Application of protein misfolding amplification techniques in prion diseases
0 Abstract
Prion diseases (PrDs) are fatal neurodegenerative disorders caused by the misfolding and aggregation of pathogenic prion protein (PrPSc). Traditional diagnostic methods have limited sensitivity and specificity, often requiring confirmation in the late stages or postmortem. In recent years, protein misfolding amplification techniques, including protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC), have achieved significant breakthroughs, enabling ultrasensitive detection of PrPSc with detection limits as low as the attogram level. These methods greatly enhance diagnostic accuracy and offer the potential for early and non-invasive detection of PrDs. Their success has extended to other seeding-based neurodegenerative diseases, such as Parkinson’s disease (PD), providing a powerful tool for early diagnosis, molecular pathology research, and clinical translation. Beyond diagnosis, these techniques play a crucial role in investigating strain characteristics and pathology, as well as in screening potential drugs for PrDs. They are also applied in research of public health security, including variant Creutzfeldt-Jakob disease (vCJD) surveillance, cross-species risk assessment, and environmental contamination monitoring and optimization of decontamination strategies. With exceptional sensitivity and specificity, these techniques are revolutionizing the landscape of neurodegenerative disease detection and intervention.
Keywords
INTRODUCTION
Prion diseases (PrDs), also known as transmissible spongiform encephalopathies (TSEs), are infectious neurodegenerative disorders that affect the central nervous system (CNS) of humans and various animal species. In humans, PrDs can be classified into sporadic, genetic, and acquired forms based on their etiology. In animals, bovine spongiform encephalopathy (BSE) can manifest as sporadic (L-type and most H-type BSE)[1], genetic (a few H-type BSE cases)[2,3] and acquired (C-type BSE). The uniqueness of these diseases lies in their pathogenic mechanism, which primarily involves a conformational transformation of the host’s prion protein (PrP). The normal cellular prion protein (PrPC) is rich in α-helical structures, non-toxic, and readily degradable by proteases. In contrast, the pathological prion protein (PrPSc) is β-sheet-rich, forming insoluble, protease-resistant aggregates[4]. PrPSc is considered the key agent in disease transmission and pathogenesis, as it promotes the conversion of PrPC into PrPSc, triggering a self-propagating cascade within the CNS.
Traditional diagnostic approaches for PrDs, particularly for sporadic and acquired forms, primarily rely on indirect biomarkers, such as hyperintensities in the cortex or basal ganglia on diffusion-weighted imaging (DWI) or fluid-attenuated inversion recovery, periodic sharp wave complexes on electroencephalography, and cerebrospinal fluid (CSF) 14-3-3 protein. However, these methods exhibit low specificity and limited predictive value in the early stages of the disease[5,6]. Current direct detection methods for pathological PrPSc, such as Western blot (WB) analysis of protease-resistant fragments, have low sensitivity, particularly in early-stage disease or peripheral samples[7]. Therefore, the development of highly sensitive and specific techniques capable of directly detecting pathological PrPSc in the early stages of disease has become an urgent priority in PrDs diagnostics.
Protein misfolding amplification techniques, primarily protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC), have emerged to address this need. While their technical approaches and applications differ, both are based on the principle that PrPSc ‘seeds’ can induce the conformational conversion of PrPC, enabling exponential amplification of minute amounts of PrPSc[8]. Compared to traditional detection methods, PMCA and RT-QuIC offer several orders of magnitude higher sensitivity in PrPSc detection, capable of identifying quantities as low as 10-18 grams[9]. Additionally, these techniques exhibit high specificity for disease-associated prion protein conformations, enabling potential detection before the onset of clinical symptoms.
This review will summarize the principles of PMCA and RT-QuIC, and focus on their applications, advantages and limitations in diagnostics, fundamental research, and therapeutic development.
PRINCIPLES OF PROTEIN MISFOLDING AMPLIFICATION TECHNIQUES
PMCA
PMCA was first developed in 2001 by Saborio and his team, simulating the ‘seed amplification’ process of prion protein aggregation in vivo[10]. The fundamental principle of PMCA involves incubating a sample containing minute amounts of PrPSc (‘seeds’) with an excess of normal PrPC (‘substrate’), allowing PrPSc to serve as a template to induce the misfolding and aggregation of PrPC. Subsequently, high-frequency ultrasound pulses are applied to the sample at brief intervals, fragmenting the newly formed PrPSc aggregates into smaller pieces, each capable of acting as a new seed. Through multiple cycles of incubation and sonication, the PrPSc concentration increases exponentially[11], enabling the amplification of initially undetectable trace amounts of PrPSc to levels that can be identified by conventional detection methods. The final products can be analyzed using WB, surround optical fiber immunoassay, or other immunoassay-based techniques.
