A30p mutation-based alpha-synuclein rapid RT-QuIC diagnostic kit for Parkinson's disease

Abstract

The application provides a Parkinson's disease rapid RT-QuIC diagnostic kit based on A30P mutant alpha-synuclein, and belongs to the technical field of detection kits. The application uses a specific A30P mutant protein as a reaction substrate, and through an optimized RT-QuIC system, can rapidly and highly sensitively amplify and detect trace amounts of misfolded alpha-synuclein in a skin sample. The kit has the characteristics of short detection period and convenient operation, provides a new tool for molecular diagnosis of Parkinson's disease, is suitable for diagnosis of suspected PD cases in the clinic, and can also be used for screening of high-risk groups. The kit has a mature preparation process, raw materials are easy to obtain, and is easy to industrialize and popularize on a large scale, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of diagnostic kit technology, and more particularly to a rapid RT-QuIC diagnostic kit for Parkinson's disease based on A30P mutant α-synuclein. Background Technology

[0002] Parkinson's disease (PD) is the second leading cause of neurodegenerative disease worldwide, with its incidence rising significantly with population aging, imposing a heavy medical and economic burden on society and families. The core pathological feature of the disease is the misfolding and abnormal aggregation of α-synuclein (α-Syn), forming Lewy bodies within neurons, ultimately leading to the progressive death of dopaminergic neurons. However, current clinical diagnosis relies primarily on the assessment of motor symptoms (such as tremor, rigidity, and bradykinesia), but by the time these symptoms appear, neuronal damage in the brain often exceeds 50-70%, missing the optimal intervention window. Furthermore, the nonspecificity of early symptoms makes it easily confused with other neurological diseases, resulting in a high misdiagnosis rate. Therefore, developing molecular diagnostic methods capable of accurate identification in the early stages of the disease has become a critical issue that urgently needs to be addressed in this field.

[0003] Current auxiliary diagnostic methods for Parkinson's disease (PD) have significant limitations. While α-Syn detection in cerebrospinal fluid (CSF) can reflect pathological changes in the central nervous system, lumbar puncture sampling is invasive, posing potential risks, and patient acceptance and compliance are generally low. Imaging techniques, such as dopamine transporter scanning (DaT-scan), can provide functional evidence, but they are costly and lack sensitivity for early lesions, making them unsuitable as routine screening tools.

[0004] Real-Time Quaking-Induced Conversion (RT-QuIC) technology provides a revolutionary tool for the ultrasensitive detection of misfolded proteins, but existing technologies have the following intractable problems: the detection cycle is too long (usually 48 to 120 hours), which cannot meet the needs of rapid clinical diagnosis; the reaction system is complex, and the requirements for reagent ratio, operating environment and personnel skills are high, resulting in poor reproducibility of results. Summary of the Invention

[0005] The purpose of this invention is to provide a rapid RT-QuIC diagnostic kit for Parkinson's disease based on A30P mutant α-synuclein, which solves the technical problems of excessively long detection cycle, complicated operation, and insufficient identification specificity in existing in vitro diagnostic methods for Parkinson's disease based on RT-QuIC technology.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a Parkinson's disease auxiliary diagnostic kit based on RT-QuIC technology for detecting misfolded α-synuclein in skin samples, comprising reaction buffer, recombinant α-synuclein protein, negative control and positive control; The recombinant α-synuclein protein contains the A30P mutation, and its amino acid sequence is shown in SEQ ID NO.2.

[0008] Preferably, the amino acid sequence of the recombinant α-synuclein protein is shown in SEQ ID NO.4.

[0009] Preferably, the recombinant α-synuclein protein is prepared by a method comprising the following steps: a) Construct the gene encoding the recombinant α-synuclein protein into an expression vector; b) Transform the expression vector into the expression host for induced expression; c) Purify the recombinant α-synuclein protein from the expression product.

[0010] Preferably, in step a), the expression vector contains the nucleotide sequence shown in SEQ ID NO.3.

[0011] Preferably, the expression vector is a pET20b(+) vector; In step b), the expression host is Escherichia coli BL21(DE3).

