Parkinson's disease auxiliary diagnosis kit based on RT-QuIC technology and application of Parkinson's disease auxiliary diagnosis kit

Abstract

The invention provides a Parkinson's disease auxiliary diagnosis kit based on an RT-QuIC technology and application of the Parkinson's disease auxiliary diagnosis kit, and belongs to the technical field of detection kits. The kit provided by the invention has remarkable minimally invasive property and high patient compliance, detection can be completed only by collecting trace skin tissues, and risks and pains of lumbar puncture or brain tissue biopsy are avoided. The kit has excellent detection performance, can realize highly sensitive and specific identification of early pathology of Parkinson's disease, has a fast detection process, can complete batch sample analysis in a short time, and powerfully supports clinical rapid diagnosis and screening requirements. Meanwhile, the overall cost is remarkably lower than that of existing methods such as imaging, a convenient, reliable and economical molecular detection means is provided for early discovery, clinical auxiliary diagnosis and treatment effect observation of the Parkinson's disease, and positive significance is achieved for improving the overall diagnosis and treatment level of the neurodegenerative diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection kits, in particular to a Parkinson's disease auxiliary diagnosis kit based on RT-QuIC technology and application thereof. BACKGROUND

[0002] Parkinson's disease is a common chronic progressive neurodegenerative disease, and its pathological features include the loss of neurons in specific parts of the midbrain and the formation of intracellular abnormal protein aggregates. The clinical manifestations of the disease mainly include motor symptoms and non-motor symptoms, but its diagnosis, especially in the early stage, is quite challenging. At present, the clinical diagnosis largely depends on the clinical experience of doctors and the subjective evaluation of symptom scales, lacking objective and accurate biological diagnostic basis. This symptom-based diagnosis method is prone to misdiagnosis or delayed diagnosis, missing the opportunity for intervention.

[0003] In the field of biomarker detection, the detection technology of the core pathological protein of the disease has always been a research hotspot. An ideal detection method should have high sensitivity and specificity, and be able to identify small pathological changes in the early stage of the disease. The existing detection methods mainly include cerebrospinal fluid analysis, imaging examination and blood detection. Although cerebrospinal fluid detection can directly reflect the changes in the central nervous system, its collection process is an invasive operation, which is poorly tolerated by patients, and there is a certain risk of complications, which is limited in routine clinical practice. Imaging methods such as positron emission tomography can assess brain function changes, but the equipment is expensive, the examination cost is high, and the accessibility is poor, making it difficult to be used for popular screening. Blood detection is of great concern because it is completely non-invasive, but the target protein concentration in blood is extremely low, making detection difficult, and the current technology is not mature, with insufficient sensitivity and stability. In addition, although an analysis technology based on the principle of protein misfolding cyclic amplification has been developed for the detection of this type of disease, it still has limitations in sample type, detection speed or operation standardization, and cannot fully meet the comprehensive needs of minimally invasive, rapid, stable and easy-to-promote in clinical practical application. Therefore, developing a diagnostic tool that can balance detection performance, patient friendliness, operation convenience and economy is of great significance to improve the diagnosis of Parkinson's disease. SUMMARY

[0004] The purpose of the present application is to provide a Parkinson's disease auxiliary diagnosis kit based on RT-QuIC technology and application thereof, which solves the problems of large sampling trauma, long detection period, low sensitivity, high cost and difficulty in popularization at the grassroots level of the existing Parkinson's disease diagnosis technology.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The application provides an auxiliary diagnostic kit for Parkinson's disease based on RT-QuIC technology for detecting misfolded alpha-synuclein in a skin sample, which comprises a reaction buffer, alpha-synuclein recombinant protein, negative control and positive control; the alpha-synuclein recombinant protein comprises an amino acid sequence shown in SEQ ID NO. 4.

[0006] Preferably, the reaction buffer of the kit comprises phosphate, sodium chloride, a surfactant and thioflavin T.

[0007] Preferably, the phosphate in the reaction buffer is selected from one or more of Na2HPO4, NaH2PO4, KH2PO4 and KCl.

