. Make sure to completely homogenize the mix.</li>
		<li>Incubate the homogenate on a rocker for 20 min at room temperature.</li>
		<li>Centrifuge the homogenate at 16,000 x g for 20 min at room temperature.</li>
		<li>Discard the supernatants by carefully pouring it off.</li>
		<li>Homogenize the pellets in the same volume of 1X lysis reagent as used in the previous step.</li>
		<li>Incubate the homogenate for 15 min on a rocker at room temperature.</li>
		<li>Add a sufficient volume of 1:10 (0.1x) lysis reagent to get a total homogenate volume of 40 mL. Mix by inversion to make sure to homogenize well.</li>
		<li>Centrifuge the homogenate at 7,900 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant by carefully pouring it.</li>
		<li>Resuspend the pellet in 40 mL of 0.1x lysis reagent.</li>
		<li>Centrifuge the homogenate at 16,000 x g for 15 min at 4 &#176;C.</li>
		<li>Discard the supernatant carefully by pouring it and store the pellet containing the inclusion bodies at -20 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Protein purification
	<ol>
		<li>Thaw the inclusion bodies at room temperature.</li>
		<li>Dissolve the thawed inclusion bodies in 14 mL of 8 M guanidine-HCl (38 g Guanidine hydrochloride in 0.1 M NaPO4 pH 8.0 and fill the suspension with H2O to 50 mL; do not adjust pH at final solution) to solubilize proteins.</li>
		<li>Make sure to homogenize well by pipetting up and down.</li>
		<li>Incubate the lysate on a rocker at room temperature for 50 min.</li>
		<li>Prepare 18 g of Ni-NTA resin beads (18 g for 3 to 4 g bacterial pellet); rinse them with 100 mL of water using vacuum and a 100 mL 0.22 &#181;m bottle top filter.
		<ol>
			<li>Place the 18 g of Ni-NTA resin beads in 50 mL tube and add sufficient volume of denaturing buffer (100 mM sodium phosphate, 10 mM Tris, pH 8.0, 6 M guanidine hydrochloride, pH 8) to make the final volume up to 50 mL.</li>
			<li>Equilibrate the beads in the denaturing buffer for 50 min on a rocker at room temperature.</li>
		</ol>
		</li>
		<li>Centrifuge the inclusion body lysate at 16,000 x g for 5 min to remove insoluble debris.</li>
		<li>Add the supernatant to the equilibrated beads and discard the pellets.</li>
		<li>Put the mix on a rocker for 40 min at room temperature to allow protein binding to Ni-NTA resin. 
		NOTE: Nickel chelate beads are used to purify PrP by its nickel affinity. The histidine-rich N-terminal region of PrP renders the affinity to nickel(II), which allows the protein to bind to nickel ions without any his-tag.</li>
		<li>Wash FPLC with water to clean out both lines (A and B).</li>
		<li>Run denaturing buffer (100 mM sodium phosphate, 10 mM Tris, pH 8.0, 6 M guanidine hydrochloride, pH 8) through both lines (A and B) to prime the system (the column is not yet attached.)</li>
		<li>Load the resin onto column. Make sure to minimize air bubbles.</li>
		<li>Attach the column to the FPLC and run a linear gradient from 100% denaturing buffer A and 0% of refolding buffer B (100 mM sodium phosphate, 10 mM Tris, pH 8.0) at first to 0% denaturing buffer and 100% refolding buffer by the end. This gradual shift is run at a flow rate of 0.75 mL/min for 240 min and is needed for proper folding of PrP. 
		NOTE: During the chromatographic purification process, the denatured PrP is first slowly refolded on the resin by applying a linear gradient of refolding buffer to replace the denaturing buffer. This step also removes the chaotropic guanidine-HCl.</li>
		<li>Make sure 100% refolding buffer continues to flow through the column for an additional 30 min at 0.75 mL/min.</li>
		<li>Rinse line A with water followed by elution buffer (100 mM sodium phosphate, 10 mM Tris, 500 mM imidazole, pH 5.8) bypassing the column. This will clean out the denaturing buffer which has been replaced now by the elution buffer in the AKTA system.</li>
		<li>Elute protein at 2 mL/min by running a linear gradient for 40 min from 100% refolding buffer B and 0% elution buffer A at first to 0% refolding buffer and 100% elution buffer. 
		NOTE: The high concentration of imidazole in elution buffer will compete PrP&#39;s binding to the nickel(II). The primary protein peak should start to elute about 1/3 of the way through the gradient.
		<ol>
			<li>Watch chromatogram for OD 280 nm to increase.</li>
		</ol>
		</li>
		<li>Collect only the tubes containing the protein at the center of the large peak. 
		NOTE: The eluted fractions of 2 mL each are collected into 15 mL tubes.</li>
		<li>Dilute the protein contained in the tubes immediately with about 1/3 volume (1 mL) of dialysis buffer (10 mM sodium phosphate, pH 5.8).</li>
		<li>Immediately place the tubes on ice.</li>
		<li>Pool the eluted protein contained in the tubes.</li>
		<li>Poor the protein into Dialysis Cassettes (molecular weight cut-off 10 kDa).</li>
		<li>Put the cassettes in pre-chilled dialysis buffer (3.6 L) for 2 h at 4 &#176;C, then transfer the cassettes into a fresh dialysis buffer (3.6 L) for overnight. Make sure that the dialysis buffer is under constant agitation.</li>
		<li>Filter protein after dialysis through a 0.2 &#181;m acrodisc syringe filter using a syringe.</li>
		<li>Measure the protein concentration using a BCA protein assay kit.</li>
		<li>Concentrate or dilute if necessary to adjust the protein concentration to 0.3 mg/mL. 
		NOTE: Confirm purity by SDS-PAGE and Coomassie Blue staining.</li>
		<li>Make 1 mL aliquots of protein in microcentrifuge tubes and immediately store at -80 &#176;C until further use as described in the RT-QuIC protocol.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