Since its development, PMCA has undergone multiple optimizations: (1) Serial PMCA (sPMCA): The amplified product is reintroduced into fresh substrate for multiple cycles, significantly enhancing amplification efficiency and sensitivity[12]; (2) Bead-assisted PMCA (PMCAb): The addition of micronized beads increases shear forces, improving amplification efficiency[13]; (3) Alternative substrates [recombinant prion protein-PMCA (rPrP-PMCA)]: In addition to traditional brain homogenate-derived PrPC, recombinant PrP has been developed as an alternative substrate to reduce background variation and enhance reproducibility[14]; (4) Cross-species detection: By optimizing reaction conditions, PMCA has been successfully adapted to detect PrPSc from various prion strains and species, including chronic wasting disease (CWD) in deer and BSE in cattle[15,16].
Optimized PMCA methods have demonstrated exceptional sensitivity, with detection limits as low as 1 attogram (10-18 g) of PrPSc[9], meaning that even a few dozen PrPSc molecules can be detected - far surpassing the sensitivity of traditional WB assays (picogram levels). This ultra-high sensitivity has enabled PMCA to be successfully applied to a wide range of clinical and experimental samples, including CSF, blood, urine, and lymphoid tissues[17], as well as non-invasive specimens such as urine[18] and nasal mucosa[19].
However, PMCA has several problems to be addressed. De novo generation of infectious prions has been reported in PrPSc-free PMCA reactions[20,21], potentially leading to false positives. Although Cosseddu et al. suggested that inadvertent cross-contamination might be the cause[22], further validation of PMCA’s diagnostic value and clinical applicability is necessary. Additionally, because PMCA-generated PrPSc retains infectivity, concerns regarding biohazard risks and cross-contamination must be carefully considered.
RT-QuIC
RT-QuIC was developed from PMCA by two groups working in parallel: Atarashi et al. applied the assay to human diagnostics[23], whereas Wilham et al. focused on animal testing[24]. Although RT-QuIC relies on the same seed-amplification concept as PMCA, its workflow is markedly different. It uses rPrP - typically of hamster or human origin - as a substrate, though bank vole rPrP can convert strains that resist standard substrates. Bank vole (BV) rPrP as substrate has also led to the conversion of certain prion strains, which were previously unresponsive to conventional rPrPs[25]. Except natural substrate, hybrid substrates (e.g. hamster-sheep chimera) have also yielded high sensitivity and specificity in sporadic Creutzfeldt-Jakob disease (sCJD) diagnosis (80%-89% and 99%-100%, respectively)[26-28]. The test sample, which may contain PrPSc ‘seeds’, is mixed with an excess amount of rPrP in a multi-well plate and incubated at 37 - 45 °C, with intermittent shaking (e.g., 60s shake/60s rest) [Table 1]. This continuous shaking provides sufficient physical energy for PrPSc seeds, if present, to convert rPrP into amyloid fibrils. Thioflavin T (ThT), added beforehand, binds β-sheet structures; its fluorescence rises as aggregation proceeds, allowing real-time tracking of the characteristic ‘lag phase → exponential growth phase → plateau phase’ on a plate reader[23,29].
Reaction conditions of RT-QuIC
| PQ-RT-QuIC[24] | IQ-RT-QuIC[31] | eQuIC[32] | IOME-RT-QuIC[33] | ||
| Features | Full-length form of rPrP as substrate | Truncated form of rPrP as substrate | SDS-PBS-bead made from 15B3-coated beads (acquired from immunoprecipitation) | Superparamagnetic iron oxide beads | |
| Substrate source | Hamster (23-231) | Hamster (90-231) | Hamster (23-231), human (23-231), hamster-sheep chimera | Hamster (90-231) | |
| Reaction buffer | Phosphate buffer | 10mM (pH 7.4) | 10mM (pH 7.4) | 10mM (pH 7.4) | 50 mM (pH 7.4) |
| NaCl | 130-500 mM | 300 mM | 130-500 mM | 320mM | |
| rPrP | 0.1 mg/mL | 0.1 mg/mL | 0.1 mg/mL | 0.1 mg/mL | |
| ThT | 10 μM | 10 μM | 10 μM | 10 μM | |
| EDTA | 10µM | 1 mM | 10 µM | 1 mM | |
| Incubation temperature | 42 °C | 55 °C | 46 °C | 42 °C | |
| Incubation time | 20-68 h | 24-60 h | 24 h, 36-60 h (after substrate replacement) | 48 h | |
| Shaking parameters | 60s shaking (700 rpm) and 60s rest | 60s shaking (700 rpm) and 60s rest | 60s shaking (700 rpm) and 60s rest | 60s shaking (700 rpm) and 15min rest | |
RT-QuIC has undergone multiple optimizations to enhance its sensitivity, detection speed, and clinical applicability. The first-generation RT-QuIC (PQ-RT-QuIC) used full-length hamster rPrP (residues 23-231) as substrate and was incubated at 42 °C. Franceschini et al. then introduced engineered rPrP (truncated hamster rPrP, residues amino acids 90-231), raised the temperature to 55 °C, and added sodium dodecyl sulfate (SDS) to the reaction system, creating the second-generation IQ-RT-QuIC [Table 1]. Compared to PQ-RT-QuIC, IQ-RT-QuIC significantly reduced the detection time for CJD CSF samples from 55-90 h to 4-14 h[30] and converted 81% of previously negative CSF samples to positive[31]. Subsequent variants have pushed sensitivity further: eQuIC incorporates immunoprecipitation to selectively enrich PrPSc using specific antibodies[32], and IOME-RT-QuIC uses iron oxide magnetic particles to extract PrPSc[33], both excelling in biofluids with low-concentration PrPSc [Table 1].