[0008] Preferably, the surfactant in the reaction buffer is selected from one or more of SDS, Triton X-100, ammonium sulfate, CHAPS, Tween-20, Tween-80, Ficoll 70 and PEG400.

[0009] Preferably, the concentration of the phosphate in the reaction buffer is 10 mM to 100 mM; the pH value of the reaction buffer is 7.2 to 8.5; the concentration of the sodium chloride is 0.1 M to 1 M; the concentration of the surfactant is 0.001% to 1.0%; and the concentration of the thioflavin T is 10 μM to 100 μM.

[0010] Preferably, the negative control of the kit is sterile normal saline, and the positive control is an alpha-synuclein pre-prepared fiber ultrasonic dilution solution.

[0011] The application also provides use of the kit in preparation of a product for diagnosing or assisting in diagnosing Parkinson's disease.

[0012] Preferably, in the use, the diagnosis or assistance in diagnosing Parkinson's disease is based on a sample collected from the skin of the neck behind the ear, the abdomen or the ankle of a subject, and more preferably the neck behind the ear.

[0013] The application also provides use of the kit in preparation of a product for detecting misfolded alpha-synuclein in a sample.

[0014] The application also provides use of the kit in preparation of a product for early screening, clinical diagnosis or treatment effect monitoring of Parkinson's disease.

[0015] The application has the following beneficial effects: The reagent kit provided by this invention is significantly minimally invasive and has high patient compliance. It requires only a small amount of skin tissue for testing, avoiding the risks and pain associated with lumbar puncture or brain biopsy. The kit boasts excellent detection performance, achieving highly sensitive and specific identification of early pathological features of Parkinson's disease. The testing process is rapid, enabling batch sample analysis in a short time, effectively supporting the needs of rapid clinical diagnosis and screening. Furthermore, its overall cost is significantly lower than existing methods such as imaging, providing a convenient, reliable, and economical molecular detection method for the early detection, clinical auxiliary diagnosis, and monitoring of treatment effects of Parkinson's disease. This has positive implications for improving the overall diagnosis and treatment of this type of neurodegenerative disease. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the technical route for a Parkinson's disease auxiliary diagnostic kit based on RT-QuIC technology; Figure 2 Figure 1 shows the purification and identification of recombinant α-synuclein protein. In the figure: M is the pre-stained molecular weight standard; 1 is the sample before recombinant protein induction; 2 is the sample after recombinant protein induction; 3 is the supernatant from recombinant protein induction expression; 4 is the precipitate from recombinant protein induction expression; 5 is the Ni-NTA affinity chromatography flow-through buffer; 6 is the 0 mM imidazole washing sample; 7 is the 10 mM imidazole washing sample; 8 is the 20 mM imidazole washing sample; 9 is the 500 mM imidazole eluted sample; 10 is the sample purified by anion exchange chromatography; 11 is the sample purified by size exclusion chromatography; and 12 is the Western Blot identification result. Figure 3 Validation image of thioflavin T fluorescence for α-synuclein pre-fibrils; Figure 4 Fluorescence kinetics curves for the aggregation of recombinant proteins induced by pre-fabricated α-synuclein fibers; Figure 5 The image shows the RT-QuIC detection results of clinical skin samples, where the horizontal axis represents incubation time and the vertical axis represents fluorescence intensity. Detailed Implementation

[0017] This invention provides a diagnostic kit for Parkinson's disease based on real-time oscillation-induced transformation (RT-QuIC) technology for detecting misfolded α-synuclein in skin samples. RT-QuIC technology is a protein misfolding cyclic amplification technique based on the principle that misfolded proteins (such as α-synuclein, prions, etc.) have the ability to self-replicate and induce misfolding and aggregation of normal conformation proteins. It detects trace amounts of misfolded proteins through in vitro amplification and labeling with fluorescent dyes (such as thioflavin T). The core components of the kit include a reaction buffer, recombinant α-synuclein protein, a negative control, and a positive control. The reaction buffer provides suitable ionic strength, pH environment, and necessary auxiliary components for the entire RT-QuIC reaction. The specific recombinant α-synuclein protein used in this invention serves as the substrate and can be induced to aggregate by misfolded α-synuclein proteins that may be present in the sample. The negative control is used to establish the background threshold for detection, and the positive control is used to verify the effectiveness of the reaction system.