RT-QuIC was previously employed to detect CWD prions in urine and fecal extracts of orally infected white-tailed deer and mule deer 38. The system shown in this manuscript is an adapted method of the RT-QuIC assay. Additional steps were incorporated into the &#34;classical&#34; RT-QuIC assay to improve the detection and sensitivity of the assay for CWD prions in fecal material of infected animals.
The low sensitivity of detection in feces extracts led us to the improvem...

Prion diseases or transmissible spongiform encephalopathies (TSE) are neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, and chronic wasting disease (CWD) in cervids 1,2. TSEs are characterized by distinctive spongiform appearance and loss of neurons in the brain. According to the &#34;protein only&#34; hypothesis, prions are mainly composed of PrPSc (&#39;Sc&#39; for scrapie) 3, a misfolded isoform of the host-encoded cellular prion protein, PrPC. PrPSc results from the conversion of PrPC into a conformation enriched in &#7526;-sheets 4,5,6 which can act as a seed to bind and convert other PrPC molecules. The newly generated PrPSc molecules are incorporated into a growing polymer 7,8 which breaks into smaller oligomers, resulting in higher numbers of infectious nuclei. PrPSc is prone to aggregation and is partially resistant to proteases 9,10.
CWD affects wild and farmed elk (Cervus canadensis), mule deer (Odocoileus hemionus), white-tailed deer (WTD; Odocoileus virginianus), moose (Alces alces), and reindeer (Rangifer tarandus tarandus) 11,12,13. It is considered the most contagious prion disease with horizontal transmission favored by cervid interactions and environmental persistence of infectivity 14,15. Unlike other prion diseases where PrPSc accumulation and infectivity are confined to the brain, in CWD these are also found in peripheral tissues and body fluids e.g. saliva, urine, and feces 16,17,18.
Immunohistochemistry is considered the gold standard for CWD diagnosis to detect PrPSc distribution and spongiform lesions 19,20. ELISA and in more rare cases, western blot are also used for CWD diagnostics. Thus, current prion disease diagnosis is mainly based on detecting prions in post-mortem tissues. Ante-mortem diagnosis for CWD is available by taking tonsils or recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies; however, this procedure is invasive and requires the capture of the animals. Thus, the use of easily accessible specimens, such as urine and feces, would be a practical way for CWD prion detection. However, those excreta harbor relatively low concentrations of prions below the detection limit of current diagnostic methods. Consequently, a more sensitive and high throughput diagnostic tool is needed. In vitro conversion systems, such as protein misfolding cyclic amplification assay (PMCA) 21, amyloid seeding assay, and real-time quaking-induced conversion (RT-QuIC) assay 22,23,24 are very powerful tools to exploit the self-propagating ability of PrPSc to mimic in vitro the prion conversion process and thereby amplify the presence of minute amounts of PrPSc to detectable levels 25,26. The RT-QuIC method, however, takes advantage of the fact that the conversion product enriched in &#946;-sheet secondary structure can specifically bind thioflavin T (Th-T). Therefore, recombinant PrP (rPrP) upon seeded conversion grows into amyloid fibrils which bind Th-T and thus can be detected in real time by measuring the fluorescence of Th-T expressed as relative fluorescence units (RFU) over time. Once monitored, the RFU can be used to evaluate relative seeding activities, and quantitative parameters such as the lag phase. The lag phase represents the time (h) required to reach the threshold, during which rPrP conversion at the early stage of the reaction is below the detection limit of Th-T fluorescence. The end of the apparent lag phase, concomitant to the formation of a sufficient amyloid nucleus (nucleation/elongation), occurs when the Th-T fluorescence exceeds the threshold level and becomes positive. The growth of amyloid fibrils can be detected in real time and the initial PrPSc or seeding activity contained in the sample is amplified by segmentation which generates more seeds. These seeds in turn induce a rapid exponential phase of amyloid fiber growth.
Because this assay is able to detect as low as 1 fg of PrPSc 24, the high sensitivity qualifies this technique to achieve ante mortem or non-invasive diagnosis by detecting PrPSc in various peripheral tissues, excreta or other kinds of specimen harboring low levels of infectivity. RT-QuIC definitely provides advantages over other assays for its reproducibility, practicality, rapidity (less than 50 h) and low costs compared to bioassays. It avoids the technical complexities such as sonication used in PMCA; also, it is performed in a tape-sealed microplate which minimizes the risk of aerosol contamination of each well. The multi-well format enables the analysis of up to 96 samples in the same experiment. To counter the recurrent problem of false positives and spontaneous