However, RT-QuIC also has its limitations. Sensitivity varies among sCJD subtypes, reflecting sequence mismatches between PrPSc strains and the chosen substrate. Additionally, Hermann et al. reported a 1.5% false-positive rate (5/371 cases) in a prospective surveillance study, with other isolated CSF RT-QuIC false positives in AD, dementia with Lewy bodies (DLB), and autoimmune encephalopathy, etc.[34]. Contributing factors may include spontaneous rPrP aggregation[35], de novo conversion[36], and excessive concentrations of salt, ThT and cleaning surfactants in the reaction mix[37].
PMCA and RT-QuIC are now the most widely used protein misfolding amplification techniques, each with distinct advantages, yet also disadvantages [Table 2]. Compared to PMCA, RT-QuIC offers higher detection efficiency, high-throughput capability, greater specificity, and lower biosafety concerns[7,29,38], making it the leading in vitro diagnostic method for PrDs. Although its sensitivity is usually slightly lower than multi-round PMCA, RT-QuIC still detects femtogram or even sub-femtogram quantities of PrPSc[9]. Ongoing improvements in substrate sequences, buffer composition, and incubation parameters continue to boost performance. Currently, RT-QuIC has been validated in CSF, skin, nasal mucosa, blood, urine, and feces, and the seeded-amplification concept is being translated to other protein-misfolding disorders.
Comparison of the reaction system between RT-QuIC and PMCA prion amplification assays
| RT-QuIC | PMCA | |
| Reaction condition | Substrate: recombinant prion protein (rPrP, BV-, hamster- or human-derived) Energy: intermittent shaking | Substrate: brain homogenate (usually hamster-derived) Energy: sonication |
| Variation | PQ-RT-QuIC, IQ-RT-QuIC, eQuIC, IOME-RT-QuIC, etc. | sPMCA, PMCAb, rPrP-PMCA, PMSA, etc. |
| Standardization | Substrates prepared in laboratory with high consistency Standardized shaking frequency and time | Difficult to ensure the continuity of substrate supply and its consistency across laboratories Low standardization of sonication |
| Efficiency | Perform in a 96-well plate. Results are available within 24-48 h | Perform in a centrifuge tube and take longer (several days to weeks for sPMCA, 72 h for PMCAb) |
| Results | Quantitative analysis: Real-time monitoring of ThT fluorescence intensity The reaction product is not conformationally identical to PrPSc and lacks infectivity | Semi-quantitative analysis: WB analysis after protein digestion Infectious products are given with identical infectivity, strain properties and species specificity |
| Disadvantages | Sequence mismatch False positivity (1.5%), possibly related to rPrP self-aggregation, de novo conversion and excessive concentrations of reaction buffer | Spontaneous generation of PrPSc, possibly lead to false positivity Biohazard and cross-contamination |
APPLICATION OF PMCA AND RT-QUIC IN DIAGNOSIS OF PRDS
Application of PMCA in diagnosis
PMCA demonstrates exceptional sensitivity, making it highly promising for early and antemortem diagnosis of PrDs, particularly in variant Creutzfeldt-Jakob disease (vCJD). Independent studies by Concha-Marambio et al. and Bougard et al. showed that optimized PMCA detects PrPSc in whole-blood samples from vCJD-infected individuals with 100% sensitivity and specificity[39,40], offering a feasible approach for screening asymptomatic carriers and blood donors. Additionally, in CSF samples from vCJD patients, PMCA likewise achieved 100% sensitivity and specificity[41], further underscoring its diagnostic value in vCJD. PMCA has also succeeded with non-invasive specimens: Moda et al. detected PrPSc in urine from vCJD patients with 92.9% sensitivity and 100% specificity[42], while Cazzaniga et al. identified PrPSc in nasal-mucosa samples with 79.3% sensitivity and 100% specificity[19], highlighting its versatility across biofluids.
Conventional PMCA, however, has not consistently amplified PrPSc from CSF and other biofluid samples of sCJD patients, limiting its diagnostic application in sCJD. Recent studies have shown that adjusting reaction conditions and substrate choice can yield low-level amplification from sCJD brain tissue, CSF, and nasal mucosa[43]. An improved PMCA protocol also detected PrPSc in sCJD urine samples, but with only 36% sensitivity and considerable subtype variation (VV1 and VV2 subtypes showed the highest detection rates, while MV1 and MM2 subtypes were not detected)[44]. PMCA efficiency depends on the compatibility between PrPSc ‘seeds’ and the reaction substrate, particularly the PRNP codon 129 genotype[38]. Further optimization is therefore required to establish ideal conditions for each sCJD subtype (MM1/MV1, MM2, MV2, VV1, VV2). Moreover, applying PMCA to non-invasive fluids such as saliva, tears, and amniotic fluid remains under investigation. If proven feasible, this would represent a major breakthrough in the early, non-invasive diagnosis of PrDs.
