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 improvement of the RT-QuIC protocol. To achieve sensitive in vitro detection of prions in feces, critical steps were added for sample preparation in order to remove components which interfere with prion conversion and/or propagation and enhance the sensitivity of detection. Incorporating substrate replacement in the protocol of RT-QuIC and NaPTA/sarkosyl treatment was critical for detecting seeding activity in pre-clinical and clinical CWD-infected elk, and for enhancing the detection limits of prion conversion in feces extracts.
NaPTA precipitation is a common technique usually accompanied by sarkosyl extraction to isolate and concentrate PrPSc to a detectable level. NaPTA is known to preferably precipitate PrPSc over PrPC 43 while sarkosyl is a detergent known to facilitate prion conversion at low concentrations in a cell free system 44. In alignment with this, using a combination of NaPTA and sarkosyl might generate a higher yield of fibrillar aggregates in vitro as shown before 45,46,47. This methodology has been incorporated previously into RT-QuIC assay to detect peripheral CWD prions successfully in specimen such as purified saliva 39 and whole blood 40. Our study provides first evidence that incorporating NaPTA/sarkosyl purification in the protocol of fecal sample preparation enables CWD prion detection by RT-QuIC. Moreover, with this methodology we were able to reduce spontaneous conversion of rPrP substrate in CWD-negative fecal homogenates in RT-QuIC assay.
A potential mechanism has been proposed to explain the effect of substrate replacement 30. In the first rounds of reaction (before the addition of fresh substrate), only a small amount of seeds is added to RT-QuIC reactions. While only a portion of rPrP is incorporated into the seeded reactions, the rest of the substrate could either be used up by interacting with the walls of reaction plate wells to form non-amyloid aggregates, or be altered to a form which is less prone to be converted by seeded RT-QuIC products rather than the original seeds. As a result, the incorporation rate of rPrP to the seeds is slow, and the fibril formation is not detectable in the lag phase. As the seeded RT-QuIC products grown in the lag phase are finally elongated to reach a &#34;fast assembly stage&#34;, prions can be amplified rapidly by segmentation through shaking or lateral addition of smaller aggregates. At this stage, the fresh substrate added to the reaction is readily incorporated into the seeded products rather than forming unspecific aggregates. The incorporation of the substrate replacement step in our protocol was a modification that increased sensitivity; it was useful to detect prion seeds in samples with a very low amount of PrPSc from pre-clinical animals and/or containing inhibitory compounds.
Using RT-QuIC assay rather than PMCA, which is the first in vitro protein misfolding amplification assay described 21, has several advantages. In PMCA the tubes are incubated and sonicated in a water bath contained within the sonicator; the tubes positioned at the periphery of the sonicator show less efficacy of amplification compared to the tubes positioned in the center 48. The RT-QuIC system quaking seems to be easier to control. The use of a 96 well format is a real advantage of RT-QuIC, however, the mb-PMCA developed by Moudjo et al. 49,50, showed similar benefits but still is less used in PMCA.
As substrate, RT-QuIC uses recombinant rPrP for conversion; in contrast, PMCA in most cases uses normal brain homogenate. In addition, in RT-QuIC no sequence homology is required between seed and substrate 51-53.
The presence of cofactors is necessary for prion replication in PMCA and the resulting product is infectious. However, in RT-QuIC assay, the amplified PrP is not infectious. In in vitro amplification assays the occurrence of false positive reactions most probably because of spontaneous conversion of PrPC is a recurring problem. Therefore, the assay and conditions used in this study were tailored to minimize such disturbances to maximize the difference between seeded rPrP-conversion and spontaneous conversion.
In conclusion, NaPTA/sarkosyl treatment enables removal of assay inhibitors in feces and reduces spontaneous conversion. Interestingly, the treatment did not hamper seeding activity of precipitatedPrPSc. In addition, the sensitivity of detection was significantly improved when substrate replacement was concomitantly adopted.