[0012] Preferably, the reaction buffer contains Tris-HCl, sodium chloride, a surfactant, and thiosulfate T; The concentration of Tris-HCl is 50 mM and the pH is 7.5; the concentration of sodium chloride is 100 mM. The surfactant is a combination of CHAPS and Triton X-100, wherein the concentration of CHAPS is 0.2% and the concentration of Triton X-100 is 0.001%. The concentration of thiosulfate T is 20 μM; The negative control was sterile physiological saline, and the positive control was α-synuclein pre-prepared fibrils solution with a concentration of 0.1 mg / mL.

[0013] The present invention also provides the application of the above-described kit in the preparation of products for the diagnosis or auxiliary diagnosis of Parkinson's disease.

[0014] Preferably, the diagnosis or auxiliary diagnosis of Parkinson's disease is based on a sample taken from the skin behind the ear on the subject's neck.

[0015] The present invention also provides the application of the above-described kit in the preparation of products for detecting misfolded α-synuclein in samples.

[0016] The present invention also provides the application of the above-mentioned kit in the preparation of products for early screening, clinical diagnosis or monitoring of treatment effects of Parkinson's disease.

[0017] The beneficial effects of this invention are: This invention has significant advantages over existing technologies, and its core beneficial effects are as follows: 1. High diagnostic specificity and sensitivity: Based on the optimized human α-synuclein detection target and RT-QuIC amplification system, the sensitivity of detection of skin biopsy samples from PD patients reaches 96.67% and the specificity is 93.33%. It can effectively distinguish PD patients from healthy people or other non-PD patients, avoid false positive and false negative results, and solve the difficulty of insufficient accuracy of existing diagnostic methods.

[0018] 2. Fast detection speed and convenient operation: This invention can preliminarily determine a positive result within 8 hours and complete the test within 8-11 hours, which is significantly shorter than the traditional RT-QuIC technology (48-96 hours). The reagent kit has a simple operation process, does not require complicated instruments, and is more suitable for rapid clinical screening.

[0019] 3. Convenient and minimally invasive sample acquisition: Using a small amount (2-3mm) of skin sample from the neck or ankle as the test object, the process is less invasive and more acceptable to patients compared to the collection of samples such as cerebrospinal fluid. It can achieve non-invasive and routine screening, breaking through the current problem of limited sample collection.

[0020] 4. High technical stability and repeatability: The buffer system has been repeatedly tested and proven to be stable. The core reagent substrate protein has undergone gene optimization, prokaryotic expression, purification verification and lyophilization, which enables this kit to be stably stored for 12 months or more under the condition of -20℃ in a conventional laboratory, reducing the risk of fluctuation in test results.

[0021] 5. Wide range of applications and controllable costs: It is suitable for both clinical diagnosis of suspected PD cases and screening of high-risk groups. The reagent kit has a mature manufacturing process, readily available raw materials, and is easy to industrialize and scale up, making it of significant clinical application value. Attached Figure Description

[0022] Figure 1The purification and identification results of α-Syn recombinant protein are shown in Figure 1. Where: M is the pre-stained molecular weight standard for the protein; 1 is the sample before recombinant protein induction; 2 is the sample after recombinant protein induction; 3 is the precipitate after recombinant protein expression induction and lysis; 4 is the supernatant after recombinant protein expression induction and lysis; 5 is the precipitate after boiling; 6 is the supernatant after boiling; 7 is the sample after acid precipitation; and 8 is the supernatant after acid precipitation. Figure 2 The results of purification and identification of α-Syn recombinant protein are shown in Figure 2, where: M is the pre-stained molecular weight standard of the protein; 1 is the supernatant sample after alkaline precipitation; 2 is the Ni-NTA affinity chromatography flow-through buffer; 3 is the 0 mM imidazole washing sample; 4 is the 10 mM imidazole washing sample; 5 is the 20 mM imidazole washing sample; 6 is the 500 mM imidazole eluted sample; and 7 is the size exclusion chromatography (SEC) purified sample. Figure 3 The image shows the Western Blot identification results of α-Syn recombinant protein, where: M is the pre-stained molecular weight standard of the protein, 1 is the sample of recombinant protein before induction, and 2 is the lyophilized powder sample of recombinant protein. Figure 4 The fluorescence signal curves were used to validate clinical samples. The horizontal axis represents incubation time (h), and the vertical axis represents fluorescence intensity (RFU). Figure 5 The graph shows the fluorescence signal curves for cross-seeding detection. The horizontal axis represents incubation time (h), and the vertical axis represents fluorescence intensity (RFU). Detailed Implementation

[0023] This invention provides a Parkinson's disease auxiliary diagnostic kit based on RT-QuIC technology for detecting misfolded α-synuclein in skin samples, comprising a reaction buffer, recombinant α-synuclein protein, a negative control, and a positive control; wherein the recombinant α-synuclein protein contains an A30P mutation, and its amino acid sequence is shown in SEQ ID NO.2.

