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Real-time quaking-induced conversion (RT-QuIC) is a highly sensitive assay for prion detection.^[1] It is nearly 100% specific for the diagnosis of Creutzfeldt-Jakob disease.^[2]

Technique

The "quaking" in the name of the technique refers to the fact that samples in the RT-QuIC assay are literally subjected to shaking. This action breaks apart aggregates of prion protein (PrP) that are then further incubated, amplifying the amount of misfolded PrP to detectable levels.^[3]^[4]

It is "an early, rapid and specific assay for prion diseases". It can sample multiple sample types, such as cerebrospinal fluid (CSF), brain, lymph nodes, blood, muscle, and skin, and so it is applicable to scrapie in sheep, chronic wasting disease (CWD) in cervids, bovine spongiform encephalopathy (BSE) in cows and sporadic Creutzfeldt–Jakob disease in humans, amongst others.^[5]

The RT-QuIC assay uses in excess recombinantly produced normally folded prions, often a truncated Syrian Hamster protein, amino acids 90-231. Samples suspected of containing misfolded prions are added, leading to misfolding and aggregation of normally folded prions. These protein aggregates can be detected by thioflavin T visible spectrum fluorescence detection.^[6]^[7]^[8] The Centers for Disease Control and Prevention includes a positive RT-QuIC result in its diagnostic criteria for the probable diagnosis of sCJD.^[9]

RT-QuIC assays can also be used to test for scrapie, BSE, and CWD. Various procedures can improve sensitivity and specificity. Iron oxide metal extraction (IOME) uses the natural metal affinity of the prion protein; a sample is incubated with magnetic beads, which bind to the prion protein. The prion-rich bead fraction is subsequently harvested and tested.^[10]

Commonly tested tissues are brain homogenates and lymph tissues; however, prions have also been detected in skin and blood samples.^[11]^[12]^[13] Certain tissues can be difficult to test for prions. For example, blood samples tend to have low levels of circulating intracellular prions and have inhibitors of the assay.^[14]