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>Materials</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 deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Calbiochem</td>
			<td>7910-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>342483</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<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>
			<td></td>
		</tr>
		<tr>
			<td>Triton-100</td>
			<td>Calbiochem</td>
			<td>9410-OP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Commercial buffers and solutions</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BugBuster Master Mix</td>
			<td>Nogagen</td>
			<td>71456-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA superflow</td>
			<td>Qiagen</td>
			<td>1018401</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) pH 7.4 (1X)</td>
			<td>Life Technoligies</td>
			<td>P5493</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Standards and commercial kits</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Express Autoinduction System 1</td>
			<td>Novagen</td>
			<td>71300-4</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</tr>
		<tr>
			<td>Equipment setup</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA protein purification systems FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman Avanti J-25 Centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman rotor JA-25.50</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beckman rotor JA-10</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</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 rPrP in RT-QuIC (Figure 1) without inhibiting amplification of the seeding activity of the reactions (Figure 2). Although better amplification was observed upon NaPTA treatment, resulting fluorescence signals of CWD-positive fecal samples were still low (Figure 1). In addition, the prion amplification of some samples reached a constant plateau at low fluorescence levels. This indicates the saturation of reactions, which can be due to the degradation/denaturation of rPrP or the formation of off-pathway aggregates which consume the rPrP pool 30. To further improve the amplification of prions and enhance the sensitivity of detection, a substrate replacement step was incorporated into the protocol. The introduction of substrate replacement to the RT-QuIC protocol (Figure 2) increased the sensitivity (77%; 14 out of 18 samples in RT-QuIC were positive) and specificity (100%; none of the negative controls in RT-QuIC turned positive) of detection (see Table 1 from Cheng et al. 27). Finally, all these steps (Figure 3) led to the optimization of a reliable and sensitive protocol for RT-QuIC detection of CWD prions, using specimen containing low levels of infectivity.
Substrate replacement was introduced after the first 25 h of RT-QuIC reaction, by replenishing reaction buffer containing fresh substrate and fresh fluorescence dye Th-T. By introducing the substrate replacement step in the protocol, the RT-QuIC reaction time was extended from 50 h to 75 h. As shown in Figure 2 with the incorporation of substrate replacement, a significant improvement of prion amplification was observed.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56373/56373fig1.jpg" /> 
Figure 1: Reduced spontaneous conversion of mouse rPrP substrate in RT-QuIC seeded with purified CWD-negative fecal homogenate. Fecal homogenates of non-infected elk (C181-006) or mule deer were homogenized in feces extract buffer to obtain a final concentration of 10% (w/v). For purification, these fecal homogenates were processed and 10-times concentrated by NaPTA precipitation. The unpurified fecal homogenates (a), NaPTA-purified and concentrated forms (b) diluted as indicated were used to seed quadruplicate RT-QuIC reactions with mouse rPrP as a substrate. The y-axes show relative Th-T fluorescence units, the x-axes depict the reaction time. A reduction of spontaneous conversions was seen in NaPTA-purified fecal samples (b). Data used from Cheng et al. 27. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56373/56373fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56373/56373fig2.jpg" /> 
Figure 2. Improved detection of CWD prions in feces using substrate replacement. Fecal homogenates of individual elk (C051-05, C052-05) orally infected with CWD prions were purified and concentrated by NaPTA precipitation. NaPTA-treated samples were diluted between 2 x 10-1 to 2 x 10-3 used to seed quadruplicate RT-QuIC reactions with mouse rPrP substrate. The RT-QuIC assay was performed without substrate replacement (a) in a regular period of 50 h or with the incorporation of substrate replacement for an extended incubation period of 75 h (b). For the latter, substrate replacement was introduced after the first 25 h of RT-QuIC reaction. Ninety percent of the reaction volume was removed and replaced by freshly prepared RT-QuIC reaction mixture containing rPrP substrate and Th-T. By introducing the substrate replacement step in the protocol, the RT-QuIC reaction time was extended from 50 h to 75 h. Data used from Cheng et al. 27. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56373/56373fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56373/56373fig3.jpg" /> 
Figure 3: Flow diagram describing PrPSc fecal extraction from CWD-infected cervids and seeding activity and prion amplification using RT-QuIC assay. Fecal samples from CWD-infected animals are homogenized in a fecal extraction buffer, submitted to NaPTA precipitation method and tested by RT-QuIC assay. The latter was performed by introducing a substrate replacement step. The incorporation of NaPTA and substrate replacement steps resulted in a reduction of spontaneous conversion and strong improvement in seeding activity and prion amplification. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56373/56373fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.5K Views.  University of Calgary. Here, we present a protocol to describe a simple, fast and efficient prion amplification technique, the real-time quaking-induced conversion (RT-QuIC) method.

