Lab Protocol Amplifies Prions from Deer and Other Animals to Detect Chronic Wasting Disease

Chronic wasting disease continues to spread through deer, elk and moose populations in North America, and one of the most sensitive tools for tracking the infectious prions behind it now has a fully updated laboratory playbook. In a protocol article published in BMC Methods, researchers describe step-by-step procedures for replicating chronic wasting disease prions in vitro using protein misfolding cyclic amplification, a cell-free technique that mimics the conversion of the normal cellular prion protein into its pathogenic, misfolded form. The protocol covers amplification from animal-derived samples and environmental fomites, newly optimized conditions for detecting the prions in non-cervid sympatric hosts, and a modified high-throughput format aimed at screening small molecule libraries for anti-prion activity.

Protein misfolding cyclic amplification, or PMCA, works in a way that its developers have likened to the polymerase chain reaction used to copy DNA. Instead of nucleic acids, the technique amplifies misfolded prion protein. Samples suspected of containing disease-associated prions are mixed with an excess of normally folded prion protein, which serves as the reaction substrate. The mixture then undergoes repeated cycles of incubation and sonication. During incubation periods, any pathogenic prion protein present acts as a seed, templating its own misfolded structure onto the normal protein. During sonication, the growing aggregates are broken into smaller fragments, each of which becomes a new seed capable of recruiting and converting additional normal protein. After each round, the number of seeds increases exponentially, and the products can be visualized with conventional methods such as western blots or dot blots.

The technique’s sensitivity is extraordinary. According to the protocol’s authors, PMCA can detect an estimated quantity as small as roughly 26 monomers of prion protein, which may correspond to a single molecule of oligomeric infectious prion protein. Multiple reports cited in the article indicate that PMCA can detect as little as a single prion polymer in specific tissues such as brain. This ultrasensitivity has allowed researchers to amplify and detect prions at pre-clinical and subclinical stages across different animal species, an ability that matters because the currently approved diagnostic methods in the United States, immunohistochemistry and enzyme-linked immunosorbent assay, exhibit limited sensitivity, cannot distinguish different prion strains, and are conducted only after death.

The substrate at the heart of the new protocol comes from transgenic mice. The authors prepared their substrate from the brains of six to eight week old homozygous Tg1536 mice, which overexpress the cervid version of the cellular prion protein at approximately tenfold the level found in wild type mice. Brains from female and male mice are pooled, homogenized at a ten percent weight-per-volume concentration, and clarified by low-speed centrifugation. Two additives are then mixed in: EDTA, a chelating agent that binds positively charged divalent metal cations, and digitonin, a non-ionic surfactant that solubilizes the membrane-bound prion protein. Both additives have been shown to improve the in vitro replication efficiency of chronic wasting disease prions compared with the classical protocol described more than a decade earlier.

The standard reaction is run in 0.2 milliliter PCR tubes, each containing three small polytetrafluoroethylene balls that help mechanically fragment the fibrils during the incubation and sonication cycles. The first round consists of 144 cycles, which the authors note is longer than strictly necessary for amplification but standardizes the procedure because additional time is needed to completely homogenize hard pellets from previous rounds. A second and third round of 96 cycles each follow, with fresh substrate replaced between rounds. After amplification, the products are treated with proteinase K, an enzyme that cleaves the N-terminal region of the prion protein. Because the pathogenic form is partially resistant to proteases, the remaining protease-resistant core can then be detected by western blot or dot blot. The full procedure, including three rounds, takes approximately seven days.

For laboratories that need to process many samples, the authors adapted the tube format into a 96-well plate system. The 96-well plate PMCA format increases the number of samples that can be analyzed while reducing processing time and the amount of reagents used, with differences in reaction volume, bead number, cycle number and detection method compared with the standard version. Dot blot detection is preferred in this format to cut overall processing times, although the authors advise confirming prion-positive readings with western blots, because incomplete proteinase K digestion can produce false positive signals in dot blots. The 96-well format also underpins a drug discovery application: small molecules are added to wells containing the reaction mixture seeded with prions, and an effective anti-prion candidate is identified when it completely inhibits the conversion of normal prion protein to the pathogenic form, producing no signal on the dot blot, while partial inhibitors yield weaker signals.

