The Generative Brain

RT-QuIC with recombinant human PrP E219K substrate for human prion strain discrimination

This is a laboratory method, not a service. It is RT-QuIC run with a different bait protein: recombinant human prion protein carrying lysine instead of glutamic acid at codon 219, on a methionine 129 background, expressed in E coli. It was reported in July 2025 by the INRAE prion team at Jouy-en-Josas with hospital collaborators in Lyon and Paris. In brain homogenates it amplified six sporadic CJD subtypes and variant CJD, and the time the reaction took to start separated variant CJD from every sporadic subtype tested, which the authors describe as a first step towards typing the strain in a living patient. Everything published so far is brain tissue. There is no cerebrospinal fluid or blood result, no clinical validation, and no sign of use outside the originating group. Information only, not medical advice.

Key facts

  • The substrate is recombinant human PrP with the E219K polymorphism on a methionine 129 allele, used at 100 micrograms per millilitre in the reaction buffer; the discussion states it was expressed in E coli, and the methods do not describe the construct or the purification steps.
  • Reaction conditions as published: 2 microlitres of diluted seed into 98 microlitres of buffer containing 20 mM sodium phosphate at pH 7.4, 300 mM sodium chloride, 10 micromolar thioflavin T and 1 mM EDTA, in a black 96 well optical bottom plate, at 46 degrees Celsius plus or minus 2, alternating one minute of orbital shaking at 600 rpm with one minute of rest, read every 30 minutes for 24 to 60 hours, in quadruplicate for samples and octuplicate for uninfected controls.
  • The seeds were brain homogenates, from humanised tg650 mice carrying human PrP M129 after four to five serial passages and from the patient brains used to inoculate those mice; dilutions spanned about eight logs for mouse material and nine logs for patient material. MM2-thalamic sporadic CJD was not tested because no sample was available.
  • Six sporadic CJD subtypes (MM1, MV1, VV1, MM2-cortical, MV2, VV2) and variant CJD amplified. In tg650 passaged material sporadic reactions started between 3 and 36 hours and variant CJD between 11 and 31 hours; in patient material sporadic reactions started between 0 and 27 hours and variant CJD between 17 and 35 hours. The patient VV1 sample amplified over only about 2 logs.
  • Discrimination was done on the area under the curve of lag time against particle concentration, with a linear model carrying a fixed strain effect, classical and permutation tests using the lmPerm package in R, and Tukey adjusted p values. Variant CJD separated from all sporadic subtypes tested; MM2-cortical separated from the other sporadic strains, and in mouse material MM1 separated from MV1.
  • Reported detection limits from the last positive dilution and PrPres quantification were about 1 femtogram for several strains, 0.2 femtograms for some, below 0.05 femtograms for tg650 MM1 and patient MV1, and about 30 femtograms for variant CJD; the paper states variant CJD was detected at an amount five times lower than previously reported with other substrates.
  • The stated advantage over earlier recombinant substrates is the absence of spontaneous conversion. No auto polymerisation was seen with uninfected brain over at least 40 hours, where the authors say other substrates often make interpretation harder; sporadic lag times were shorter than the 7 to 20 hours they cite for hamster or human PrP substrates, and variant CJD shorter than the more than 30 hours often needed with hamster PrP.
  • The paper's own framing is cautious. It says RT-QuIC does not yet reliably differentiate all human prion strains, that variant CJD often shows reduced sensitivity and may need more than one substrate to be told apart from MM1 sporadic CJD, and that a larger clinical cohort and condition optimisation may be needed for the rarer subtypes.
  • Cerebrospinal fluid and blood are named by the authors as matrices still to be evaluated, which is what would be needed for any use before death.
  • The reason for choosing codon 219 is a human genetic observation, not a technical one: the paper calls E219K a dominant negative polymorphism known to protect against sporadic CJD in Asian populations.
  • That protection claim traces to a 1998 Annals of Neurology report, in which no codon 219 glutamic acid or lysine heterozygote was found among 85 people with sporadic CJD, while 4 of 43 people with non-CJD dementia carried it at about the frequency of the general Japanese population, where the allele runs at roughly 6 percent.
  • The published work sits in a line of substrate engineering for strain reading. An earlier and still cited approach uses recombinant bank vole PrP as a broad substrate that detects prions across species and allows some strain discrimination.
  • Funding recorded on the journal record is a French medicines agency grant (2014S033 HAP ANSM 2014/iPDB), a French National Research Agency grant (ANR-21-CE15-0011-01) and a Fundacion Ramon Areces fellowship.
  • Authorship spans a basic research unit and two clinical sites: INRAE Jouy-en-Josas for most authors, the INRAE AgroParisTech animal genetics unit for the statistician, the Hospices Civils de Lyon neurological hospital with the BIORAN team in Lyon, and Sorbonne Universite with the Paris Brain Institute.

Timeline

  1. 1998Report in Annals of Neurology that the codon 219 lysine allele was absent from 85 sporadic CJD cases in Japan, the genetic observation the substrate choice rests on.
  2. 2024Preprint posted on bioRxiv (10.1101/2024.07.31.605973), corresponding author Davy Martin, Universite Paris-Saclay, category pathology.
  3. 2025Manuscript received by Microbiology Spectrum.
  4. 2025Manuscript accepted.
  5. 2025Discrimination of human prion strains by RT-QuIC presented at the 10th French national study day on prion diseases at the Paris Brain Institute, by the INRAE Jouy-en-Josas team with Lyon and Paris co-authors.
  6. 2025First publication in Microbiology Spectrum 13(8):e0029225, DOI 10.1128/spectrum.00292-25, PMID 40637424, PMC12323364, open access under CC BY; print issue dated August 2025.
  7. 2026Search for later use or citation of the substrate found no 2026 paper and no laboratory adoption record.
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