Name: real-time quaking-induced conversion assay for detection of cwd prions in fecal material
Uploaded: 2017-09-29T15:00:00
Description: Watch this Scientific Journal Video about Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material at JoVE.com

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 (...

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>
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		<tr>
			<td>Materials</td>
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			<td>Acrodisc seringe filters</td>
			<td>PALL</td>
			<td>4652</td>
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			<td>amicon Ultra-15 Centrifugal filter Unit</td>
			<td>Millipore</td>
			<td>UCF901024</td>
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			<td>BD 10 ml seringe</td>
			<td>VWR</td>
			<td>CA75846-842</td>
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			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C0378</td>
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			<td>Corning bottle-top vacuum filters</td>
			<td>Sigma-Aldrich</td>
			<td>431118</td>
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			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E4884</td>
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			<td>gentleMACS M Tube</td>
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			<td>130-093-236</td>
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			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
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			<td>Imidazole</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
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			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>I9516</td>
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			<td>Kanamycin sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
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			<td>Luria-Bertani (LB) broth</td>
			<td>ThermoFisher Scientific</td>
			<td>12780029</td>
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			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
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			<td>N2 supplement (100X)</td>
			<td>ThermoFisher Scientific</td>
			<td>15502048</td>
			<td></td>
		</tr>
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			<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>
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			<td>Nunc sealing tapes</td>
			<td>ThermoFisher Scientific</td>
			<td>232702</td>
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			<td>Parafilm M</td>
			<td>VWR</td>
			<td>52858-000</td>
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			<td>phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
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			<td>Protease inhibitor tablet</td>
			<td>Roche</td>
			<td>4693159001</td>
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			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
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			<td>Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
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			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Calbiochem</td>
			<td>7910-OP</td>
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			<td>sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>342483</td>
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			<td>Sodium phosphate dibasic anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S9763</td>
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			<td>Sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
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			<td>Sodium phosphotungstate hydrate (NaPTA)</td>
			<td>Sigma-Aldrich</td>
			<td>496626</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin T</td>
			<td>Sigma-Aldrich</td>
			<td>T3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-Hydroxy-Methyl-Amino-Methan (Tris)</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
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			<td>Triton-100</td>
			<td>Calbiochem</td>
			<td>9410-OP</td>
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			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949</td>
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		<tr>
			<td>Name</td>
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			<td>BugBuster Master Mix</td>
			<td>Nogagen</td>
			<td>71456-4</td>
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			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td>1018401</td>
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			<td>Phosphate-buffered saline (PBS) pH 7.4 (1X)</td>
			<td>Life Technoligies</td>
			<td>P5493</td>
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			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
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			<td>Name</td>
			<td>Company</td>
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			<td>Standards and commercial kits</td>
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			<td>Express Autoinduction System 1</td>
			<td>Novagen</td>
			<td>71300-4</td>
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			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
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			<td>Equipment setup</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>AKTA protein purification systems FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
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			<td>Beckman Avanti J-25 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
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		<tr>
			<td>Beckman rotor JA-25.50</td>
			<td>Beckman Coulter</td>
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		<tr>
			<td>Beckman rotor JA-10</td>
			<td>Beckman Coulter</td>
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		</tr>
		<tr>
			<td>FLUOstar Omega microplate reader</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Sofware</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MARS Data Analysis</td>
			<td>BMG Labtech</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism6</td>
			<td>GraphPad software</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

Watch this Scientific Journal Video about Real-time Quaking-induced Conversion Assay for Detection of CWD Prions in Fecal Material at JoVE.com

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

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 ...