conversion of rPrP in the in vitro conversion assays the implementation of a threshold (cut-off) in RT-QuIC is very useful. Indeed, based on the results of the negative control (average RFU of negative samples +5 SD 27), a baseline is set up from which discrimination between positive and negative samples can be done. The use of four replicates for each sample can thus help to define a sample as positive when at least 50% of the replicates show a positive signal, i.e. cross the cut-off 28. The homology between seed and substrate is not required in RT-QuIC, as e.g. in a previous study, hamster rPrP was found to be a more sensitive substrate compared to the homologous substrate in human PrPvCJD seeded and sheep scrapie seeded reactions 29. Hamster-sheep chimeric rPrP was also suggested to be a more well-suited substrate than human rPrP to detect human variant CJD prions 30. Thus, the use of rPrP substrates from different species is very common in this assay. This assay has been successfully applied to several prion diseases, such as sporadic CJD 31,32,33, genetic prion diseases 34, BSE 35,36,37, scrapie 23,36, and CWD 38,39,40,41,42. Studies using processed cerebrospinal fluid, whole blood, saliva, and urine as seeds in RT-QuIC were all successful to detect PrPSc 38,39,40,41,42. To foster the detection ability in samples such as blood plasma that may contain inhibitors of amyloid formation, Orr&#250; et al. (2011) developed a strategy to remove potential inhibitors of amyloid formation by combing PrPSc immunoprecipitation (IP) step and RT-QuIC, named &#34;enhanced QuIC&#34; assay (eQuIC). In addition, a substrate replacement step was employed after ~24 h of reaction time in order to improve the sensitivity. Ultimately, as low as 1 ag of PrPSc was detectable by eQuIC 30.
In order to purify feces extracts and remove possible assay inhibitors in feces, fecal samples collected at preclinical and clinical stages from elk upon experimental oral infection were homogenized in buffer containing detergents and protease inhibitors. The feces extracts were further submitted to different methodologies to concentrate PrPSc in the samples utilizing protein precipitation via sodium phosphotungstic acid (NaPTA) precipitation. The NaPTA precipitation method, first described by Safar et al.43, is used to concentrate PrPSc in test samples. The incubation of NaPTA with the sample results in preferential precipitation of PrPSc rather than PrPC. However, the molecular mechanism is still unclear. This step also helped containing and preventing the spontaneous conversion of rPrP, which is observed in some cases. Finally, the feces extracts were tested by optimized RT-QuIC using mouse rPrP (aa 23-231) as a substrate and including substrate replacement in the protocol to improve the sensitivity of detection.
The results here demonstrate that this improved method can detect very low concentrations of CWD prions and increases the sensitivity of detection and specificity in fecal samples compared to a protocol without NaPTA precipitation and substrate replacement. This method potentially can be applied to other tissues and body fluids and can be of great use for CWD surveillance in wild and captive cervids.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acrodisc seringe filters</td><td>PALL</td><td>4652</td><td></td></tr><tr><td>amicon Ultra-15 Centrifugal filter Unit</td><td>Millipore</td><td>UCF901024</td><td></td></tr><tr><td>BD 10 ml seringe</td><td>VWR</td><td>CA75846-842</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C0378</td><td></td></tr><tr><td>Corning bottle-top vacuum filters</td><td>Sigma-Aldrich</td><td>431118</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td>E4884</td><td></td></tr><tr><td>gentleMACS M Tube</td><td>Miltenyi Biotec</td><td>130-093-236</td><td></td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>G4505</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I5513</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>I9516</td><td></td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td></td></tr><tr><td>Luria-Bertani (LB) broth</td><td>ThermoFisher Scientific</td><td>12780029</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich</td><td>M9272</td><td></td></tr><tr><td>N2 supplement (100X)</td><td>ThermoFisher Scientific</td><td>15502048</td><td></td></tr><tr><td>N-lauroylsarcosine sodium salt (sarkosyl)</td><td>Sigma-Aldrich</td><td>ML9150</td><td></td></tr><tr><td>Nanosep centrifugal devices with omega membrane 100K</td><td>PALL</td><td>OD100C34</td><td></td></tr><tr><td>Nunc sealing tapes</td><td>ThermoFisher Scientific</td><td>232702</td><td></td></tr><tr><td>Parafilm M</td><td>VWR</td><td>52858-000</td><td></td></tr><tr><td>phenylmethylsulfonyl fluoride (PMSF)</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Protease inhibitor tablet</td><td>Roche</td><td>4693159001</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td></td></tr><tr><td>Sodium 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real-time quaking-induced conversion assay for detection of cwd prions in fecal material