[0018] Specifically, the recombinant α-synuclein protein comprises the amino acid sequence shown in SEQ ID NO. 4. α-synuclein is a presynaptic terminal protein mainly composed of 140 amino acids; its misfolding and aggregation are core pathological features of synuclein-related diseases such as Parkinson's disease and Lewy body dementia. The recombinant protein of this invention is prepared by genetic engineering methods, and its sequence contains the full-length sequence of wild-type human α-synuclein (as shown in SEQ ID NO. 2). It exhibits high reactivity and can be efficiently induced to aggregate by misfolded α-synuclein seeds, generating a detectable fluorescent signal.

[0019] In a preferred embodiment of the invention, the reaction buffer of the kit comprises phosphate, sodium chloride, a surfactant, and thioflavin T. The phosphate buffer system is a buffer system used in biochemical experiments to maintain pH stability. Sodium chloride is used to provide appropriate ionic strength. The surfactant helps maintain protein solubility and reduces nonspecific adsorption. Thioflavin T is an amyloid cellulose-specific fluorescent dye that exhibits significantly enhanced fluorescence upon binding to β-sheet structures. The reaction buffer is preferably a 2× concentration premixed solution for easy dilution before use and to ensure batch-to-batch consistency.

[0020] Furthermore, the phosphate in the reaction buffer can be selected from common phosphates in the art, such as one or more of Na₂HPO₄, NaH₂PO₄, KH₂PO₄, and KCl. Combinations of these salts can be used to formulate buffer solutions with the desired pH range and buffering capacity. The concentration range of the phosphate can be adjusted within a wide range, for example from 5 mM to 200 mM, preferably from 20 mM to 80 mM, more preferably from 30 mM to 60 mM, and most preferably 50 mM as described in the examples.

[0021] Similarly, the surfactant in the reaction buffer can be selected from a variety of nonionic or amphoteric surfactants, such as one or more of SDS (sodium dodecyl sulfate), Triton X-100, ammonium sulfate, CHAPS (3-[(3-cholamidopropyl)dimethylamino]propanesulfonate), Tween-20, Tween-80, Ficoll 70 (a synthetic sucrose polymer), and PEG400 (polyethylene glycol 400). The concentration of the surfactant can vary from 0.0005% to 2.0%, preferably from 0.005% to 0.5%, more preferably from 0.01% to 0.1%, and most preferably 0.02% as described in the examples.

[0022] Other parameters regarding the reaction buffer: The pH of the reaction buffer can be adjusted within a near-neutral to weakly alkaline range, for example, from pH 7.0 to pH 9.0, preferably from pH 7.5 to pH 8.2, more preferably from pH 7.8 to pH 8.0, and most preferably from pH 8.0 as described in the examples. The concentration of the sodium chloride can range from 0.05M to 2.0M, preferably from 0.1M to 0.5M, more preferably from 0.12M to 0.18M, ​​and most preferably from 150mM (i.e., 0.15M) as described in the examples. The concentration of the thioflavin T can range from 1μM to 200μM, preferably from 5μM to 50μM, more preferably from 15μM to 25μM, and most preferably from 20μM as described in the examples.

[0023] In another preferred embodiment of the present invention, the negative control of the kit is sterile physiological saline, which is typically an aqueous solution containing 0.9% sodium chloride and may contain preservatives such as 0.01% sodium azide. The positive control is an ultrasonically diluted solution of pre-prepared α-synuclein fibers (PFFs), which are misfolded α-synuclein fibers prepared in vitro to simulate pathological samples and validate the detection system.

[0024] 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 "product" may be a detection system comprising the kit, a detection service package, or diagnostic report generation software, etc. Currently, the diagnosis of Parkinson's disease mainly relies on clinical symptoms. This kit, by providing objective molecular pathological evidence (detection of misfolded α-Syn in the skin), can be used to assist clinical diagnosis and improve diagnostic accuracy, especially in early stages of the disease or in atypical cases.

