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.