Application of RT-QuIC in diagnosis
In patients clinically suspected of CJD, a positive CSF RT-QuIC result is considered strong evidence for clinical confirmation. Large-scale clinical cohort studies have demonstrated that the first-generation RT-QuIC assay for detecting PrPSc seeds in CSF has a sensitivity of 73%-89%[45]. In contrast, the second-generation RT-QuIC has shown improved sensitivity, ranging from 92% to 97%[31,46], with specificity approaching 100%, highlighting its high diagnostic accuracy for detecting PrPSc in the CSF of sCJD patients[7,30,31,47].
The molecular subtypes of sCJD are defined by the polymorphism at codon 129 of the PRNP gene [methionine (M) or valine (V)] and the PrPSc glycotype (type 1 or type 2), resulting in distinct subtypes such as MM1 and MV1. Among sCJD patients, MM1/MV1 and VV2 are the most common subtypes and exhibit relatively high detection sensitivity. In contrast, the detection sensitivity is lower for VV1 and MM2 subtypes, with VV1 ranging from 0% to 100% and MM2 ranging from 44% to 78%[48]. Additionally, RT-QuIC shows significant variability in diagnostic value across different types of genetic PrDs. In a retrospective study of 218 genetic PrDs patients, Shi et al. reported RT-QuIC shows high positivity in carriers of P102L mutation (63.6%), followed by E200K (44.0%), E196A (37.5%), and T188K (25.7%), with the lowest positivity in D178N (15.8%)[49]. This suggests that while RT-QuIC provides excellent diagnostic performance for certain PrDs subtypes, its application in rare subtypes and specific genetic PrDs remains limited. In vCJD, human-derived substrates usually failed to amplify vCJD PrPSc effectively, while BV-derived rPrP has yielded positive results, albeit with limited efficacy (one in two patients tested positive)[25]. Further optimization and refinement of RT-QuIC are necessary to improve its applicability and accuracy across different subtypes.
CSF has traditionally been the primary sample for RT-QuIC-based PrPSc detection. However, approximately 10% of patients are unable to undergo lumbar puncture due to various contraindications. This has prompted researchers to explore alternative body fluids and tissues, leading to successful PrPSc detection in skin, olfactory mucosa, tears, and blood.
In 2017, Orrú et al. first applied RT-QuIC to human skin samples for the diagnosis of PrDs[50]. Subsequent animal studies confirmed that PrPSc could be detected in the skin even before the onset of clinical symptoms[51], providing a theoretical basis for skin RT-QuIC in early PrDs diagnosis. Postmortem skin RT-QuIC has shown comparable sensitivity and specificity to CSF RT-QuIC in patients with PrDs[48,52]. Furthermore, Chen et al. demonstrated that in living patients, the RT-QuIC positivity rate at a single skin site was comparable to that of CSF RT-QuIC, and that combining samples from multiple skin sites significantly improved detection rates[53].
Due to the early and abundant PrPSc deposition in the olfactory mucosa, nasal brush RT-QuIC has gained attention as a non-invasive and convenient sample type. Studies have reported high detection rates (95%-100%) in confirmed sCJD cases, with no false positives[54,55]. Combining RT-QuIC testing of olfactory mucosa and CSF could potentially achieve near-perfect diagnostic sensitivity for clinical CJD[55]. Moreover, in the same patient samples, olfactory mucosa-based RT-QuIC exhibited a faster reaction time and higher fluorescence intensity compared to CSF-based RT-QuIC[29,56].
In 2024, Schmitz et al. modified the RT-QuIC by using E200K-mutant recombinant human PrP as a substrate to detect PrPSc in tears from PrDs patients, PRNP mutation carriers, and healthy controls[57]. Their findings revealed an 84% positivity rate in patients with sCJD and genetic PrDs, with sensitivity comparable to CSF RT-QuIC. Notably, PrPSc seeding activity was also detected in asymptomatic PRNP mutation carriers, suggesting that PrPSc formation may begin in the early, preclinical stages of the disease[57].
