Amplification Assay Research Articles

Dual-Recognition-Mediated Autocatalytic Amplification Assay for the Subpopulations of PD-L1 Positive Extracellular Vesicle.

The PD-L1 protein on extracellular vesicles (EVs) is a promising biomarker for tumor immunotherapy. However, PD-L1+ EVs have various cell origins, so further analysis of the subpopulations is essential to help understand better their relationship with tumor immunotherapy. Different from the previous work which focus on the level of total PD-L1+ EVs expression, we, herein, report a dual-recognition mediated autocatalytic amplification (DRMAA) assay to detect the PD-L1 derived from tumors (EpCAM+), immune T cells (CD3+), and total (Lipids) EVs, respectively. The DRMAA assay employed proximity hybridization to construct a complete trigger sequence and then catalyzed the cross-hybridization of three hairpin probes, producing a three-way DNA junction (3-WJ) structure carrying the newly exposed trigger sequence. The 3-WJ complex subsequently initiated an autocatalytic amplification reaction and higher sensitivity than the traditional catalytic hairpin assembly assay was obtained. It was found that the EpCAM+ and PD-L1+ EVs were more effective than others in distinguishing lung cancer patients from healthy people. Surprisingly, the CD3+ and PD-L1+ EVs in lung cancer patients were also upregulated, indicating that immune cell-derived PD-L1+ EVs are also non-negligible marker in a tumor microenvironment. Our results suggested that the DRMAA assay would improve the study of subpopulations of PD-L1+ EVs to provide new insights for cancer immunotherapies.

DNA repair-retarded isothermal CRISPR amplification for rapid, sensitive base excision repair enzyme assay

BackgroundAs a core enzyme in the base excision repair system, uracil DNA glycosylase (UDG) is indispensable in maintaining genomic integrity and normal cell cycles. Its abnormal activity intervenes in cancers and neurodegerative diseases. Previous UDG assays based on isothermal amplification and Clustered Regularly Interspaced Short Palindromic Repeats/Cas (CRISPR/Cas) system were fine in sensitivity, but exposed to complications in assay flow, time, and probe design. After isothermal amplification, a CRISPR/Cas reagent should be separately added with extra manual steps and its guide RNA (gRNA) should be designed, considering the presence of protospacer adjacent motif (PAM) site. ResultsWe herein describe a UDG-REtarded CRISPR Amplification assay, termed ‘URECA’. In URECA, isothermal nucleic acid (NA) amplification and CRISPR/Cas12a system were tightly combined to constitute a one-pot, isothermal CRISPR amplification system. Isothermal NA amplification for a UDG substrate (US) with uracil (U) bases was designed to activate and boost CRISPR/Cas12a reaction. Such scheme enabled us to envision that UDG would halt the isothermal CRISPR amplification reaction by excising U bases and messing up the US. Based on this principle, the assay detected the UDG activity down to 9.17 x 10−4 U/mL in 50 min. With URECA, we fulfilled the recovery test of UDG activities in plasma and urine with high precision and reproducibility and reliably determined UDG activities in cell extracts. Also, we verified its capability to screen candidate UDG inhibitors, showing its potentials in practical application as well as drug discovery. SignificanceURECA offers further merits: i) the assay is seamless. Following target recognition, the reactions proceed in one-step without any intervening steps, ii) probe design is simple. Unlike the conventional CRISPR/Cas12a-based assays, URECA does not consider the PAM site in probe design as Cas12a activation relies on instantaneous gRNA binding to single-stranded DNA strands. By rationally designing an enzyme substrate probe to be specific to other enzymes, while keeping a role as a template for isothermal CRISPR amplification, the detection principle of URECA will be expanded to devise biosensors for various enzymes of biological, clinical significance.

