Few research laboratories have validated and utilized prion amplification (Real-Time Quaking Induced Conversion, RT-QuIC) technologies for detecting Chronic Wasting Disease (CWD) in white-tailed deer (Odocoileus virginianus). This collaborative project among research laboratories in New York, Michigan, Minnesota, Wisconsin, Missouri, and Pennsylvania will expand the number of laboratories capable of using the technology through formal validation the of the techniques using commercial or in-house synthesized (by the University of Minnesota) RT-QuIC substrate (recombinant prion protein, rPrP). The collaboration will interact with the United States Department of Agriculture (USDA), National Institutes of Health (NIH), and other research laboratories to ensure that state diagnostic labs can successfully implement RT-QuIC. We will evaluate retropharyngeal lymph nodes (RPLN) and obex samples to determine which has the greatest potential for RT-QuIC applications and will validate the RT-QuIC amplification products in a subset of experiments by Western blotting. The collaboration will use this information to develop, evaluate, optimize, and share protocols for use by stakeholders to collect and submit samples for RT-QuIC testing.