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

Protein Misfolding Cyclic Amplification of Prions

Prions are infectious agents that cause the inevitably fatal transmissible spongiform encephalopathy (TSE) in animals and humans9,18. The prion protein has two distinct isoforms, the non-infectious host-encoded protein (PrPC) and the infectious protein (PrPSc), an abnormally-folded isoform of PrPC 8.
One of the challenges of working with prion agents is the long incubation period prior to the development of clinical signs following host inoculation13. This traditionally mandated long and expensive animal bioassay studies. Furthermore, the biochemical and biophysical properties of PrPSc are poorly characterized due to their unusual conformation and aggregation states.
PrPSc can seed the conversion of PrPC to PrPSc in vitro14. PMCA is an in vitro technique that takes advantage of this ability using sonication and incubation cycles to produce large amounts of PrPSc, at an accelerated rate, from a system containing excess amounts of PrPC and minute amounts of the PrPSc seed19. This technique has proven to effectively recapitulate the species and strain specificity of PrPSc conversion from PrPC, to emulate prion strain interference, and to amplify very low levels of PrPSc from infected tissues, fluids, and environmental samples6,7,16,23 .
This paper details the PMCA protocol, including recommendations for minimizing contamination, generating consistent results, and quantifying those results. We also discuss several PMCA applications, including generation and characterization of infectious prion strains, prion strain interference, and the detection of prions in the environment.

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.

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Detection of MicroRNAs in Microglia by Real-time PCR in Normal CNS and During Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many diseases associated with neuroinflammation such as multiple sclerosis (MS)2. Microglia in a normal CNS express macrophage marker CD11b and exhibit a resting phenotype by expressing low levels of activation markers such as CD45. During pathological events in the CNS, microglia become activated as determined by upregulation of CD45 and other markers3. The factors that affect microglia phenotype and functions in the CNS are not well studied. MicroRNAs (miRNAs) are a growing family of conserved molecules (~22 nucleotides long) that are involved in many normal physiological processes such as cell growth and differentiation4 and pathologies such as inflammation5. MiRNAs downregulate the expression of certain target genes by binding complementary sequences of their mRNAs and play an important role in the activation of innate immune cells including macrophages6 and microglia7. In order to investigate miRNA-mediated pathways that define the microglial phenotype, biological function, and to distinguish microglia from other types of macrophages, it is important to quantitatively assess the expression of particular microRNAs in distinct subsets of CNS-resident microglia. Common methods for measuring the expression of miRNAs in the CNS include quantitative PCR from whole neuronal tissue and in situ hybridization. However, quantitative PCR from whole tissue homogenate does not allow the assessment of the expression of miRNA in microglia, which represent only 5-15% of the cells of neuronal tissue. Hybridization in situ allows the assessment of the expression of microRNA in specific cell types in the tissue sections, but this method is not entirely quantitative. In this report we describe a quantitative and sensitive method for the detection of miRNA by real-time PCR in microglia isolated from normal CNS or during neuroinflammation using experimental autoimmune encephalomyelitis (EAE), a mouse model for MS. The described method will be useful to measure the level of expression of microRNAs in microglia in normal CNS or during neuroinflammation associated with various pathologies including MS, stroke, traumatic injury, Alzheimer's disease and brain tumors.

Microglia are resident macrophages that provide the first line of defense and immune surveillance of the central nervous system. MicroRNAs are regulatory molecules that play an important role in many physiological processes including activation and differentiation of macrophages. In this article, we describe the method for measurement of microRNAs in microglia.