Compared to PMCA, which can directly detect PrPSc in blood, conventional RT-QuIC exhibits lower sensitivity in complex biological matrices such as blood[33]. To overcome this limitation, enhanced RT-QuIC methods have been developed, including enhanced QuIC (eQuIC) and immunomagnetic enrichment RT-QuIC (IOME-RT-QuIC). eQuIC uses antibody-mediated enrichment of PrPSc before RT-QuIC amplification, significantly increasing sensitivity[32]. Studies have shown that this method can detect PrPSc in vCJD brain tissue diluted to 10-14 - approximately 1 attogram of PrPSc - representing a 10000-fold sensitivity increase over standard RT-QuIC, with no false positives reported[32,58]. IOME-RT-QuIC employs superparamagnetic iron oxide nanoparticles to capture PrP amyloid aggregates, followed by magnetic separation and concentration before RT-QuIC analysis. Denkers et al. demonstrated that this approach improves RT-QuIC detection limit for brain-derived PrPSc by approximately 100-fold[33]. These advancements have significantly enhanced RT-QuIC’s ability to detect low-level PrPSc in complex biological samples, paving the way for large-scale, non-invasive screening applications. However, blood-based RT-QuIC for sCJD diagnosis remains experimental and requires further validation and optimization.
Although RT-QuIC demonstrated extremely high specificity, it has been reported with reduced sensitivity in certain PrDs subtypes. To address this, Kraft et al. combined PMCA and RT-QuIC by applying the product of PMCA directly into RT-QuIC as substrate, successfully amplifying low PrPSc concentrations in muscle tissue of CWD-infected deer, whereas RT-QuIC alone yielded a markedly low amplification rate[59]. This finding highlights the potential of integrating protein amplification techniques to significantly enhance diagnostic sensitivity, particularly in samples with low PrP concentration. Therefore, RT-QuIC results should be interpreted in conjunction with clinical presentation, neuroimaging findings, and other biomarkers, and ideally used in combination with other protein amplification techniques (e.g., PMCA), to minimize the risk of misdiagnosis due to reliance on a single test result.
POTENTIAL OF PMCA AND RT-QUIC IN OTHER NEURODEGENERATIVE DISEASES
The successful research on PrDs has provided a model for other protein misfolding disorders. Many researchers are now attempting to apply PMCA and RT-QuIC to more common neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and related disorders. Although the hallmark proteins of these diseases - including β-amyloid (Aβ), α-synuclein (α-syn), tau, and TAR DNA-binding protein 43 (TDP-43) - are not classically infectious agents, they exhibit self-propagating and cell-to-cell spreading behaviors similar to PrPSc[60]. Therefore, developing in vitro seed amplification assays for these proteinopathies promises greater diagnostic sensitivity, in vivo diagnosis, and even treatment monitoring.
In AD, Aβ deposition and tau neurofibrillary tangles are the primary pathological hallmarks. In 2014, Soto et al. introduced an Aβ-PMCA assay that incubated synthetic Aβ with patient CSF under shaking while tracking fibril formation[61]. It detected Aβ oligomers at concentrations as low as 3 fmol and differentiated AD from controls with 90% sensitivity and 92% specificity[61]. This ultrasensitive ‘seed amplification’ approach reveals low-level Aβ aggregates invisible to ELISA, making it attractive for early diagnosis. Nevertheless, recently developed blood biomarkers - especially plasma phosphorylated tau (p-tau 217, p-tau 181, and p-tau 231) - reach comparable accuracy and can stage disease and predict progression[62-66], potentially limiting RT-QuIC’s added value for AD.
Meanwhile, significant advancements have been made with α-synucleinopathies, including PD, multiple system atrophy (MSA), and DLB. In recent years, αSyn-RT-QuIC has been widely applied to detect α-syn aggregates in CSF[67,68], skin biopsies[69,70], olfactory mucosa[71-73], duodenal and gastric mucosa[74]. Furthermore, by combining immunoprecipitation with RT-QuIC (IP-RT-QuIC), researchers have successfully amplified α-syn aggregates in blood[75] and saliva samples[76], expanding the potential for non-invasive or minimally invasive testing. In the newly proposed biological diagnostic framework for PD (SynNeurGe), pathological α-syn aggregation is a core diagnostic marker, underscoring the crucial role of in vitro amplification techniques such as RT-QuIC in PD diagnosis[77].
Tauopathies - including AD, progressive supranuclear palsy (PSP), frontotemporal degeneration (FTD), and Pick’s disease (PiD) - feature prion-like spread of hyperphosphorylated tau. However, reliable biomarkers that reflect tau seeding in peripheral tissues are scarce. Recently, Kraus et al. described an improved dual-substrate tau-RT-QuIC assay capable of detecting 3-repeat (3R) and 4-repeat (4R) tau aggregates, successfully identifying 3R tau seeds in PiD patients and 4R tau seeds in PSP patients’ CSF[78]. Postmortem skin tau-RT-QuIC has also shown 75%-80% sensitivity and 95%-100% specificity in AD, PSP, corticobasal degeneration (CBD), and PiD[79]. These suggest tau-RT-QuIC as a promising tool for in vivo diagnosis of various tauopathies. Yet, discriminating among individual tauopathies remained difficult: Though K12 RT-QuIC [using a C-terminally extended recombinant 3R tau substrate (K12CFh) as substrate] differentiated PiD (3R tau) from AD (3R/4R tau) through the quantitative differences in ThT responses, it failed to separate mixed 3R/4R tau subtypes (e.g., AD, and primary age-related tauopathy)[80]. Future research may focus on more disease-specific substrates to further sharpen specificity.