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant prion protein (PrP) substrate using prion seeds as a template for the conversion. RT-QuIC is a novel high-throughput technique which is analogous to real-time polymerase chain reaction (PCR). Detection of amyloid fibril growth is based on the dye Thioflavin T, which fluoresces upon specific interaction with &#7526;-sheet rich proteins. Thus, amyloid formation can be detected in real time. We attempted to develop a reliable non-invasive screening test to detect chronic wasting disease (CWD) prions in fecal extract. Here, we have specifically adapted the RT-QuIC technique to reveal PrPSc seeding activity in feces of CWD infected cervids. Initially, the seeding activity of the fecal extracts we prepared was relatively low in RT-QuIC, possibly due to potential assay inhibitors in the fecal material. To improve seeding activity of feces extracts and remove potential assay inhibitors, we homogenized the fecal samples in a buffer containing detergents and protease inhibitors. We also submitted the samples to different methodologies to concentrate PrPSc on the basis of protein precipitation using sodium phosphotungstic acid, and centrifugal force. Finally, the feces extracts were tested by optimized RT-QuIC which included substrate replacement in the protocol to improve the sensitivity of detection. Thus, we established a protocol for sensitive detection of CWD prion seeding activity in feces of pre-clinical and clinical cervids by RT-QuIC, which can be a practical tool for non-invasive CWD diagnosis.