[0024] In this invention, the "reagent kit" refers to a set of reagents, controls, consumables, and instruction materials packaged together to achieve a specific detection purpose, facilitating standardized operation. RT-QuIC (Real-Time Oscillator-Induced Conversion) technology is an ultrasensitive detection technique based on the principle of protein misfolding cyclic amplification. It can exponentially amplify trace amounts of misfolded protein "seeds" in a sample, thereby detecting them through fluorescence signals. Skin samples, as peripheral tissue samples, are minimally invasive to obtain, have high patient compliance, and have been shown to contain α-synuclein deposits associated with central nervous system pathologies. α-Synuclein is a protein abundantly expressed in the presynaptic terminals of the central nervous system; its misfolding and abnormal aggregation are core pathological markers of synuclein-related diseases such as Parkinson's disease and Lewy body dementia. The A30P mutation is a point mutation in which alanine at position 30 of the protein is replaced by proline. SEQ ID NO.2 provides the specific sequence information of a 140-amino acid mutant of human α-synuclein protein that includes this mutation.

[0025] Preferably, the amino acid sequence of the recombinant α-synuclein protein is shown in SEQ ID NO.4.

[0026] The amino acid sequence shown in SEQ ID NO.4 is based on SEQ ID NO.2, with a purification tag sequence consisting of 6 histidine (His) residues fused to the C-terminus, and an additional vector expression-related short peptide sequence may be included at the N-terminus. The histidine tag (6×His tag) is an affinity tag widely used in recombinant protein purification; it binds to nickel (Ni... 2+ ) or cobalt (Co) 2+ The specific binding of ion chelating media allows for rapid and efficient purification of target proteins via metal ion affinity chromatography (such as Ni-NTA chromatography). Recombinant proteins with this tag retain their biological activity while significantly simplifying downstream purification processes.

[0027] Preferably, the recombinant α-synuclein protein is prepared by a method comprising the following steps: a) Construct the gene encoding the recombinant α-synuclein protein into an expression vector; b) Transform the expression vector into the expression host for induced expression; c) Purify the recombinant α-synuclein protein from the expression product.

[0028] Step a) involves gene construction, which involves inserting the target gene sequence into a suitable expression vector using molecular cloning techniques (such as restriction endonuclease digestion and ligation, homologous recombination, Gibson assembly, etc.) to form a recombinant expression plasmid. Step b) describes the expression host, typically a prokaryotic system (such as *E. coli*) or a eukaryotic system (such as yeast, insect cells, mammalian cells), selected based on factors including expression level, protein solubility, and post-translational modification requirements. Induced expression is initiated by adding a chemical inducer (such as IPTG) or altering culture conditions (such as temperature) to initiate the transcription and translation of the exogenous gene. Step c) describes the purification process, which may include cell disruption, centrifugation, chromatography (such as affinity chromatography, ion exchange chromatography, size exclusion chromatography), concentration, and dialysis to obtain a high-purity target protein.

[0029] Preferably, in step a), the expression vector contains the nucleotide sequence shown in SEQ ID NO.3.

[0030] SEQ ID NO.3 provides the complete or partial nucleotide sequence of a specific recombinant expression vector for expressing the α-synuclein recombinant protein of the present invention. An expression vector is a DNA molecule capable of autonomous replication within a host cell and directing the expression of a foreign gene, typically containing a replication origin, selection markers (such as antibiotic resistance genes), multiple cloning sites, and transcriptional-translational regulatory elements (such as promoters, ribosome binding sites, and terminators). This specific sequence information uniquely defines the specific DNA construct used to produce the protein of the present invention.

[0031] Preferably, the expression vector is a pET20b(+) vector.