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

Real-time Live Imaging of T-cell Signaling Complex Formation

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating the activation and responses of multiple immune cells 3,4. T-cell activation is dependent on the recognition of specific antigens displayed by antigen presenting cells (APCs). The T-cell antigen receptor (TCR) is specific to each T-cell clone and determines antigen specificity 5. The binding of the TCR to the antigen induces the phosphorylation of components of the TCR complex. In order to promote T-cell activation, this signal must be transduced from the membrane to the cytoplasm and the nucleus, initiating various crucial responses such as recruitment of signaling proteins to the TCR;APC site (the immune synapse), their molecular activation, cytoskeletal rearrangement, elevation of intracellular calcium concentration, and changes in gene expression 6,7. The correct initiation and termination of activating signals is crucial for appropriate T-cell responses. The activity of signaling proteins is dependent on the formation and termination of protein-protein interactions, post translational modifications such as protein phosphorylation, formation of protein complexes, protein ubiquitylation and the recruitment of proteins to various cellular sites 8. Understanding the inner workings of the T-cell activation process is crucial for both immunological research and clinical applications.
Various assays have been developed in order to investigate protein-protein interactions; however, biochemical assays, such as the widely used co-immunoprecipitation method, do not allow protein location to be discerned, thus precluding the observation of valuable insights into the dynamics of cellular mechanisms. Additionally, these bulk assays usually combine proteins from many different cells that might be at different stages of the investigated cellular process. This can have a detrimental effect on temporal resolution. The use of real-time imaging of live cells allows both the spatial tracking of proteins and the ability to temporally distinguish between signaling events, thus shedding light on the dynamics of the process 9,10. We present a method of real-time imaging of signaling-complex formation during T-cell activation. Primary T-cells or T-cell lines, such as Jurkat, are transfected with plasmids encoding for proteins of interest fused to monomeric fluorescent proteins, preventing non-physiological oligomerization 11. Live T cells are dropped over a coverslip pre-coated with T-cell activating antibody 8,9, which binds to the CD3/TCR complex, inducing T-cell activation while overcoming the need for specific activating antigens. Activated cells are constantly imaged with the use of confocal microscopy. Imaging data are analyzed to yield quantitative results, such as the colocalization coefficient of the signaling proteins.

We describe a live-cell imaging method that provides insight into protein dynamics during the T-cell activation process. We demonstrate the combined usage of the T-cell spreading assay, confocal microscopy and imaging analysis to yield quantitative results to follow signaling complex formation throughout T-cell activation.

Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

Following in Real Time the Impact of Pneumococcal Virulence Factors in an Acute Mouse Pneumonia Model Using Bioluminescent Bacteria

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. Despite advances in knowledge of this illness, the availability of intensive care units (ICU), and the use of potent antimicrobial agents and effective vaccines, the mortality rates remain high1. Streptococcus pneumoniae is the leading pathogen of community-acquired pneumonia (CAP) and one of the most common causes of bacteremia in humans. This pathogen is equipped with an armamentarium of surface-exposed adhesins and virulence factors contributing to pneumonia and invasive pneumococcal disease (IPD). The assessment of the in vivo role of bacterial fitness or virulence factors is of utmost importance to unravel S. pneumoniae pathogenicity mechanisms. Murine models of pneumonia, bacteremia, and meningitis are being used to determine the impact of pneumococcal factors at different stages of the infection. Here we describe a protocol to monitor in real-time pneumococcal dissemination in mice after intranasal or intraperitoneal infections with bioluminescent bacteria. The results show the multiplication and dissemination of pneumococci in the lower respiratory tract and blood, which can be visualized and evaluated using an imaging system and the accompanying analysis software.

Streptococcus pneumoniae is the leading pathogen causing severe community-acquired pneumonia and responsible for over 2&#160;million deaths worldwide. The impact of bacterial factors implicated in fitness or virulence can be monitored in real-time in an acute mouse pneumonia or bacteremia model using bioluminescent bacteria.

Pneumonia is one of the major health care problems in developing and industrialized countries and is associated with considerable morbidity and mortality. ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological malignancies such as childhood acute lymphocytic leukemia. Efforts are currently underway to extend this promising therapy to solid tumors in addition to other hematological cancers. Here, we describe the development and production of potent CAR T cells targeting antigens with unique or preferential expression on solid and liquid tumor cells. The in vitro potency of these CAR T cells is then evaluated in real-time using the highly sensitive impedance-based xCELLigence assay. Specifically, the impact of different costimulatory signaling domains, such as glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR), on the in vitro potency of CAR T cells is examined. This report includes protocols for: generating CAR T cells for preclinical studies using lentiviral gene transduction, expanding CAR T cells, validating CAR expression, and running and analyzing xCELLigence potency assays.

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Chimeric antigen receptor (CAR) T-cell therapy for cancer has achieved significant clinical benefit for resistant and refractory hematological ...

