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[Display omitted] • An innovative method for prion recovery from swabs was developed. • Recovery of prions decreased as swab-drying time was increased. • Recovery of CWD prions from stainless steel and glass was approximately 30%. • RT-QuIC enhanced CWD prion detection by 4 orders of magnitude. • Surface-recovered CWD prion was sufficient for efficient RT-QuIC detection. Chronic wasting disease (CWD) has been identified in 30 states in the United States, four provinces in Canada, and recently emerged in Scandinavia. The association of CWD prions with environmental materials such as soil, plants, and surfaces may enhance the persistence of CWD prion infectivity in the environment exacerbating disease transmission. Identifying and quantifying CWD prions in the environment is significant for prion monitoring and disease transmission control. A systematic method for CWD prion quantification from associated environmental materials, however, does not exist. In this study, we developed an innovative method for extracting prions from swabs and recovering CWD prions swabbed from different types of surfaces including glass, stainless steel, and wood. We found that samples dried on swabs were unfavorable for prion extraction, with the greatest prion recovery from wet swabs. Using this swabbing technique, the recovery of CWD prions dried to glass or stainless steel was approximately 30% in most cases, whereas that from wood was undetectable by conventional prion immunodetection techniques. Real-time quake-induced conversion (RT-QuIC) analysis of these same samples resulted in an increase of the detection limit of CWD prions from stainless steel by 4 orders of magnitude. More importantly, the RT-QuIC detection of CWD prions recovered from stainless steel surfaces using this method was similar to the original CWD prion load applied to the surface. This combined surface swabbing and RT-QuIC detection method provides an ultrasensitive means for prion detection across many settings and applications. [ABSTRACT FROM AUTHOR]