Our multidisciplinary group is trying to understand how an infectious protein, a prion, can perform complex biological tasks. We're also interested in how prions can persist in the environment for years to decades. Following prion decontamination procedures, a robust method for determining the efficacy of prion inactivation was not available.
We are trying to address this issue with our research. The swabbing method, coupled with RT-QuIC, allows for sampling a wide range of surfaces to detect prion seeding activities. This can be used for to detect surface associated prions in laboratory, clinical, or environmental settings.
I am interested in understanding how prions interact with surfaces and the requirements of surface bound prions to establish infection. To begin, identify and label appropriate surveillance areas for swabbing. Prepare two 1.5 milliliter micro centrifuge tubes for each identified area and add 250 microliters of DPBS to the first tube, leaving the second one empty.
Retrieve foam tipped swabs from storage in an area free of prion contamination. Prepare the positive and negative controls using appropriate brain homogenates or BH in DPBS. Using clean gloves, retrieve the required number of foam swabs from the packaging and place them handle side down into a tube rack.
Ensuring the tips do not contact other surfaces. Holding a clean foam swab by the handle, apply 50 microliters of the respective positive and negative control samples to the swab tips. Using scissors, cut off the excess handle of the swab to approximately half of its length and place the swab in the preloaded micro centrifuge tube to submerge the foam tip in DPBS.
Holding a clean foam tip swab by the handle, pre-wet the foam tip with molecular grade water and shake off the excess water. Place the moistened foam tip on the area chosen for surveillance and swab the area back and forth approximately 10 times while simultaneously rotating the swab tip on the surface. Using scissors, cut off the excess handle of the swab to approximately half its length and place the swab into the preloaded micro centrifuge tube to submerge the foam tip in DPBS, ensuring the lid can close completely.
Discard gloves and replace them with new ones before proceeding to the next swabbing site to minimize cross contamination. For swab extraction, place the micro centrifuge tube into a circular tube rack and submerge the rack into the cup horn sonicater water bath. Ensure the foam swab in DPBS within the tube is below the water surface, leaving the handle portions unsubmerged.
Sonicate the sample for 15 seconds in total with five seconds on and five seconds off cycles. Following sonication, centrifuge the tube for approximately 15 seconds to collect the DPBS at the bottom of the tube prior to transfer. Using a P 1000 pipette set to 250 microliters, carefully collect all the liquid swab extract from the bottom into the corresponding pre-labeled empty tube.
Discard the used tube containing the swab. Concentrate the sample in a vacuum concentrator, ensuring the tube cap is open, upon cycle completion, ensure that the sample is completely concentrated with only a pellet remaining. Store the pellet at minus 80 degrees Celsius until further use.
For RT-QuIC analysis, prepare a negative and positive plate controls with corresponding brain homogenates diluted in tissue dilution solution. Now thaw the stored swab extract pellet from the minus 80 degrees Celsius freezer and resuspend the dried swab extract with 50 microliters of molecular grade water by pipetting. Vortex briefly, and let the sample sit at room temperature.
Load two microliters of negative and positive plate controls followed by swab extracts into the respective RT-QuIC plate wells. Negative control swabs did not cross the positive fluorescence threshold, confirming no contamination during procedures. Positive control swab extracts exhibited consistent seeding in all replicate wells, demonstrating proper detection sensitivity.
Surface swab samples from non contaminated areas failed to show seeding above the threshold, confirming prion negativity. Prion contaminated surfaces, exhibited seeding above the threshold with varied maximum point ratios and fluorescence times compared to controls. Bleach-treated prion-contaminated surfaces failed to seed RT-QuIC, demonstrating effective disinfection.
Some surface artifacts exhibited altered kinetic curves and longer fluorescence times indicating potential false positives, likely due to the presence of dust or residual chemicals on a surface.