In amyotrophic lateral sclerosis (ALS) and FTD, the primary pathological protein involved is TDP-43. Scialò et al. demonstrated that recombinant human TDP-43 could serve as substrate for RT-QuIC, detecting minute amounts of TDP-43 seeds (as low as 15pg) and distinguishing ALS and FTD cohorts from controls with an overall sensitivity of 94% and specificity of 85%[81]. Notably, TDP-43 RT-QuIC also yielded positive results in the CSF of a GRN mutation carrier, hinting at presymptomatic utility[81].
Taken together, these advances demonstrate that PMCA and RT-QuIC, originally established for PrDs, can be effectively extended to a wide spectrum of neurodegenerative diseases [Table 3]. While challenges remain, particularly in achieving disease-specific discrimination and optimizing substrates, these ultrasensitive protein amplification techniques already hold considerable promise for early diagnosis, differential diagnosis, and longitudinal monitoring across multiple neurodegenerative disorders.
Applicability of RT-QuIC and PMCA in detecting other pathogenic proteins
| Protein | Aβ | αSyn | Tau | TDP-43 |
| Diseases | AD | PD, MSA, DLB | AD, PSP, FTD, PiD | ALS, FTD |
| Sensitivity | 90% (CSF PMCA)[61] | 92%-95.3% (CSF RT-QuIC)[67,68] 87.7%-94% (skin RT-QuIC)[69,70] 64.1%-96.4% (serum RT-QuIC)[75] 48%-81% (olfactory mucosa RT-QuIC)[71-73] 40.9% (duodenal RT-QuIC)[74] 68.4% (gastric RT-QuIC)[74] 83.78% (salivary RT-QuIC)[76] | 75%-80% (skin RT-QuIC)[79] | 94% (CSF RT-QuIC)[81] |
| Limitation | Superseded by blood biomarkers | Differential diagnosis between different αSyn-related diseases and their subtypes remains limited | Difficult to distinguish specific tauopathies by RT-QuIC alone | Further validation on preclinical detection is needed |
APPLICATION OF PMCA AND RT-QUIC IN OTHER FIELDS
Investigation of prion strain characteristics
In the study of PrPSc strains, PMCA provides an in vitro method for replicating and characterizing different strains, offering a crucial foundation for investigating their molecular properties. By amplifying PrPSc from different phenotypic variants, researchers can assess whether the amplified products retain the characteristics of the original strains. For instance, Castilla et al. demonstrated that PMCA-amplified PrPSc shared similar electrophoretic mobility, glycosylation profiles, and pathogenicity with those original strains, which are derived from the brains of animals with PrDs[82]. This finding, corroborated by multiple studies[11,18,83], supports the notion that the structural conformation of PrPSc dictates strain specificity and that these conformational properties can be faithfully propagated in a cell-free system. With this capability, PMCA enables researchers to compare the biochemical characteristics, replication kinetics, and drug responsiveness of different prion strains in vitro. Additionally, for prion strains that are challenging to maintain in animal models, PMCA-based amplification offers an alternative approach for strain preservation and serial propagation for further analysis. In 2020, Shahnawaz et al. demonstrated that PMCA could be applied to detect α-syn aggregates in CSF samples to distinguish the conformational strains associated with PD and MSA, achieving a detection sensitivity of 95.4%[84]. This study further validated the potential of PMCA in the molecular classification of neurodegenerative diseases and suggested its future application in the non-invasive or minimally invasive diagnosis of PrDs. Compared to PMCA, RT-QuIC currently lacks the ability to directly discriminate between prion strains. However, it remains a valuable tool for investigating the seeding properties of novel or atypical PrDs. For example, some studies have used RT-QuIC to examine variably protease-sensitive prionopathy, comparing its pathogenic PrPSc with that of classical CJD to elucidate molecular differences and similarities between these conditions[85,86].
Research on public health security
Monitoring of vCJD
Though primarily caused by consuming meat products contaminated with BSE, vCJD has also been reported to be transmissible between individuals through blood transfusion[87] and potentially via organ or tissue transplantation from asymptomatic carriers[88]. Consequently, there is an urgent need for highly sensitive detection methods to screen biological samples and organ donors for PrPSc, thereby minimizing the risk of vCJD transmission. Concha-Marambio et al. demonstrated that PMCA could detect PrPSc in blood samples from vCJD patients with 100% sensitivity and specificity, requiring only a few microliters of sample for analysis[39]. Additionally, PMCA has been successfully employed to screen asymptomatic vCJD carriers[39] and detect PrPSc in appendix samples[88]. These findings suggest that PMCA could be implemented for blood and blood product screening[89] to enhance transfusion safety, as well as for large-scale screening of asymptomatic vCJD carriers[90], thereby providing critical insights for public health policy. RT-QuIC, however, only amplifies PrPSc in vCJD samples with low efficacy; thus, it has not been applied to routine sample detection and monitoring of vCJD.