High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Single-tube colorimetric loop-mediated isothermal amplification (LAMP) assay for high-sensitivity detection of SLCMV in cassava from southern India

Sri Lankan cassava mosaic virus (SLCMV) is a major cause for mosaic infections in cassava leaves, resulting in significant economic losses in southern India. SLCMV leads to growth retardation, leaf curl, and chlorosis in the host, with rapid transmission through whitefly insect vectors. Detecting SLCMV promptly is crucial, and the study introduces a novel and efficient colorimetric Loop-mediated isothermal amplification (LAMP) assay for successful detection in 60 min. Three primer sets were designed to target the conserved region of the SLCMV genome, specifically the coat protein gene, making the assay highly specific. The LAMP assay offers rapid and sensitive detection, completing within 60 min in a temperature-controlled water bath or thermal cycler. Compared to PCR techniques, it demonstrates 100 times superior sensitivity. The visual inspection of LAMP tube results using a nucleic acid dye and observing ladder-like pattern bands in a 2 % agarose gel confirms the presence of SLCMV. The assay is specific to SLCMV, showing no false positives or contaminations when tested against other virus. The standardized SLCMV LAMP assay proves technically efficient, providing a rapid, specific, simple, and low-cost solution, streamlining the detection and management of SLCMV.

Analysis of Replication, Cell Division-Mediated Spread, and HBV Envelope Protein-Dependent Pseudotyping of Three Mammalian Delta-like Agents.

The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.

DNA-Functionalized Gold Nanorods for One-Step Simultaneous Determination of Multiple microRNAs Based upon Plasma-Enhanced Fluorescence

MicroRNAs (miRNAs) are endogenous, noncoding, and sequence-specific single-stranded RNA regulatory factors whose abnormal expression levels are closely related to various diseases, especially cancer, and are considered effective biomarkers for disease diagnosis and prognosis. Since the concentrations of miRNAs in the body are low and disease diagnosis by detecting single miRNAs may result in a high false positive, it is essential to construct a sensitive biosensor for the determination of multiple miRNAs. In this work, an enzyme-free signal amplification assay based on plasma-enhanced fluorescence (PEF) is reported for the one-step simultaneous determination of two miRNAs. Gold nanorods (AuNRs) were functionalized with fluorescein isothiocyanate (FAM)-tagged hairpin DNA1 (H1) and cyanine 5.5 (Cy5.5)-tagged hairpin DNA2 (H2). The fluorescence signals of FAM and Cy5.5 were quenched due to their proximity to the surface of the AuNRs. In the presence of target miRNA-21 and miRNA-155, H1 and H2 complemented miRNA-21 and miRNA-155, respectively, and the fluorescence signals of FAM and Cy5.5 were not only restored but amplified due to the plasma-enhanced fluorescence. A suitable linear range (0.1 to 10 nM) and low limits of detection for miRNA-21 (23 pM) and miRNA-155 (1.6 pM) were achieved. The developed assay for the one-step simultaneous measurement of two miRNAs is simple, fast and sensitive, exhibiting great potential for monitoring these biomarkers.

Real-World Efficacy of Ensitrelvir in Hospitalized Patients With COVID-19 in Japan: A Retrospective Observational Study.

The coronavirus disease 2019 (COVID-19) pandemic necessitates continuously evaluating antiviral treatments, especially for high-risk groups, including older individuals. This study aimed to compare the efficacy of three antiviral drugs, including remdesivir, molnupiravir, and ensitrelvir, in hospitalized patients as measured by our own institution's antigen test, focusing on outcomes, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen levels, hospitalization duration, and fever resolution. This retrospective observational study was conducted at Yoshida Hospital, Asahikawa City, Japan, enrolling 154 patients who received antiviral treatment upon COVID-19 diagnosis from July 1, 2022, to September 15, 2023. The diagnosis was confirmed by proprietary antigen tests or loop-mediated isothermal amplification assays. Patients who received treatment outside the hospital or with consistently negative antigen results were excluded. Drug administration was determined by attending physicians, considering oral administration challenges and renal dysfunction. The data were statistically analyzed using an unpaired two-tailed Student's t-test and one-way analysis of variance complemented by the Tukey post-hoc test for detailed group comparisons. No significant differences were observed in the initial antigen levels among the treatment groups. By day 10, the ensitrelvir group showed lower antigen levels than the other groups, but not significantly. The ensitrelvir group had a higher antigen-negative conversion rate and a significantly shorter hospital stay than the molnupiravir group. However, no significant differences were noted in the fever resolution time among the groups. This study suggests the potential benefits of ensitrelvir in reducing antigen levels and hospitalization duration. However, the overall efficacy of the antiviral agents for symptomatic relief appears similar. These findings underscore the need for further research to optimize COVID-19 management by considering personalized treatment approaches and long-term outcomes.