Because chronic wasting disease prions are shed into the environment, the protocol also details how to prepare diverse sample types beyond brain tissue. Soft tissues are homogenized in tubes with ceramic beads, while harder materials require different homogenizing tubes. Two pretreatment routes help remove inhibitors or concentrate prions before amplification. Ultracentrifugation, which takes roughly four to five hours, enriches the misfolded protein from fluids and tissues such as semen, blood, and lymph nodes by pelleting it at 100,000 times gravity after sarkosyl treatment. Alternatively, sodium phosphotungstate precipitation, preferred for increasing seeding activity detection in plant tissues, uses a carefully pH-adjusted polyoxometalate solution whose hydrolysis products facilitate the precipitation of the misfolded prion protein, followed by overnight incubation and centrifugation before the enriched pellet is resuspended in fresh reaction mixture.

The protocol is not without constraints, and the authors are explicit about them. PMCA outcomes are largely qualitative, and the technique depends on substrate prepared from brain tissue. Reaction efficiency can be influenced by factors present in the sample, particularly specimens containing blood and environmental samples such as soils. Because the products of PMCA are themselves infectious, diagnostic screenings must be conducted in laboratories with appropriate biosafety levels, BSL2 or BSL2+, and with strict prion disposal protocols. As a cell-free technique, PMCA also cannot assess complex biological processes at the cell or tissue level, so results must be interpreted strictly from protein-protein interaction perspectives. Detection relies on endpoint immunolabeling rather than real-time data, and the authors recommend running each sample set at least in triplicate and by different investigators to reduce variability and bias.

Cross-contamination poses a particular hazard given the technique’s ultrasensitivity, and the protocol devotes considerable attention to preventing it. Decontamination solutions containing sodium hydroxide and sodium dodecyl sulfate are used to clean the sonicator’s microplate horn, tube racks, forceps and scissors, with metal instruments soaked overnight in a two normal sodium hydroxide solution. The authors note that most positive signals in negative controls arise from cross-contamination between samples, and while this is infrequent among experienced manipulators, it can become a recurrent problem for researchers new to the method. They also report that although several groups have documented spontaneous, de novo conversion of the normal protein with this technique, their experienced team does not observe it with substrates from mice expressing deer, elk, mouse, hamster or pig prion protein, though it occurs in roughly half of reactions using meadow vole brain substrate.

Beyond diagnostics and drug screening, the protocol situates PMCA within a broader research toolkit. The technique retains the biological properties of the infectious agent, including infectivity and strain identity, which has made it critical for studying interspecies transmission, strain diversity, and the temporal distribution of prions in tissues and fluids. Materials amplified by PMCA have been shown to generate bona fide infectivity that reproduces the same neurodegenerative phenotypes and clinical signs as brain-derived infectious material in bioassays. The conceptual framework has also been adapted to study the misfolding of other proteins implicated in human neurodegenerative diseases, including alpha-synuclein, amyloid beta and tau. With prion diseases, including chronic wasting disease, remaining without cure or treatment, the authors argue that optimizing PMCA for drug discovery may importantly advance efforts in that direction, while the same ultrasensitive amplification continues to sharpen surveillance of a disease they describe as currently manifesting at epidemic proportions in North American cervids.

Subject of Research: An updated protein misfolding cyclic amplification protocol for amplifying and detecting chronic wasting disease prions in animal and environmental samples and for screening anti-prion molecules

Article Title: In vitro amplification of prions from deer and other animal species through the protein misfolding cyclic amplification (PMCA) technique

Article References: Bravo-Risi, F., Benavente, R., & Morales, R. (2026). In vitro amplification of prions from deer and other animal species through the protein misfolding cyclic amplification (PMCA) technique. BMC Methods, 3(1), Article 38. https://doi.org/10.1186/s44330-026-00091-6

Image Credits: AI Generated

DOI: 10.1186/s44330-026-00091-6

Keywords: prions, chronic wasting disease, PMCA, protein misfolding, cervids, diagnostics, drug screening, prion detection, transmissible spongiform encephalopathies, laboratory protocol, PrPSc, biosafety