The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases

Article metrics


The development and adaption of in vitro misfolded protein amplification systems has been a major innovation in the detection of abnormally folded prion protein scrapie (PrPSc) in human brain and cerebrospinal fluid (CSF) samples. Herein, we describe a fast and efficient protein amplification technique, real-time quaking-induced conversion (RT-QuIC), for the detection of a PrPSc seed in human brain and CSF. In contrast to other in vitro misfolded protein amplification assays—such as protein misfolding cyclic amplification (PMCA)—which are based on sonication, the RT-QuIC technique is based on prion seed–induced misfolding and aggregation of recombinant prion protein substrate, accelerated by alternating cycles of shaking and rest in fluorescence plate readers. A single RT-QuIC assay typically analyzes up to 32 samples in triplicate, using a 96-well-plate format. From sample preparation to analysis of results, the protocol takes 87 h to complete. In addition to diagnostics, this technique has substantial generic analytical applications, including drug screening, prion strain discrimination, biohazard screening (e.g., to reduce transmission risk related to prion diseases) and the study of protein misfolding; in addition, it can potentially be used for the investigation of other protein misfolding diseases such as Alzheimer's and Parkinson's disease.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Schematic diagram of PrP seed amplification by RT-QuIC assay.
Figure 2: Influence of serial dilution of brain homogenates on the RT-QuIC response.
Figure 3: Influence of serial dilution of CSF on the RT-QuIC response.


  1. 1

    Otto, M. et al. S-100 protein concentration in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. J. Neurol. 244, 566–570 (1997).

  2. 2

    Otto, M. et al. Elevated levels of tau-protein in cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. Neurosci. Lett. 225, 210–212 (1997).

  3. 3

    Zerr, I. et al. Detection of 14-3-3 protein in the cerebrospinal fluid supports the diagnosis of Creutzfeldt-Jakob disease. Ann. Neurol. 43, 32–40 (1998).

  4. 4

    Gawinecka, J. et al. Desmoplakin as a potential candidate for cerebrospinal fluid marker to rule out false 14-3-3 positive rates in sporadic Creutzfeldt-Jakob disease differential diagnosis. Neurodegener. Dis. 9, 139–144 (2012).

  5. 5

    Schmitz, M. et al. Validation of 14-3-3 protein as a marker in sporadic Creutzfeldt-Jakob diagnostic. Mol. Neurobiol. 53, 2189–2199 (2016).

  6. 6

    Llorens, F. et al. Quantification of CSF biomarkers using an electrochemiluminescence-based detection system in the differential diagnosis of AD and sCJD. J. Neurol. 262, 2305–2311 (2015).

  7. 7

    Beaudry, P. et al. 14-3-3 protein, neuron-specific enolase, and S-100 protein in cerebrospinal fluid of patients with Creutzfeldt-Jakob disease. Dement. Geriatr. Cogn. Disord. 10, 40–46 (1999).

  8. 8

    Zerr, I. et al. Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease. Lancet 345, 1609–1610 (1995).

  9. 9

    Parchi, P. et al. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann. Neurol. 46, 224–233 (1999).

  10. 10

    Zerr, I. et al. Updated clinical diagnostic criteria for sporadic Creutzfeldt-Jakob disease. Brain 132, 2659–2668 (2009).

  11. 11

    World Health Organization. in WHO Manual for Surveillance of Human Transmissible Spongiform Encephalopathies including variant Creutzfeldt-Jakob Disease 51-9 (World Health Organization, Geneva, 2003).

  12. 12

    Cramm, M. et al. Stability and reproducibility underscore utility of RT-QuIC for diagnosis of Creutzfeldt-Jakob disease. Mol. Neurobiol. 53, 1896–1904 (2016).

  13. 13

    McGuire, L.I. et al. Real time quaking-induced conversion analysis of cerebrospinal fluid in sporadic Creutzfeldt-Jakob disease. Ann. Neurol. 72, 278–285 (2012).

  14. 14

    Atarashi, R. et al. Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion. Nat. Med. 17, 175–178 (2011).

  15. 15

    Orrú, C.D. et al. A test for Creutzfeldt-Jakob disease using nasal brushings. N. Engl. J. Med. 371, 519–529 (2014).

  16. 16

    McGuire, L.I. et al. CSF RT-QuIC is a robust and reliable test for sporadic CJD: an international study. Ann. Neurol. 80, 160–165 (2016).

  17. 17

    Saborio, G.P., Permanne, B. & Soto, C. Sensitive detection of pathological prion protein by cyclic amplification of protein misfolding. Nature 411, 810–813 (2001).