References

^ Zerr, Inga; Cramm, Maria; da Silva Correia, Susana Margarida; Zafar, Saima; Villar-Piqué, Anna; Llorens, Franc; Schmitz, Matthias (2020-11-19). "Optimization of the Real-Time Quaking-Induced Conversion Assay for Prion Disease Diagnosis". Frontiers in Bioengineering and Biotechnology. 8: 586890. doi:10.3389/fbioe.2020.586890. ISSN 2296-4185. PMC 7710546. PMID 33330419.
^ Zerr, Inga (7 April 2022). "Laboratory Diagnosis of Creutzfeldt–Jakob Disease". New England Journal of Medicine. 386 (14): 1345–1350. doi:10.1056/NEJMra2119323. PMID 35388668.
^ Lewczuk, Piotr; Riederer, Peter; O’Bryant, Sid E.; Verbeek, Marcel M.; Dubois, Bruno; Visser, Pieter Jelle; Jellinger, Kurt A.; Engelborghs, Sebastiaan; Ramirez, Alfredo; Parnetti, Lucilla; Jack, Clifford R.; Teunissen, Charlotte E.; Hampel, Harald; Lleó, Alberto; Jessen, Frank; Glodzik, Lidia; de Leon, Mony J.; Fagan, Anne M.; Molinuevo, José Luis; Jansen, Willemijn J.; Winblad, Bengt; Shaw, Leslie M.; Andreasson, Ulf; Otto, Markus; Mollenhauer, Brit; Wiltfang, Jens; Turner, Martin R.; Zerr, Inga; Handels, Ron; Thompson, Alexander G.; Johansson, Gunilla; Ermann, Natalia; Trojanowski, John Q.; Karaca, Ilker; Wagner, Holger; Oeckl, Patrick; van Waalwijk van Doorn, Linda; Bjerke, Maria; Kapogiannis, Dimitrios; Kuiperij, H. Bea; Farotti, Lucia; Li, Yi; Gordon, Brian A.; Epelbaum, Stéphane; Vos, Stephanie J. B.; Klijn, Catharina J. M.; Van Nostrand, William E.; Minguillon, Carolina; Schmitz, Matthias; Gallo, Carla; Lopez Mato, Andrea; Thibaut, Florence; Lista, Simone; Alcolea, Daniel; Zetterberg, Henrik; Blennow, Kaj; Kornhuber, Johannes (27 October 2017). "Cerebrospinal fluid and blood biomarkers for neurodegenerative dementias: An update of the Consensus of the Task Force on Biological Markers in Psychiatry of the World Federation of Societies of Biological Psychiatry". The World Journal of Biological Psychiatry. 19 (4): 244–328. doi:10.1080/15622975.2017.1375556. PMC 5916324. PMID 29076399.
^ Cramm, Maria; Schmitz, Matthias; Karch, André; Mitrova, Eva; Kuhn, Franziska; Schroeder, Bjoern; Raeber, Alex; Varges, Daniela; Kim, Yong-Sun; Satoh, Katsuya; Collins, Steven; Zerr, Inga (1 April 2015). "Stability and Reproducibility Underscore Utility of RT-QuIC for Diagnosis of Creutzfeldt-Jakob Disease". Molecular Neurobiology. 53 (3): 1896–1904. doi:10.1007/s12035-015-9133-2. PMC 4789202. PMID 25823511.
^ Atarashi, R.; Sano, K.; Satoh, K.; Nishida, N. (2011). "Real-time quaking-induced conversion: A highly sensitive assay for prion detection". Prion. 5 (3): 150–3. doi:10.4161/pri.5.3.16893. PMC 3226039. PMID 21778820.
^ Haley NJ, Hoover EA (2015). "Chronic wasting disease of cervids: current knowledge and future perspectives". Annual Review of Animal Biosciences. 3: 305–25. doi:10.1146/annurev-animal-022114-111001. PMID 25387112.
^ Atarashi R, Satoh K, Sano K, Fuse T, Yamaguchi N, Ishibashi D, Matsubara T, Nakagaki T, Yamanaka H, Shirabe S, Yamada M, Mizusawa H, Kitamoto T, Klug G, McGlade A, Collins SJ, Nishida N (February 2011). "Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion" (PDF). Nature Medicine. 17 (2): 175–78. doi:10.1038/nm.2294. hdl:10069/25642. PMID 21278748. S2CID 11381303.
^ Orrú CD, Bongianni M, Tonoli G, Ferrari S, Hughson AG, Groveman BR, Fiorini M, Pocchiari M, Monaco S, Caughey B, Zanusso G (August 2014). "A test for Creutzfeldt–Jakob disease using nasal brushings". The New England Journal of Medicine. 371 (6): 519–29. doi:10.1056/NEJMoa1315200. PMC 4186748. PMID 25099576.
^ "CDC's Diagnostic Criteria for Creutzfeldt–Jakob Disease (CJD), 2018". Centers for Disease Control and Prevention. September 17, 2018. Retrieved November 22, 2018. Adapted from:
a) Global surveillance, diagnosis, and therapy of human transmissible spongiform encephalopathies: Report of a WHO consultation, February 1998, Geneva, Switzerland;
b) Zerr et al. 2009
c) National CJD Research & Surveillance Unit. Protocol: Surveillance of CJD in the UK. Item c accessed 15 Aug 2018.
Zerr I, Kallenberg K, Summers DM, Romero C, Taratuto A, Heinemann U, Breithaupt M, Varges D, Meissner B, Ladogana A, Schuur M, Haik S, Collins SJ, Jansen GH, Stokin GB, Pimentel J, Hewer E, Collie D, Smith P, Roberts H, Brandel JP, van Duijn C, Pocchiari M, Begue C, Cras P, Will RG, Sanchez-Juan P (October 2009). "Updated clinical diagnostic criteria for sporadic Creutzfeldt-Jakob disease". Brain. 132 (Pt 10): 2659–68. doi:10.1093/brain/awp191. PMC 2759336. PMID 19773352.
^ Denkers ND, Henderson DM, Mathiason CK, Hoover EA (August 2016). "Enhanced prion detection in biological samples by magnetic particle extraction and real-time quaking-induced conversion". J Gen Virol. 97 (8): 2023–2029. doi:10.1099/jgv.0.000515. PMC 5903251. PMID 27233771.
^ Mammana A, Baiardi S, Rossi M, Franceschini A, Donadio V, Capellari S, Caughey B, Parchi P (April 2020). "Detection of prions in skin punch biopsies of Creutzfeldt-Jakob disease patients". Ann Clin Transl Neurol. 7 (4): 559–564. doi:10.1002/acn3.51000. PMC 7187701. PMID 32141717.
^ Concha-Marambio L, Pritzkow S, Moda F, Tagliavini F, Ironside JW, Schulz PE, Soto C (December 2016). "Detection of prions in blood from patients with variant Creutzfeldt-Jakob disease". Sci Transl Med. 8 (370): 370ra183. doi:10.1126/scitranslmed.aaf6188. PMC 5538786. PMID 28003548.
^ Orrú CD, Yuan J, Appleby BS, Li B, Li Y, Winner D, Wang Z, Zhan YA, Rodgers M, Rarick J, Wyza RE, Joshi T, Wang GX, Cohen ML, Zhang S, Groveman BR, Petersen RB, Ironside JW, Quiñones-Mateu ME, Safar JG, Kong Q, Caughey B, Zou WQ (November 2017). "Prion seeding activity and infectivity in skin samples from patients with sporadic Creutzfeldt-Jakob disease". Sci Transl Med. 9 (417). doi:10.1126/scitranslmed.aam7785. PMC 5744860. PMID 29167394.
^ Orrú CD, Wilham JM, Raymond LD, Kuhn F, Schroeder B, Raeber AJ, Caughey B (2011). "Prion disease blood test using immunoprecipitation and improved quaking-induced conversion". mBio. 2 (3): e00078–11. doi:10.1128/mBio.00078-11. PMC 3101782. PMID 21558432.