Pan-lyssavirus Real Time RT-PCR for Rabies Diagnosis

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to confirm rabies diagnosis but have only recently been accepted by the OIE (World Organisation for Animal Health) as a primary method to detect rabies infection. Real-time RT-PCR assays provide real-time data, and are closed-tube systems, minimizing the risk of contamination during setup. DNA intercalating fluorochrome real-time RT-PCR assays do not require expensive probes, minimizing the cost per sample, and when the primers are designed in conserved regions, assays that are specific across virus genera rather than specific to just one virus species are possible. Here we describe a pan-lyssavirus SYBR real-time RT-PCR assay that detects lyssaviruses across the Lyssavirus genus, including the most divergent viruses IKOV, WCBV and LLEBV. In conjunction with dissociation curve analysis, this assay is sensitive and specific, with the advantage of detecting all lyssavirus species. The assay has been adopted in many diagnostic laboratories with quality assured environments, enabling robust, rapid, sensitive diagnosis of animal and human rabies cases.

This real-time RT-PCR using dsDNA intercalating dye is suitable to diagnose lyssavirus infections. The method begins with RNA extracted from rabies suspected ante-mortem or post-mortem samples, detailing master mix preparation, RNA addition, setup of the real-time machine and correct interpretation of results.

Molecular assays are rapid, sensitive and specific, and have become central to diagnosing rabies. PCR based assays have been utilized for decades to ...

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician and the patient. A culture is also prone to false negative results from random loss of viability during specimen handling, overgrowth of other fecal flora, and poor growth of several pathogenic Campylobacter species on traditional media. These problems can confound clinical decisions on patient treatment and have limited the field from answering fundamental questions on Campylobacter growth and infections. We describe a procedure that estimates the lower limit of bacterial numbers that can be detected by a culture and a method for quantifying survival of C. jejuni in media used for transport of this fragile organism. Knowing this information, it becomes possible to set clinically relevant detection thresholds for diagnostic tests and address unstudied issues of whether non-symptomatic colonization is prevalent, if co-infection with other enteric pathogens is common, or if bacterial load correlates with symptoms or serious sequelae. The study also included testing of 1,552 prospectively collected patient diarrheal fecal specimens that were initially classified by conventional culture and further tested by a new enzyme immunoassay. Positive and discrepant specimens were then screened by four molecular methods to assign true-positive or true-negative status. The 5 non-culture methods showed complete agreement on all 48 positive and discrepant specimens, while the culture mis-identified 14 (28%). The specimens that were incorrectly identified by culture included 13 false negative and 1 false positive sample. This basic protocol can be used with multiple Campylobacter spp. and will allow the numbers of Campylobacter bacteria that produce symptoms of gastroenteritis in humans to be determined and for prevalence rates to be updated.

Culture Methods to Determine the Limit of Detection and Survival in Transport Media of Campylobacter Jejuni in Human Fecal Specimens

Although stool culture for Campylobacter is imprecise, it is still considered the gold standard for identification. Methods to determine the limit of detection and survival in transport media of C. jejuni in human stool are described and compared with a new immunoassay with better accuracy.

A culture from human stool for diagnosis of Campylobacter-based intestinal illness takes several days, a wait that taxes the fortitude of the physician ...

Fecal (micro) RNA Isolation

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs from fecal samples of animal and human subjects. The aim is to isolate total RNA with high purity and quantity for downstream analyses such as RNA sequencing, RT-PCR, and micro-array. The advantages of this optimized protocol in the miRNA isolation are capabilities of isolating highly purified RNA products with additional washing steps described, increased quantity of RNA obtained with an improved method in the resuspension of sample, and important tips of decontamination. One limitation is the inability to process and purify larger sample of more than 200 mg as these sample sizes would cause a difficulty in the clear formation of the interphase. Consequently, the large sample size may contaminate the aqueous phase to be extracted as described in the protocol with organic matters that affect the quality of RNA isolated in the end. However, RNA isolates from a sample of up to 200 mg are sufficient for most of downstream analyses.

Fecal micro RNA Isolation

This protocol isolates high quality total RNA from fecal samples of animal and human subjects. A commercial miRNA isolation kit is used with significant adaption to isolate pure RNA with optimized quantity and quality. The RNA isolates are good for most downstream RNA assays such as sequencing, micro-array, and RT-PCR.

It is becoming clear that RNA exists in the gut lumen and feces in animals and humans. The protocol described below isolates total RNA including microRNAs ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...