Development and clinical evaluation of a real-time multiple cross displacement amplification assay for rapid and sensitive detection of Mycobacterium tuberculosis

Molecular techniques of nucleic acid testing recommended by the World Health Organization (WHO) for the Mycobacterium tuberculosis (MTB) detection were considered to have the potential access to the accurate tuberculosis (TB) notifications. In this study, a new method, which coupled real-time (rt) fluorescence technique with multiple cross displacement amplification (MCDA), was developed for the rapid, sensitive and specific detection of MTB (termed MTB-rt-MCDA). According to the principle of the rt-MCDA test, a set of ten primers were designed for the MCDA reaction, of which one was engineered with a restrictive endonuclease recognition site, a fluorophore and a quencher for achieving the real-time fluorescence detection. MTB-rt-MCDA test was conducted under the optimized conditions (67 °C, 40 min) on the real-time fluorescence platform. The MTB-rt-MCDA assay accurately identified the MTB strains with no cross reaction with other bacteria. The lowest detectable genomic DNA concentration of the MTB-rt-MCDA assay was 25 fg/μl. We employed the genomic DNA templates extracted from sputum of clinical cases for validating the practical applicability of this assay, and the detection power of the MTB-rt-MCDA assay was comparable to that of the Xpert method and MCDA-based biosensor detection and superior to smear microscope method. The complete process of the MTB-rt-MCDA assay, including rapid extraction of DNA and rt-MCDA test, takes less than 1 h. In conclusion, the presented MTB-rt-MCDA assay provided an effective and simple option for the rapid screening of MTB infection.

Parvalbumin Gene: A Valuable Marker for Pike Authentication and Allergen Risk Assessment.

Fish from the pike (Esox) genus are valued in gastronomy for their superior meat quality. However, they can cause allergic reactions in sensitive consumers. This work aimed to fill the gap in the detection of pike allergens using molecular-biological techniques. New, fast, and accurate loop-mediated isothermal amplification (LAMP) and real-time PCR (qPCR) assays were designed to detect pike DNA using the parvalbumin gene as a marker. LAMP was assessed by electrophoresis, SYBR green optical detection, and real-time fluorescence detection. The latter was the most sensitive, detecting as little as 0.78 ng of pike DNA; the qPCR detection limit was 0.1 ng. The LAMP analysis took 20-70 min, which is significantly faster than qPCR. The study provides reliable detection and quantification of the parvalbumin gene in both fresh and processed samples and further highlights the versatility of the use of the parvalbumin gene for the authentication of food products and consumer protection via refined allergen risk assessment that is independent of the type of tissue or food processing method used.

A cross-sectional study of α-synuclein seed amplification assay in Alzheimer's disease neuroimaging initiative: Prevalence and associations with Alzheimer's disease biomarkers and cognitive function.

Alzheimer's disease (AD) pathology is defined by β-amyloid (Aβ) plaques and neurofibrillary tau, but Lewy bodies (LBs; 𝛼-synuclein aggregates) are a common co-pathology for which effective biomarkers are needed. A validated α-synuclein Seed Amplification Assay (SAA) was used on recent cerebrospinal fluid (CSF) samples from 1638 Alzheimer's Disease Neuroimaging Initiative (ADNI) participants, 78 with LB-pathology confirmation at autopsy. We compared SAA outcomes with neuropathology, Aβ and tau biomarkers, risk-factors, genetics, and cognitive trajectories. SAA showed 79% sensitivity and 97% specificity for LB pathology, with superior performance in identifying neocortical (100%) compared to limbic (57%) and amygdala-predominant (60%) LB-pathology. SAA+ rate was 22%, increasing with disease stage and age. Higher Aβ burden but lower CSF p-tau181 associated with higher SAA+ rates, especially in dementia. SAA+ affected cognitive impairment in MCI and Early-AD who were already AD biomarker positive. SAA is a sensitive, specific marker for LB-pathology. Its increase in prevalence with age and AD stages, and its association with AD biomarkers, highlights the clinical importance of α-synuclein co-pathology in understanding AD's nature and progression. SAA shows 79% sensitivity, 97% specificity for LB-pathology detection in AD. SAA positivity prevalence increases with disease stage and age. Higher Aβ burden, lower CSF p-tau181 linked with higher SAA+ rates in dementia. SAA+ impacts cognitive impairment in early disease stages. Study underpins need for wider LB-pathology screening in AD treatment.