[0032] The pET series of vectors is one of the commonly used vector systems in *E. coli* for high-level expression of recombinant proteins. pET20b(+) is a specific vector in this series, characterized by a T7 / lac heterozygous promoter, efficient induction by IPTG, and the presence of a pelB signal peptide sequence (for periplasmic expression) and a C-terminal 6×His tag coding sequence. Using the pET20b(+) vector facilitates soluble or secretory expression of the target protein and allows for purification via the His tag.

[0033] Preferably, in step b), the expression host is Escherichia coli BL21(DE3).

[0034] Escherichia coli BL21(DE3) is a classic host strain for protein expression. This strain lacks the lon and ompT proteases, reducing the degradation of recombinant proteins. Simultaneously, its genome integrates the λ phage DE3 lysoma, which contains the T7 RNA polymerase gene controlled by the lacUV5 promoter. Therefore, it can perfectly cooperate with vectors containing the T7 promoter, such as pET, enabling efficient expression of target proteins after the addition of IPTG. BL21(DE3) and its derivatives (such as Rosetta™) are widely used in the production of recombinant proteins due to their rapid growth, ease of culture, and high expression levels.

[0035] Preferably, the reaction buffer comprises Tris-HCl, sodium chloride, a surfactant, and thiamine T; the concentration of Tris-HCl is 50 mM and the pH is 7.5; the concentration of sodium chloride is 100 mM; the surfactant is a combination of CHAPS and Triton X-100, wherein the concentration of CHAPS is 0.2% and the concentration of Triton X-100 is 0.001%; ​​the concentration of thiamine T is 20 μM; the negative control is sterile physiological saline, and the positive control is an α-synuclein pre-prepared fibrous solution with a concentration of 0.1 mg / mL.

[0036] In this invention, the reaction buffer provides a suitable physicochemical environment for the entire RT-QuIC detection reaction. Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride is a commonly used biological buffer used to maintain the stability of the pH value of the reaction system. In some embodiments of this invention, the concentration of Tris-HCl can be selected between 10 mM and 200 mM, for example, 20 mM, 30 mM, 50 mM, or 100 mM; preferably 30 mM to 80 mM; more preferably 40 mM to 60 mM; and most preferably 50 mM. Its pH value can be adjusted between 6.8 and 8.5, for example, 7.0, 7.2, 7.5, 7.8, or 8.0; preferably 7.2 to 7.8; more preferably 7.4 to 7.6; and most preferably 7.5. Sodium chloride is used to provide a suitable ionic strength, and its concentration range can be from 0.05M to 0.5M, for example 0.08M, 0.1M, 0.15M or 0.2M; preferably from 0.08M to 0.2M; most preferably 0.1M.

[0037] In this invention, surfactants can reduce surface tension, prevent nonspecific protein adsorption, and influence protein aggregation kinetics. CHAPS (3-[(3-cholamidopropyl)dimethylamino]propanesulfonate) is a zwitterionic detergent with mild properties; Triton X-100 is a nonionic surfactant. Their combined use helps optimize reaction specificity. The total concentration of surfactants can range from 0.001% to 1% (w / v or v / v), preferably from 0.01% to 0.5%. The concentration of CHAPS can be independently from 0.05% to 1%, preferably from 0.1% to 0.3%, and most preferably 0.2%. The concentration of Triton X-100 can be independently from 0.0001% to 0.1%, preferably from 0.0005% to 0.01%, and most preferably 0.001%.

[0038] In this invention, thiosulfate T (ThT) is a benzothiazole fluorescent dye that specifically binds to amyloid fibers rich in β-sheet structures, significantly enhancing their fluorescence intensity after binding. It is commonly used for real-time monitoring of protein fibrillation processes. Its working concentration can be between 1 μM and 100 μM, for example, 5 μM, 10 μM, 20 μM, or 50 μM; preferably 10 μM to 40 μM; further preferably 15 μM to 25 μM; and most preferably 20 μM.