The overall goal of this protocol for real-time quaking-induced conversion assay is to allow sensitive detection of chronic wasting disease prions in cervid fecal samples. This method can help answer key questions in the CWD research field such as when are infected cervid CWD prions in feces. The main advantage of this technique is that it is specific, sensitive, and it enables high throughput of samples.Demonstrating the procedure will be Ginny Cheng, a lab technician from my laboratory. To begin this procedure, add one gram of fecal material to a tube containing 10 milliliters feces extract buffer. Using a dissociator with a preset program for proteins, homogenize the fecal pellets for one minute at room temperature.Repeat this two to three times until the fecal samples are completely homogenized. Then use parafilm to seal the tubes and place them onto a rocking platform or rotary shaker for a one hour incubation at room temperature. After this, centrifuge at 18, 000 g's for five minutes at room temperature.Collect the supernatants and aliquot them into 1.5 milliliter tubes. Store at minus 80 degrees Celsius until ready to use. To begin the sodium phosphotungstic acid precipitation method, add 250 microliters of sarkosyl to one milliliter of fecal protein extract.Using parafilm, seal the tube. Place the tube in a thermal mixer and incubate at 37 degrees Celsius for 30 minutes with constant shaking at 1, 400 rpm. After this, add a stock solution containing 10%sodium phosphotungstic acid and 170 millimolar magnesium chloride such that the final concentration of sodium phosphotungstic acid in the samples is 3%Transfer the samples to the thermal mixer and incubate at 37 degrees Celsius for two hours with constant shaking at 400 rpm.Next, centrifuge at 15, 800 g's for 30 minutes at 14 degrees Celsius. Carefully remove the supernatants and wash the pellets by resuspending them in wash buffer. Then centrifuge at 15, 800 g's for 15 minutes at 14 degrees Celsius.Carefully remove the supernatants and resuspend the pellets in 100 microliters of RT-QuIC dilution buffer. Store the treated samples at minus 20 degrees Celsius until ready to perform the RT-QuIC assay. To begin, add 0.032 grams of Thioflavin T to 10 milliliters of double distilled water to prepare a fresh 10 millimolar Thioflavin T stock solution.Next, use double distilled water to make a one to 10 dilution of the stock solution resulting in a final concentration of one millimolar Thioflavin T.Using a syringe equipped with a 0.2 micrometer Acro disk filter, filter the one millimolar Thioflavin T solution. Then thaw the most recombinant prion protein substrate. Load 500 microliters of recombinant prion protein substrate at a time into 100 kilo Dalton size exclusion filter microtubes.Centrifuge at 14, 000 g for five minutes at room temperature. After this, transfer the eluted substrate into a sterile 1.5 milliliter tube. Store at four degrees Celsius until ready to use.Next, thaw the stored RT-QuIC dilution buffer. Dilute the seed and prepare the stock solutions and RT-QuIC reaction mixture as outlined in the text protocol. Using a pipette with a filter tip, mix the reaction mixture by pipetting up and down.Pour the mixture into a 50 milliliter disposal pipetting reservoir. Using a multichannel pipette, load 98 microliters of RT-QuIC reaction mixture into each well of a 96-well optical bottom plate. To each reaction, add two microliters of either undiluted or 10 fold serially diluted fecal protein extract.Then use sealing tape to seal the plate. Incubate in a plate reader at 42 degrees Celsius for 25 hours with repeated cycles of one minute of double orbital shaking at 700 rpm and one minute of resting throughout the incubation. Meanwhile, define the fluorescence measurement settings as outlined in the text protocol.After 25 hours, remove the plate from the plate reader. Spin down the plate at 3, 000 g's for three minutes to avoid potential contamination between the wells. Next, remove the seal from the plate.Prepare a new plate by adding 90 microliters of RT-QuIC reaction mixture containing fresh substrate and fresh Thioflavin T fluorescence dye. Transfer 10 microliters from each well of the first plate to the newly prepared 96-well plate. Use sealing tape to seal the new plate.After defining the fluorescence measurement settings, continue the assay for an additional 50 hours. Read and document the Thioflavin T fluorescence signal for each well every 15 minutes. Then collect and plot the data as outlined in the text protocol.In this study, fecal material from noninfected elk or mule deer are homogenized in feces extract buffer to obtain a final sample concentration of 10%Some homogenates are then processed and 10 times concentrated by sodium phosphotungstic acid precipitation. While a reduction of spontaneous conversions is seen in purified samples, all samples showed low fluorescence signals in CWD positive fecal samples. To improve the amplification of prions and enhance the sensitivity of detection, a substrate replacement step is incorporated.Samples that underwent substrate replacement as part of the RT-QuIC protocol show an increase in the sensitivity and specificity of detection with 14 of the 18 samples being positive and none of the negative controls registering as false positive. Once mastered, fecal extract preparation and PRP/SCRP concentration can be done in about four hours and the RT-QuIC assay can be set up in about 1/1-2 hours if it is performed properly. While attempting the procedure, it is important to remember to filter the substrate but not never vortex it.Following the procedure, other methods such as western blotting can be used to answer additional questions like how resistant proteases are the resulting products. After its development, this technique paved the way for scientists in the field of prion research to explore the presence of prions in live animals by testing fecal samples. After watching this video, you should have a good understanding of how to prepare and concentrate PRP/SCRP from fecal extracts and how to perform a modified RT-QuIC assay to obtain sensitive and specific prion detection.Don't forget that working with prions can be extremely hazardous and that it is important to wear proper PPEs and to work under a biosafety cabinet while performing the procedure.

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During activation, the metabolism of T cells adapts to changes that impact their fate. An increase in mitochondrial oxidative phosphorylation is ...

Cell-Based Electrical Impedance Platform to Evaluate Zika Virus Inhibitors in Real Time

Cell-based electrical impedance (CEI)&#160;technology measures changes in impedance caused by a growing or manipulated adherent cell monolayer on culture ...