Assessment of species barriers and zoonotic risks
Beyond biological sample detection, PMCA and RT-QuIC have also proven valuable for assessing species barriers and zoonotic transmission risks of PrDs. Traditionally, interspecies transmission studies have relied on cross-species animal infection models, which are time-consuming and expensive. Recently, PMCA and RT-QuIC have emerged as rapid alternative methods for evaluating the cross-species transmissibility of PrDs. These techniques enable in vitro amplification by combining PrPSc ‘seeds’ and PrPC substrates from different species, thereby simulating prion compatibility across species and predicting the potential and conditions for cross-species transmission[83,91,92]. For example, Barria et al. utilized PMCA with human PrP substrates to investigate the cross-species transmission of CWD prions from deer. Their findings indicated that after multiple cross-species passages, prion conformation could adapt in a way that may increase infectivity in humans[91]. Furthermore, in vitro amplification experiments demonstrated that PMCA-generated PrPSc retained the biochemical and pathogenic characteristics of the original strain, including glycosylation patterns and infectivity, while preserving species-specific transmission properties[93].
Despite the potential of PMCA and RT-QuIC for evaluating cross-species transmission risks, the abilities of various animal prion strains to cross species barriers remain debated. This discrepancy may arise from the inherent limitations of in vitro amplification techniques in fully replicating the complex physiological conditions of PrDs transmission in vivo. Future research should focus on refining PMCA conditions, expanding its application to a broader range of prion strains, and standardizing its protocols and result interpretation, which will be essential for accurately assessing species barriers, ultimately contributing to animal disease surveillance and public health management.
Detection of contaminated surface and evaluation of decontamination
PrPSc has been shown to persist on various environmental surfaces - including surgical forceps, electroencephalography electrodes, soil, stainless steel, and plant debris - while retaining its infectivity. This persistence may further enhance its environmental stability, potentially exacerbating disease transmission. Studies have demonstrated that PMCA and RT-QuIC can detect extremely low levels of PrPSc[11,94-96], underscoring their potential for environmental surveillance. Compared to traditional bioassays using cell cultures or animal models, in vitro amplification techniques offer significantly higher sensitivity, shorter assay time[24,97,98], and high-throughput capabilities, making them preferred methods for detecting prion seeding activity.
Prions exhibit remarkable resistance to conventional sterilization protocols, making the decontamination of surgical instruments contaminated with PrPSc particularly challenging. Consequently, evaluating and improving decontamination procedures is critical for preventing the transmission of PrDs. Due to its ultra-high sensitivity, PMCA has been employed to detect residual PrPSc on instrument surfaces and to evaluate the effectiveness of different sterilization methods[99]. Belondrade et al. developed the surface PMCA (Surf-PMCA) method, a technique capable of detecting PrPSc residues equivalent to a 10-8 brain homogenate dilution on a single stainless steel wire, achieving sensitivity several orders of magnitude higher than traditional bioassays[94]. Based on such studies, the UK has recommended incorporating alkaline detergents and high-temperature, high-pressure sterilization for high-risk surgical instruments to maximize PrPSc clearance. Except for surgical instruments, PMCA and RT-QuIC have also been used to evaluate prion decontamination in environmental samples, such as farm soil, feeding troughs, and hospital wastewater, as well as to assess the residual infectivity of PrPSc in the environment[99].
In summary, protein misfolding amplification technologies provide an unprecedentedly sensitive approach for quantifying otherwise undetectable PrPSc contamination. These techniques facilitate the continuous refinement of prion decontamination strategies, thereby mitigating the risk of both iatrogenic and environmental transmission.
Drug screening
Currently, there are no effective treatments for PrDs; however, protein amplification techniques provide a highly efficient platform for screening anti-prion aggregation compounds. Instead of the traditional infected cell or animal models, RT-QuIC enables the rapid assessment of compounds that inhibit PrPSc formation by monitoring the extension of the lag phase and/or the reduction in ThT fluorescence amplitude[100]. This enables direct in vitro observation of PrPSc aggregation and inhibition, making RT-QuIC a powerful tool for evaluating the impact of compounds on prion seed propagation.
For instance, a study conducted by the Korean CDC utilized RT-QuIC to test known anti-prion compounds, including propidine derivatives, polyanions, and tannic acid. The results mirrored the PrPSc reduction observed in cell-based models. Notably, research by Hyeon et al. demonstrated that these compounds may either prevent PrPSc formation or promote the degradation of preformed aggregates. This highlights RT-QuIC’s potential to classify compounds as either aggregation inhibitors or disassemblers, a distinction that is challenging to achieve using traditional screening approaches[101].
Furthermore, in prion-infected mouse models, skin RT-QulC seeding activity was counteracted after treatment with anti-prion compounds that extended survival time, suggesting its potential as a biomarker for therapeutic efficacy[102]. So far, high-throughput RT-QuIC screenings have already been applied to dozens of compounds, identifying promising lead molecules that significantly inhibit PrP aggregation in vitro, thus informing subsequent cell-based and animal studies.