  18. 18

    Colby, D.W. et al. Prion detection by an amyloid seeding assay. Proc. Natl. Acad. Sci. USA 104, 20914–20919 (2007).

  19. 19

    Atarashi, R. et al. Simplified ultrasensitive prion detection by recombinant PrP conversion with shaking. Nat. Methods 5, 211–212 (2008).

  20. 20

    Wilham, J.M. et al. Rapid end-point quantitation of prion seeding activity with sensitivity comparable to bioassays. PLoS Pathog. 6, e1001217 (2010).

  21. 21

    Bessen, R.A. et al. Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 375, 698–700 (1995).

  22. 22

    Atarashi, R. et al. Ultrasensitive detection of scrapie prion protein using seeding conversion of recombinant prion protein. Nat. Methods 4, 645–650 (2007).

  23. 23

    Schmitz, M . et al. Application of an in vitro-amplification assay as a novel pre-screening test for compounds inhibiting the aggregation of prion protein scrapie. Sci. Rep. 6, 28711 (2016).

  24. 24

    Orrú, C.D. et al. Detection and discrimination of classical and atypical L-type bovine spongiform encephalopathy by real-time quaking-induced conversion. J. Clin. Microbiol. 53, 1115–1120 (2015).

  25. 25

    Orrú, C.D. et al. Rapid and sensitive RT-QuIC detection of human Creutzfeldt-Jakob disease using cerebrospinal fluid. MBio 6, e02451–e02414 (2015).

  26. 26

    Orrú, C.D. et al. Prion disease blood test using immunoprecipitation and improved quaking induced conversion. MBio 2, e00078–e00011 (2011).

  27. 27

    Orrú, C.D., Wilham, J.M., Vascellari, S., Hughson, A.G. & Caughey, B. New generation QuIC assays for prion seeding activity. Prion 6, 147–152 (2012).

  28. 28

    Orrú, C.D. et al. Bank vole prion protein as an apparently universal substrate for RT-QuIC-based detection and discrimination of prion strains. PLoS Pathog. 11, e1004983 (2015).

  29. 29

    Sano, K. et al. Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay. PLoS One 8, e54915 (2013).

  30. 30

    Zanusso, G., Bongianni, M. & Caughey, B. A test for Creutzfeldt-Jakob disease using nasal brushings. N. Engl. J. Med. 371, 1842–1843 (2014).

  31. 31

    Cali, I. et al. Classification of sporadic Creutzfeldt-Jakob disease revisited. Brain 129, 2266–2277 (2006).

  32. 32

    Bishop, M., Will, R.G. & Manson, J.C. Defining sporadic Creutzfeldt-Jakob disease strains and their transmission properties. Proc. Natl. Acad. Sci. USA 107, 12005–12010 (2010).

  33. 33

    Cramm, M. et al. Characteristic CSF prion-seeding efficiency in humans with prion diseases. Mol. Neurobiol. 51, 396–405 (2015).

  34. 34

    Masujin, K. et al. Detection of atypical H-type bovine spongiform encephalopathy and discrimination of bovine prion strains by real-time quaking-induced conversion. J. Clin. Microbiol. 54, 676–686 (2016).

  35. 35

    Prusiner, S.B. Novel proteinaceous infectious particles cause scrapie. Science 216, 136–144 (1982).

  36. 36

    Prusiner, S.B. Prion encephalopathies of animals and humans. Dev. Biol. Stand. 80, 31–44 (1993).

  37. 37

    Groveman, B.R. et al. Charge neutralization of the central lysine cluster in prion protein (PrP) promotes PrP(Sc)-like folding of recombinant PrP amyloids. J. Biol. Chem. 290, 1119–1128 (2015).

  38. 38

    Johnson, C., Gilbert, P., McKenzie, D., Pedersen, J. & Aiken, J. Ultraviolet-ozone treatment reduces levels of disease-associated prion protein and prion infectivity. BMC Res. Notes 2, 121–125 (2009).

  39. 39

    Krasemann, S. et al. Preclinical deposition of pathological prion protein in muscle of experimentally infected primates. PLoS One 5, e13906 (2010).

  40. 40

    Kloehn, P.C., Stoltze, L., Flechsig, E., Enari, M. & Weissmann, C. A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie prions. Proc. Natl. Acad. Sci. USA 100, 11666–11671 (2003).

  41. 41

    Fischer, M. et al. Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie. EMBO J. 15, 1255–1264 (1996).