[0025] In a more preferred embodiment, in the above application, 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. Skin, as a peripheral tissue, also contains pathological deposits of α-synuclein, which are correlated with the degree of pathology in the central nervous system. The postauricular region of the neck, due to its high density of nerve distribution and potential accumulation of pathological proteins, allows for higher detection rates and better patient acceptance in this test. In practical applications, other sites such as the abdomen and ankle can also be used for testing.

[0026] The present invention also provides the application of the above-described kit in the preparation of products for detecting misfolded α-synuclein in samples.

[0027] 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" here 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: A commercial in vitro diagnostic (IVD) test kit: that is, the components, consumables and instructions of the present invention are packaged directly into a complete kit for use in clinical laboratories or testing departments.

[0028] An integrated detection system or platform: This kit is integrated with automated sample processing equipment (such as automated tissue homogenizers and liquid handling workstations), fluorescence detection instruments (such as fluorescence microplate readers), and supporting control and analysis software to form a complete solution from sample to report.

[0029] A data analysis software product: specifically designed to process and analyze fluorescence kinetic curve data generated by this kit, automatically calculate fluorescence thresholds, determine positive / negative results, and generate standardized test reports.

[0030] 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.

[0031] This application clarifies three core clinical application scenarios for the kit of this invention. The form of the "product" is also diverse, and it can be any of the aforementioned testing products, but its intended use and scenario are specific: Early screening for Parkinson's disease: This refers to large-scale, rapid preliminary testing using the product in target populations (such as high-risk individuals with a family history of Parkinson's disease, those experiencing prodromal symptoms like decreased sense of smell or REM sleep behavior disorder, or individuals undergoing health checkups within specific age groups) before they develop typical motor symptoms or with very mild symptoms. The aim is to identify individuals with potential pathological changes (misfolded α-synuclein in the skin) and recommend further in-depth clinical evaluation. The product may be offered as a high-throughput, convenient screening kit or a community screening service package.

[0032] Clinical diagnosis of Parkinson's disease: This refers to the process by which clinicians, after a patient has developed clinical symptoms (such as tremor, bradykinesia, etc.), use the test results of the aforementioned product as objective laboratory evidence to confirm the diagnosis, differentiate the diagnosis (such as distinguishing it from other diseases like essential tremor and multiple system atrophy), or confirm the pathophysiological process. This evidence is combined with clinical symptoms, imaging findings, and other results to support the diagnosis of Parkinson's disease. The product is typically a diagnostic-grade reagent kit or laboratory test that meets clinical laboratory quality standards.

[0033] Parkinson's disease treatment efficacy monitoring: This refers to the repeated testing of the same patient periodically (e.g., every 6 months or 1 year) using the product after the patient has received disease-modifying therapy (a treatment designed to delay or halt disease progression) or symptomatic treatment. By dynamically observing changes in the "quantity" or "activity" of misfolded α-synuclein in the skin (e.g., delayed signal onset, decreased fluorescence intensity increase), it indirectly assesses whether the treatment may have affected the aggregation process of underlying pathological proteins, providing molecular-level reference information for efficacy evaluation and treatment regimen adjustments. The product formulation requires particular attention to the quantitative or semi-quantitative comparability of the test results and good batch-to-batch consistency to ensure the reliability of longitudinal monitoring data.

[0034] In summary, the kit of the present invention, through its unique sample source (skin) and optimized RT-QuIC detection system, provides a core technical foundation and material guarantee for the development of in vitro diagnostic products related to Parkinson's disease for different scenarios (screening, diagnosis, monitoring) and in different forms (kits, systems, services, software).

[0035] 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.