[0039] In this invention, negative controls are used to establish the background fluorescence threshold for detection, and are typically matrix solutions without the target analyte. Sterile physiological saline (containing approximately 0.9% sodium chloride) is one of the most commonly used negative controls; PBS buffer or the reaction buffer itself can also be used. Positive controls are used to verify the effectiveness of the entire detection system, ensuring that the reagents and instruments are functioning correctly. α-synuclein pre-fabricated filaments (PFFs) are fibrous aggregates formed by the spontaneous aggregation of recombinant α-synuclein in vitro, which can mimic the "seeds" in pathological samples. Their concentration can be adjusted according to the required detection sensitivity, for example, from 0.01 mg / mL to 1 mg / mL; preferably from 0.05 mg / mL to 0.5 mg / mL; and most preferably 0.1 mg / mL.

[0040] This invention also provides the application of the above-described kit in the preparation of products for the diagnosis or auxiliary diagnosis of Parkinson's disease. The term "product" as used herein can refer to a complete in vitro diagnostic (IVD) system, a testing service package, or diagnostic reporting software containing the test results, with the kit as its core component. "Diagnosis" of Parkinson's disease refers to the process of ultimately determining the disease through a comprehensive assessment (including clinical symptoms, medical history, imaging, and laboratory tests); while "auxiliary diagnosis" refers to providing objective laboratory evidence to support a clinician's diagnostic decision, especially in cases of atypical symptoms or early stages of the disease. This kit provides an objective molecular pathological basis for the diagnosis of Parkinson's disease by detecting the presence of misfolded α-synuclein in the skin.

[0041] This invention also provides the application of the above-described kit in the preparation of products for detecting misfolded α-synuclein in samples. The core of this application lies in utilizing the composition and reaction system of the kit to specifically and sensitively detect the target biomarker—misfolded α-synuclein. The term "product" as used herein is not limited to the kit itself, but can be extended to any commercial or standardized form that incorporates the core function of detection using this kit. For example, the product could be an integrated platform combining an automated detection instrument and a kit, or a software algorithm specifically designed to analyze the data generated by this kit.

[0042] This invention also provides the application of the above-mentioned kit in the preparation of products for early screening, clinical diagnosis, or treatment efficacy monitoring of Parkinson's disease. This application clarifies three core clinical application scenarios for the kit of this invention. "Early screening" refers to large-scale preliminary testing in target populations (such as those with a family history or prodromal symptoms) before the development of typical motor disorders, to identify high-risk individuals. "Clinical diagnosis" refers to using the test results as one of the laboratory criteria for diagnosis and differential diagnosis (such as differentiating essential tremor, multiple system atrophy, etc.) after the onset of symptoms. "Treatment efficacy monitoring" refers to periodically repeating the test after the patient receives intervention (such as medication or surgery), indirectly assessing whether the treatment has affected the core pathological processes of the disease by observing the dynamic changes in the activity of misfolded α-synuclein "seeds" in the skin, providing a reference for efficacy evaluation and treatment plan adjustment. Products used for monitoring require particular attention to the stability and comparability of the test results.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] The main sequences involved in the embodiments are as follows: SEQ ID NO.1: Codon-optimized human SNCA gene nucleotide sequence (420 bp in length) ATGGACGTGTTCATGAAAGGTCTGTCTAAAGCGAAAGAGGTGTTGTTGCTGCGGCTGAGAAGACCAAACAGGGTGTTGCTGAAGCTCCGGGTAAGACTAAAGAAGGCGTTCTGTACGTTGGTTCTAAGACCAAAGAAGGTGTTGTTCACGGTGTGCGACTGTTGCGGAAAAAACCAAAGAACAGGTTACCAACGTTGGTGGTGCGGTT GTTACTGGTGTTACCGCAGTTGCGCAGAAGACCGTTGAAGGTGCTGGTTCCATCGCAGCAGCTACCGGTTTCGTTAAGAAAGACCAGCTGGGTAAGAACGAAGAAGGTGCTCCGCAAGAAGGTATCTTGGAAGATATGCCGGTAGATCCGGACAACGAAGCATACGAAATGCCGTCTGAAGAAGGTTACCAAGACTACGAACCGGAAGCA SEQ ID NO.2: Amino acid sequence of mutant A30P human α-Syn protein (length 140aa) MDVFMKGLSKAKEGVVAAAEKTKQGVAEAPGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA SEQ ID NO.3: Nucleotide sequence of recombinant vector pET20b-α-Syn (containing SNCA gene and 6×His tag, length ~5000bp) SEQ ID NO.4: The amino acid sequence of the α-Syn protein expressed by the recombinant vector used in this invention (containing a 6×His tag, 157 aa in length). MDIGINSDPMDVFMKGLSKAKEGVVAAAEKTKQGVAEAPGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH

[0045] Example Preparation and identification of α-Syn recombinant protein 1. Plasmid construction Based on the wild-type sequence of the human α-synuclein gene (GenBank: 6622), synonymous mutation optimization was performed using E. coli codon bias to specifically eliminate rare codons, potential secondary structures, and redundant restriction enzyme sites in the sequence, thereby improving subsequent prokaryotic expression efficiency. The optimized full-length gene with the A30P mutation point was artificially synthesized, and its optimized nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The synthesized gene and the pET 20b(+) vector (containing a C-terminal 6×His tag) were double-digested with enzymes, and the target fragment was purified by agarose gel electrophoresis. In vitro directional ligation was performed using T4 DNA ligase to construct the recombinant plasmid pET 20b-α-Syn. The recombinant plasmid was confirmed to be correctly constructed and free of base mutations, and its nucleotide sequence is shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.4.

[0046] 2. Prokaryotic expression The recombinant plasmid pET 20b-α-Syn, verified by sequencing, was transformed into *E. coli* BL21(DE3) competent cells. The cells were plated on LB agar plates containing Amp resistance and incubated at 37°C for 16 hours to screen for positive single colonies. Single colonies were picked and inoculated into self-induction medium and cultured at 37°C with shaking at 220 rpm until OD200 reached. 600 After reaching a molecular weight of 0.6, the temperature was adjusted to 30℃ and cultured continuously for 16 hours. The bacterial culture was collected and centrifuged, and the supernatant and precipitate samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed a clear and specific band at a relative molecular mass of approximately 14 kDa, consistent with the theoretical molecular weight of the α-synuclein recombinant protein, confirming that the target protein was expressed in a highly efficient and soluble manner.

[0047] 3. Protein purification 1. Cell disruption: Collect 1L of induced bacterial culture, centrifuge to collect the cells, resuspend in 50mL of lysis buffer, homogenize in an autoclave for 15min, and centrifuge to collect the supernatant; 2. Boiling treatment: Boil the supernatant of the bacterial solution in a 100℃ water bath for 10 minutes, then allow it to cool naturally; 3. Acid-base precipitation: Add 1M HCl to adjust the pH to 3.5, stir and mix at 4°C for 30 min, then centrifuge at 4°C and 12000 rpm for 45 min; collect the supernatant, add 1M NaOH to adjust the pH of the supernatant to 7.6, centrifuge again at 4°C and 12000 rpm for 45 min, and transfer the supernatant to a new centrifuge tube (SDS-PAGE showed a purity of 80%). 4. Ni-NTA column chromatography purification: Load the sample into a pre-equilibrated Ni-NTA purification column, wash with 0 mM, 10 mM, and 20 mM imidazole buffers sequentially, and elute the target protein with 500 mM imidazole buffer (SDS-PAGE showed a purity of 90%). 5. Size exclusion chromatography: The dialyzed sample was concentrated to 10 mL using a 3 kDa ultrafiltration tube; the sample was loaded onto a Superdex 75 gel filter column, and the monomeric protein peak was collected (SDS-PAGE showed a purity of 95%). 6. Lyophilization: Dispense into 0.4mg vials and lyophilize, then store at -80℃.

[0048] Surfactant screening To optimize the RT-QuIC reaction system, this embodiment screened various surfactants to improve the sensitivity and specificity of the detection.

[0049] 1. Surfactant type and concentration settings The surfactants tested included: SDS, Triton X-100, ammonium sulfate, CHAPS, Tween-20, Tween-80, Ficoll 70, and PEG400. The concentrations were set as follows: SDS concentration: 0-0.001% Triton X-100, CHAPS, Tween-20, Tween-80 concentrations: 0-2.0% Ammonium sulfate, Ficoll 70, and PEG 400 concentrations: 0-20% 2. Incubation temperature setting The incubation temperature was set at four gradients: 37℃, 45℃, 50℃, and 55℃.