  42. 42

    Murayama, Y. et al. Protein misfolding cyclic amplification as a rapid test for assessment of prion inactivation. Biochem. Biophys. Res. Commun. 348, 758–762 (2006).

  43. 43

    Pritzkow, S. et al. Quantitative detections and biological propagation of scrapie seeding activity in vitro facilitate use of prions as moedel pathogens for disinfection. PLoS One 6, e20384 (2011).

  44. 44

    Eisele, Y.S. et al. Peripherally applied Abeta-containing inoculates induce cerebral beta-amyloidosis. Science 330, 980–982 (2010).

  45. 45

    Kim, J. & Holtzman, D.M. Medicine. Prion-like behavior of amyloid-beta. Science 330, 918–919 (2010).

  46. 46

    Meyer, V., Dinkel, P.D., Rickman Hager, E. & Margittai, M. Amplification of Tau fibrils from minute quantities of seeds. Biochemistry 53, 5804–5809 (2014).

  47. 47

    Salvadores, N., Shahnawaz, M., Scarpini, E., Tagliavini, F. & Soto, C. Detection of misfolded Ab oligomers for sensitive biochemical diagnosis of Alzheimer's disease. Cell Rep. 7, 261–268 (2014).

  48. 48

    Soto, C., Saborio, G.P. & Anderes, L. Cyclic amplification of protein misfolding: application to prion-related disorders and beyond. Trends Neurosci. 25, 390–394 (2002).

  49. 49

    Morales, R., Duran-Aniotz, C., Diaz-Espinoza, R., Camacho, M.V. & Soto, C. Protein misfolding cyclic amplification of infectious prions. Nat. Protoc. 28, 1397–1409 (2012).

  50. 50

    Gonzalez-Montalban, N. et al. Highly efficient protein misfolding cyclic amplification. PLoS Pathog. 7, e1001277 (2011).

  51. 51

    Peden, A.H. et al. Sensitive and specific detection of sporadic Creutzfeldt-Jakob disease brain prion protein using real-time quaking-induced conversion. J. Gen. Virol. 93, 438–449 (2012).

  52. 52

    Saá, P., Castilla, J. & Soto, C. Ultra-efficient replication of infectious prions by automated protein misfolding cyclic amplification. J. Biol. Chem. 281, 35245–35252 (2006).

  53. 53

    Maddison, B.C. et al. Prions are secreted into the oral cavity in sheep with preclinical scrapie. J. Infect. Dis. 201, 1672–1676 (2010).

  54. 54

    Chen, B., Morales, R., Barria, M.A. & Soto, C. Estimating prion concentration in fluids and tissues by quantitative PMCA. Nat. Methods 7, 519–520 (2010).

  55. 55

    Haley, N.J. et al. Detection of chronic wasting disease prions in salivary, urinary and intestinal tissues of deer: potential mechanisms of prion sheeding and transmission. J. Virol. 85, 6309–6318 (2011).

  56. 56

    Castilla, J. et al. Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions. Cell 134, 757–768 (2008).

Download references


This study was performed as part of the Clinical Dementia Center at the University Medical Center Göttingen and was partly supported by grants from the EU Joint Program—Neurodegenerative Disease Research (JPND-DEMTEST (Biomarker-based diagnosis of rapid progressive dementias—optimization of diagnostic protocols, 01ED1201A) and by the Robert Koch Institute through funds from the Federal Ministry of Health (grant no. 1369-341). This work was also supported in part by the Intramural Research Program of the NIAID. S.C. is supported by a NHMRC Practitioner Fellowship (identification no. APP1005816) and by the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR), which is funded by the Commonwealth Department of Health.

Author information

M.S. was involved in study concept and design, and validated the protocol, interpreted data and wrote the manuscript. M.C. performed experiments, analyzed and interpreted data, prepared figures and wrote the manuscript. F.L. provided samples and interpreted data.

D.M.-C. designed Figure 1. S.C., R.A., K.S., C.D.O., B.R.G., S.Z. and B.C. critically revised the manuscript. W.J.S.-S. provided samples. I.Z. supervised the study and critically revised the manuscript.

Correspondence to Matthias Schmitz.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Methods and Supplementary Tables 1 and 2 (PDF 424 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Schmitz, M., Cramm, M., Llorens, F. et al. The real-time quaking-induced conversion assay for detection of human prion disease and study of other protein misfolding diseases. Nat Protoc 11, 2233–2242 (2016) doi:10.1038/nprot.2016.120

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.