The CWD fecal extracts prepared at 10% (w/v) were able to seed RT-QuIC reaction, yet the sensitivity of detection was low 27. Using a specific buffer for fecal homogenization was a critical step to avoid high background fluorescence in RT-QuIC reactions concomitant to the use of mouse rPrP substrate rather than deer rPrP which allowed to get more specific results 27. The addition of NaPTA precipitation reduced the spontaneous conversion of r...

Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

The RT-QuIC technique is a sensitive in vitro cell-free prion amplification assay based mainly on the seeded misfolding and aggregation of recombinant ...

real-time-quaking-induced-conversion-assay-for-detection-cwd-prions

Research

JoVE Journal

Immunology and Infection

Here we describe a novel assay for monitoring prion uptake and trafficking by immune cells immediately following intraperitoneal inoculation by purifying and fluorescently labeling aggregated prion rods from infected brain material then monitoring their uptake and movement from the injection site and characterizing the cells mediating these events.

Monitoring Immune Cells Trafficking Fluorescent Prion Rods Hours after Intraperitoneal Infection

Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers.  It can also be used as a prion detection assay.

Protein Misfolding Cyclic Amplification of Prions

Scavengers have potential to translocate infectious transmissible spongiform encephalopathy prions in their feces to disease-free areas.  We detail methods used to determine if mouse-adapted scrapie prions remain infectious after passage though the digestive tract of American crows (Corvus brachyrhynchos), a common consumer of dead animals.

Procedures for Identifying Infectious Prions After Passage Through the Digestive System of an Avian Species

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Measuring the barrier to the interspecies transmission of prion diseases is challenging and typically involves animal challenges or biochemical assays. Here, we present an in vitro prion protein conversion assay with the ability to predict species barriers.

Assessing Transmissible Spongiform Encephalopathy Species Barriers with an In Vitro Prion Protein Conversion Assay

Provided is a protocol for developing a real-time recombinase polymerase amplification assay to quantify initial concentration of DNA samples using either a thermal cycler or a microscope and stage heater. Also described is the development of an internal positive control. Scripts are provided for processing raw real-time fluorescence data.

Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control

Rapid and accurate detection of plant pathogens on-site, especially soil-borne pathogens, is crucial to prevent further inoculum production and proliferation of plant diseases in the field. The method developed here using a portable real-time PCR detection system enables on-site diagnosis under field conditions.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

This protocol presents a qRT-PCR-based approach for determining the rabies virus nucleoprotein (N) gene copy number within various bovine brain anatomical structures using in vitro transcription.

Quantitation of Rabies Virus in Various Bovine Brain Structures

Immunolabeling abnormal prion protein using immunohistochemistry protocols requires specific sample and anti-PrP antibody preparation methodologies. The present protocol describes the key steps in epitope demasking to ensure proper PrP immunolabeling and to minimize non-specific background staining. Also, this approach considers biosafety measures when conducting immunohistochemistry studies with the prion-infected tissues.

Detection of Abnormal Prion Protein by Immunohistochemistry

A method to assess commonly used areas in laboratory settings for prion contamination and effective decontamination is lacking. The protocol described here provides key fundamentals for implementing a laboratory prion safety swipe test that can easily be modified to meet the individual needs of specific laboratories.

Prion Safety Laboratory Swipe Test

Learn the RT-QuIC assay for detecting chronic wasting disease prions in cervid fecal samples. This sensitive, high-throughput method enables researchers to study prion shedding timing and transmission dynamics in wildlife populations.

Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material

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