[0050] 3. Test Results Comparing the detection effects at different incubation temperatures and surfactant concentrations, the results showed that a rapid and accurate detection could be achieved at an incubation temperature of 50℃ using a 0.2% CHAPS + 0.001% Triton X-100 system, exhibiting high sensitivity and specificity. Therefore, this invention selected the CHAPS + Triton X-100 combination as the surfactant component in the reaction buffer.

[0051] Reagent kit composition and detection process: 1. Core components and specifications of the reagent kit The kit provided in this embodiment contains the following main components, and the detailed composition and specifications of each component are shown in Table 1 below: Table 1. Kit Components

[0052] 2. Detection methods (steps and key parameters) Step 1: Skin Sample Collection and Preprocessing Sampling site: The preferred location is the skin at C7 behind the ear on the neck. Sampling procedure: Use a disposable 2mm biopsy needle to take 5-10mg of skin tissue (depth ≤1mm, avoid damaging the deep dermis), and immediately place it into a sterile centrifuge tube containing physiological saline; Pretreatment process: ① Rinse 3 times with physiological saline (1 mL each time, shake for 10 s) to remove surface contaminants; ② Add 1 mL of a rapid skin tissue protein extraction reagent containing 1×PMSF, the main components of which are Triton X-100, Tween-20, EDTA, phosphate-buffered saline (PBS), octylphenoxypolyethoxyethanol (NP-40), etc.; incubate at room temperature for 5-10 min (mix gently once every 2 min). ③ Manual grinding with a disposable grinding stick for 1-5 minutes is sufficient to completely break down the tissue; ④ Centrifuge at 4℃ and 10000g for 1 min, and collect the supernatant; ⑤ Dilute the supernatant 10 times with sterile water and store in portions at -80℃.

[0053] Step 2: Preparation of RT-QuIC reaction solution Prepare the RT-QuIC reaction solution according to the proportions shown in Table 2 below. The required amounts of each component to be added to each well are as follows: Table 2 RT-QuIC reaction solution

[0054] Step 3: 96-well plate sample loading and incubation reaction Add 98 μL of the above RT-QuIC reaction solution to each well of a pre-loaded black 96-well plate containing silica beads. Then add 2 μL each of the negative control, positive control, and test sample, with three replicates per sample group. After sample addition, cover the plate with a sealing film to prevent evaporation. Place the 96-well plate in a multi-functional microplate reader (QF-pro100) and set the detection program according to the parameters shown in Table 3 below: Table 3 Detection Parameters

[0055] Step 4: Fluorescence detection and result interpretation After sample testing, the fluorescence intensity change curve is analyzed using ELISA reader software. A ROC threshold is set (determined based on clinical sample validation). If the fluorescence intensity of at least two replicate reactions exceeds the set threshold, the sample is judged as positive; conversely, if the fluorescence intensity of all replicate reactions is below the threshold, the sample is judged as negative.

[0056] Clinical sample validation 1. Sample Selection Sixty skin biopsy samples were selected, including 30 clinically diagnosed PD samples (meeting the 2015 MDS diagnostic criteria) and 30 non-PD control samples (including 5 healthy controls and 25 patients with other neurological diseases).

[0057] 2. Detection Method The detection method described in this invention includes sample pretreatment, reaction solution preparation, sample addition, incubation reaction, and result determination.

[0058] 3. Test Results The negative control test result was negative, and the positive control test result was positive, proving that the detection system is stable and reliable. As the reaction proceeds, the fluorescence signal of the PD sample begins to increase at approximately 4 hours, reaches its peak at approximately 9-12 hours, and then tends to stabilize, exhibiting an S-shaped fluorescence curve, indicating the presence of α-synuclein seeding activity in the PD sample. The fluorescence value of non-PD samples did not increase or only slightly increased, indicating that non-PD samples lack α-synuclein seeding activity. The statistics of each detection index are shown in Table 4 below: Table 4 Detection Indicators

[0059] Cross-seeding test 1. Sample Selection A total of 30 skin biopsy samples were collected, including 3 clinically diagnosed PD samples, 10 transthyretin amyloidosis (TTR) samples, 8 multiple system atrophy (MSA) samples, 7 Alzheimer's disease (AD) samples, and 2 healthy controls.

[0060] 2. Detection Method The α-synuclein seeding activity was detected using the kit described in this invention.