High Diversity of Curtobacterium flaccumfaciens in Poinsettia and Detection of Three Pathogenicity Plasmids for Identification of C. flaccumfaciens pv. poinsettiae

Outbreaks in Europe have raised concerns about poinsettia bacterial canker caused by Curtobacterium flaccumfaciens pv. poinsettiae, described in the United States. Using a semiselective medium containing aztreonam and fosfomycin and selection during isolation based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectra, 424 Curtobacterium strains were isolated from Belgian poinsettias of African and European origin. Different populations coexisted: 130 strains were identified as C. flaccumfaciens with scores ≥2.0 and 294 with scores <2.0 or as another Curtobacterium. MALDI-TOF MS libraries constructed using similar medium and extraction procedure and a pathogenicity test on poinsettia were used to screen collections from poinsettia and wheat, a possible alternative host, for C. flaccumfaciens pv. poinsettiae. The concatenated recA- gyrB partial sequences showed that 114 poinsettia or wheat strains belonged to different Curtobacterium species. Eighty-eight nonpathogenic strains and four U.S. strains from litter were intermingled with bean pathogenic C. flaccumfaciens pv. flaccumfaciens strains in the three genetic groups of C. flaccumfaciens but lacked their pathogenicity markers. Four new European recA- gyrB sequatypes of C. flaccumfaciens pv. poinsettiae, obtained from symptomatic and asymptomatic poinsettias, were distinguished from three American sequatypes. Six sequatypes were pathogenic in tests, belonged to genetic groups related to two different genomospecies, and possessed a plasmid. Sequencing of six plasmids revealed three related plasmids containing proteases and a polygalacturonase found only in strains pathogenic in pathogenicity tests and specifically identified by polygalacturonase-based PCR and loop-mediated isothermal amplification assays. Similarities between plant and litter Curtobacterium and the role of plasmids in pathogenicity and punctual transmission of pathogenicity among heterogeneous C. flaccumfaciens are suggested.

Rapid detection of grapevine berry inner necrosis virus using a simple reverse transcription loop-mediated amplification method

To develop a simple method for the detection of grapevine berry inner necrosis (GINV) that does not depend on expensive instruments, a reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assay was developed. A set of primers was used in RT-LAMP to specifically detect GINV and different GINV variants belonging to groups 1, 2 and 3. The sensitivity of the RT-LAMP was about 100 times greater than that of conventional RT-PCR. Furthermore, a rapid “pin-prick” assay without RNA extraction was developed to detect GINV in RT-LAMP. The assay performed well for the detection of GINV from different parts of field grapevine plants and micropropagated grapevine plantlets, and had a higher detection rate of GINV than RT-PCR in the tested grapevine samples.

Loop-mediated isothermal amplification (LAMP) assay targeting STY2879 gene for rapid detection of Salmonella enterica serovar Typhi in blood

Loop-mediated isothermal amplification (LAMP) assay targeting STY2879 gene for rapid detection of Salmonella enterica serovar Typhi in blood

Loop‐mediated isothermal amplification (LAMP) assays for Aedes and Culex species and evaluation of a simple DNA preparation method for field application

Loop‐mediated isothermal amplification (<scp>LAMP</scp>) assays for <i>Aedes</i> and <i>Culex</i> species and evaluation of a simple <scp>DNA</scp> preparation method for field application

Real-time fluorescence loop-mediated isothermal amplification assays for detection of zoonotic malaria Plasmodium parasites

BackgroundNatural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique. MethodsBy using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal. ResultsThere was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 109 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 1010 copies/µL. ConclusionThis is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.