In summary, PMCA and RT-QuIC offer a rapid, efficient and non-infectious approach to anti-prion drug discovery, enabling the identification and characterization of potential therapeutic agents in a controlled environment [Figure 1]. These advances pave the way for the development of effective treatments for PrDs, accelerating progress against these devastating disorders.
Figure 1. The applications of RT-QuIC and PMCA (Created in BioRender. LIU, R. (2025). https://BioRender.com/104rlef). They can be utilized for the diagnosis and monitoring of prion diseases, prion strain investigation, assessment of species barriers and zoonotic risks, drug screening, detection of surface contamination, and evaluation of decontamination protocols. They also hold potential as auxiliary diagnostic tools for other neurodegenerative disorders. CSF: Cerebrospinal fluid; AD: Alzheimer’s disease; PD: Parkinson’s disease; RT-QuIC: real-time quaking-induced conversion; PMCA: protein misfolding cyclic amplification.
CHALLENGES AND PROSPECTS
Despite the significant advancements in PMCA and RT-QuIC, several challenges remain, along with opportunities for further innovation.
First, improving the sensitivity and reliability of detecting low PrPSc concentration samples, such as blood, saliva, and urine, is crucial for achieving large-scale, non-invasive screening. While PMCA has demonstrated 100% sensitivity for detecting vCJD blood samples under laboratory settings[39-41], sCJD accounts for approximately 85% of all PrDs, and no stable or reliable non-invasive detection method has yet been established. Therefore, future research should prioritize optimizing PMCA and RT-QuIC for detecting PrPSc in non-invasive biofluids from sCJD patients, improving both sensitivity and specificity to enhance clinical applicability.
Second, current PrPSc-PMCA and RT-QuIC assays are primarily qualitative, indicating only the presence or absence of PrPSc, while quantitative analysis remains a major challenge. Srivastava et al. recently optimized end-point dilution (ED) RT-QuIC by refining dilution factors, replicate counts, and data analysis algorithms, enabling quantification of α-syn seed concentrations. The refined assay could discriminate as little as 2-fold difference in α-syn seed concentration, and detected approximately 2- to 16-fold reductions in seeding activity[103]. Applying similar approaches to PrPSc quantification could provide a valuable tool for early, dynamic monitoring of PRNP mutation carriers and help determine optimal timing for clinical interventions. Furthermore, quantifying PrPSc seeding activity could facilitate pre- and post-treatment evaluations of therapeutic efficacy, providing an objective biomarker for assessing antibody- and small molecule-based therapies, thereby advancing precision medicine and drug discovery.
Third, ongoing improvements in amplification methods may further enhance assay performance. For example, researchers have proposed replacing the ultrasound step in PMCA with a more controlled oscillation process, termed ‘Protein Misfolding Shaking Amplification (PMSA)’[104], which may reduce protein conformational damage and improve amplification stability. Additionally, microfluidic chip technology has emerged as a promising tool for accelerating amplification reactions and enabling real-time detection, thereby improving efficiency while reducing operational complexity. These technological advancements will enhance the robustness and clinical practicality of PMCA and RT-QuIC, and further broaden their applications in the diagnosis and research of neurodegenerative diseases.
CONCLUSION
The invention and application of PMCA and RT-QuIC represent a major breakthrough in PrD research. These technologies enable researchers to precisely replicate the key pathogenic processes of PrDs in vitro, providing experimental validation for the proteinopathy hypothesis and transforming this pathogenic mechanism into highly sensitive detection methods. Clinically, PMCA and RT-QuIC have significantly improved detection rate of PrDs, enabling earlier, non-invasive diagnosis and valuable time for clinical management. In basic research, these technologies deepen our understanding of PrD transmission mechanisms, strain characteristics, and species barriers, while also accelerating the screening of potential therapeutic agents. Moreover, they play a crucial role in public health research and disease prevention and control, such as monitoring vCJD, detecting environmental contamination, and optimizing decontamination strategies. More broadly, the success of protein misfolding amplification technologies is informing work on other ‘seeding-type’ neurodegenerative diseases: RT-QuIC assays targeting α-syn and tau are already reshaping the diagnostic landscape of PD and related disorders.
In summary, protein misfolding amplification technologies, with their exceptional sensitivity and specificity, form a crucial bridge between molecular pathology and clinical application, bringing new hope for tackling PrDs and other protein misfolding disorders.
DECLARATIONS
Authors’ contributions
Writing and drafting: Liu R, Chen Z, Wang Y, Wu L
Editing: Wu L
Availability of data and materials
Not applicable.
Financial support and sponsorship
This work was supported by grants from ‘Yangfan 3.0’ Diagnostic and Therapeutic Capability Enhancement Project of Beijing Municipal Hospital Administration (ZLRK202515), National Natural Science Foundation of China (82271464, 81971011), and Capital’s Funds for Health Improvement and Research (2024-2-2018).
Conflicts of interest
All authors declared that there are no conflicts of interest.
Ethical approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Copyright
© The Author(s) 2025.
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