[0061] 3. Test Results The negative control showed a negative result, and the positive control showed a positive result, demonstrating the stability and reliability of the detection system. As the reaction proceeded, all 10 PD samples exhibited strong α-synuclein seeding activity; while 7 AD samples, 10 TTR samples, 8 MSA samples, and 2 healthy controls showed no enhancement of fluorescence signal, indicating that the kit of this invention has no cross-seeding reaction and good specificity for PD.

[0062] The purification and identification results of the α-Syn recombinant protein are shown in the figure. Figures 1-2 The Western Blot results are shown below. Figure 3 The purification and identification results of α-Syn recombinant protein showed that the purified α-Syn recombinant protein had a single specific band at a relative molecular mass of about 14 kDa, and the protein purity was ≥95%.

[0063] The fluorescence signal curves for clinical sample validation are shown below. Figure 4 The results showed that the fluorescence signal in PD patient samples increased significantly after 4-5 hours and reached a plateau at 8-10 hours, both of which were higher than the threshold; the fluorescence signal in non-PD samples and negative controls was lower than the threshold.

[0064] The fluorescence signal curve for cross-seeding detection is shown in the figure. Figure 5 The results showed that the fluorescence signal of PD patient samples increased significantly after 3-5 hours and reached a plateau period after 8-10 hours, all of which were higher than the threshold; the fluorescence signals of AD, TTR and MSA samples were lower than the threshold.

[0065] As demonstrated by the above embodiments, this invention provides a Parkinson's disease auxiliary diagnostic kit based on a specific protein and an optimized RT-QuIC reaction system. This kit uses minimally invasive skin samples, can complete the detection in a short time, and shows high recognition and discrimination ability for samples from Parkinson's disease patients. Experimental results show that the kit is stable and easy to operate, providing a new potential tool for the early detection and clinical auxiliary diagnosis of Parkinson's disease.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A diagnostic kit for Parkinson's disease based on RT-QuIC technology for detecting misfolded α-synuclein in skin samples, characterized in that, This includes reaction buffer, recombinant α-synuclein protein, negative control, and positive control; The recombinant α-synuclein protein contains the A30P mutation, and its amino acid sequence is shown in SEQ ID NO.2; The reaction buffer contains Tris-HCl, sodium chloride, surfactant, and thiosulfate T; The concentration of Tris-HCl is 50 mM and the pH is 7.5; the concentration of sodium chloride is 100 mM. The surfactant is a combination of CHAPS and Triton X-100, wherein the concentration of CHAPS is 0.2% and the concentration of Triton X-100 is 0.001%. The concentration of thiosulfate T is 20 μM.

2. The reagent kit according to claim 1, characterized in that, The amino acid sequence of the recombinant α-synuclein protein is shown in SEQ ID NO.

4.

3. The reagent kit according to claim 1, characterized in that, The recombinant α-synuclein protein was prepared by a method comprising the following steps: a) Construct the gene encoding the recombinant α-synuclein protein into an expression vector; b) Transform the expression vector into the expression host for induced expression; c) Purify the recombinant α-synuclein protein from the expression product.

4. The reagent kit according to claim 3, characterized in that, In step a), the expression vector contains the nucleotide sequence shown in SEQ ID NO.

3.

5. The reagent kit according to claim 4, characterized in that, The expression vector is the pET20b(+) vector; In step b), the expression host is Escherichia coli BL21(DE3).

6. The reagent kit according to claim 1, characterized in that, The negative control was sterile physiological saline, and the positive control was α-synuclein pre-prepared fibrils solution with a concentration of 0.1 mg / mL.

7. The use of the kit according to any one of claims 1 to 6 in the preparation of products for the diagnosis or auxiliary diagnosis of Parkinson's disease.

8. The application according to claim 7, characterized in that, The diagnosis or auxiliary diagnosis of Parkinson's disease is based on a sample taken from the skin behind the ear on the subject's neck.

9. Use of the kit according to any one of claims 1 to 6 in the preparation of a product for detecting misfolded α-synuclein in a sample.

10. The use of the kit according to any one of claims 1 to 6 in the preparation of a product for early screening, clinical diagnosis or monitoring of treatment efficacy for Parkinson's disease.

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