Rapid on-site authentication of three cockle species using real-time loop-mediated isothermal amplification (LAMP) assay

Cockles, crucial for commercial fishing and aquaculture, emerge as highly marketable shellfish. However, the morphological similarities among various cockle species are frequently exploited for shellfish fraud. In this study, we developed LAMP assays (end-point LAMP and real-time LAMP) to detect three commercially important cockle species: Tegillarca granosa, Anadara broughtonii, and Anadara kagoshimensis. The primers were designed based on mitochondrial DNA and used to develop LAMP assays. Exclusivity was assessed using 17 shellfish species, and no cross-reactivity was confirmed. The estimate of the detection limit was determined to be 0.0001 ng. The established assays were further refined to create an on-site real-time LAMP assay by combining fast DNA isolation with a real-time fluorometer for field application. The effectiveness of the on-site real-time LAMP assay was successfully verified using 38 commercial samples, with analysis accuracy confirmed using a DNA extraction kit. Consequently, the on-site real-time LAMP assay can complete the whole procedure, including DNA extraction, real-time LAMP reaction, and data analysis within a 45 min timeframe, maintaining the accuracy of detecting three cockle species from processed cockle samples in the field. Our method proves valuable for detecting shellfish fraud by identifying cockle species using samples of both raw and processed products in the field.

An enzymatic recombinase amplification assay combined with CRISPR-Cas12a for the rapid detection of acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

An enzymatic recombinase amplification assay combined with CRISPR-Cas12a for the rapid detection of acute hepatopancreatic necrosis disease in shrimp Penaeus vannamei

Development of an isothermal reverse transcriptase-recombinase polymerase amplification assay for the rapid diagnosis of four viroids in different grape cultivars

Development of an isothermal reverse transcriptase-recombinase polymerase amplification assay for the rapid diagnosis of four viroids in different grape cultivars

A Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Bremia lactucae in the Field.

A real-time loop-mediated isothermal amplification (LAMP) assay for the detection of Bremia lactucae, the causal pathogen of lettuce downy mildew, was developed and validated to aid in-field detection of airborne inoculum. Assay specificity was confirmed against a range of other pathogenic oomycete and fungal spp., and sensitivity of the assay for the detection of DNA extracted from sporangia was evaluated. The B. lactucae LAMP assay reliably detected DNA equivalent to 1 spore/reaction (16.7 pg DNA/reaction). Following extraction of DNA from Rotorod air samplers, to which sporangial suspensions were added, the assay reliably detected 25 sporangia/Rotorod. Detection of airborne inoculum of B. lactucae collected through the season from air samplers deployed in-field in plots infected with B. lactucae and in commercial lettuce fields in Scotland over two growing seasons was assessed. The method can be deployed on samples collected from commercial lettuce production to inform disease management strategies and limit the use of unnecessary prophylactic pesticide applications.

Detection of α-Synuclein in Oral Mucosa by Seed Amplification Assay in Synucleinopathies and Isolated REM Sleep Behavior Disorder.

Evidence of abnormal α-synuclein (α-Syn) deposition in the brain is required for definitive diagnosis of synucleinopathies, which remains challenging. The seed amplification assay (SAA) is an innovative technique that can detect the seeding activity of misfolded α-Syn, enabling the amplification and detection of minute quantities of pathogenic α-Syn aggregates. This study aimed to evaluate oral mucosa α-Syn SAA as possible diagnostic and prodromal biomarkers for synucleinopathies. A total of 107 Parkinson's disease (PD) patients, 99 multiple system atrophy (MSA) patients, 33 patients with isolated rapid eye movement sleep behavior disorder (iRBD) and 103 healthy controls (HC) were included. The SAA was applied to detect the seeding activity of α-Syn from oral mucosa. A combination of morphological, biochemical, and biophysical methods was also used to analyze the fibrils generated from the oral mucosa α-Syn SAA. Structured illumination microscopy images revealed the increased α-Syn species in oral mucosa of PD, MSA, and iRBD patients than in HCs. Oral mucosa α-Syn SAA distinguished patients with PD from HC with 67.3% sensitivity and 90.3% specificity. Oral mucosa was α-Syn SAA positive in 53.5% MSA patients and 63.6% iRBD patients. Furthermore, the α-Syn fibrils generated from MSA demonstrated greater resistance to proteinase K digestion and exhibited stronger cytotoxicity compared to those from PD patients. Oral mucosa α-Syn seeding activity may serve as novel non-invasive diagnostic and prodromal biomarkers for synucleinopathies. The α-Syn aggregates amplified from the oral mucosa of PD and MSA exhibited distinct biochemical and biophysical properties. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.