[0036] Key sequences involved in the embodiments: SEQ ID NO.1: Codon-optimized human SNCA gene nucleotide sequence (420 bp in length); ATGGACGTGTTCATGAAAGGTCTGTCTAAAGCGAAAGAGGTGTTGTTGCTGCGGCTGAGAAGACCAAACAGGGTGTTGCTGAAGCTGCTGGTAAGACTAAAGAAGGCGTTCTGTACGTTGGTTCTAAGACCAAAGAAGGTGTTGTTCACGGTGTGCGACTGTTGCGGAAAAAACCAAAGAACAGGTTACCAACGTTGGTGGTGCGGTT GTTACTGGTGTTACCGCAGTTGCGCAGAAGACCGTTGAAGGTGCTGGTTCCATCGCAGCAGCTACCGGTTTCGTTAAGAAAGACCAGCTGGGTAAGAACGAAGAAGGTGCTCCGCAAGAAGGTATCTTGGAAGATATGCCGGTAGATCCGGACAACGAAGCATACGAAATGCCGTCTGAAGAAGGTTACCAAGACTACGAACCGGAAGCA SEQ ID NO.2: Wild-type human α-Syn protein amino acid sequence (length 140aa); MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA; SEQ ID NO.3: Nucleotide sequence of recombinant vector pET22b-α-Syn (containing SNCA gene and 6×His tag, length 5886bp); 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, 159aa in length).

[0037] MDIGINSDPNSMDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH*.

[0038] Example The schematic diagram of this embodiment is as follows: Figure 1 As shown.

[0039] 1. Preparation and identification of α-Syn recombinant protein: 1.1 Gene Synthesis and Vector Construction Gene optimization: Based on the human SNCA gene (GenBank: 6622), codon optimization was performed according to the codon preference of E. coli. The optimized gene sequence is shown in SEQ ID NO.1 and was synthesized by Anhui General Biotechnology Co., Ltd.

[0040] Vector construction: The optimized SNCA gene was homologously recombinated with the pET22b(+) vector (containing a C-terminal 6×His tag) to construct the recombinant vector pET22b-α-Syn; it was transformed into Escherichia coli DH5α competent cells, and single colonies were picked for sequencing verification. The correctly cloned nucleotide sequence is shown in SEQ ID NO.3.

[0041] 1.2 Prokaryotic Expression Optimization Transformation and culture: 10 ng pET22b-α-Syn plasmid was transformed into 100 μL of E. coli BL21(DE3) competent cells (cell density 102). 7 (CFU / mL), spread onto LB solid medium containing ampicillin (100 μg / mL), and incubate upside down at 37°C for 14 h; Expression condition screening: The expression effects of different induction temperatures (25℃, 30℃, 37℃), induction times (8h, 12h, 16h), and inducers (IPTG 0.5mM, self-induction medium containing 0.2% lactose) were compared. The results showed that the recombinant protein expression level was the highest (40mg / L) when cultured at 37℃ in self-induction medium for 16h, and the soluble ratio reached 90% (SDS-PAGE showed a clear band at 14kDa with no significant inclusion bodies).

[0042] 1.3 Protein purification: After overnight induction culture of the recombinant substrate protein, the expressing bacterial cells were collected and purified.

[0043] (1) Centrifuge at 4℃ and 4000 rpm for 25 min and collect the bacterial cells expressing the protein.

[0044] (2) After resuspending the bacterial cells in 50 mL of lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1× protease inhibitor), the bacterial cells were homogenized using a high-pressure homogenizer at a pressure of 700~850 Pa for 10~20 min. After homogenization, the cells were centrifuged at 4 °C and 12000 rpm for 30 min, and the supernatant was collected.

[0045] (3) Boil the supernatant in boiling water for 15 min, centrifuge at 4℃ and 12000 rpm for 30 min, and collect the supernatant. (4) Ni-NTA column chromatography purification: The purification column was equilibrated with equilibration buffer (50mM Tris pH7.5, 150mM NaCl), impurities were washed with low concentration imidazole purification buffer, and the target protein was eluted with 500mM imidazole purification buffer (50mM Tris pH7.5, 150mM NaCl, 500mM imidazole). Then, overnight dialysis was performed for 16 hours (50mM Tris pH7.5, 150mM NaCl).

[0046] (5) Anion exchange column (IEX) purification: The target protein was eluted with linear salt concentration (50mM Tris pH7.5, 0-1M NaCl) to obtain a purer α-Syn recombinant protein; (6) Size exclusion chromatography (SEC): Further purification was carried out by SEC column (50mM Tris pH7.5, 150mM NaCl) to obtain pure target protein. The protein concentration was determined and adjusted to about 1mg / mL. The protein lyophilization amount per tube was 0.5mg.

[0047] 1.4 Protein Identification Purity testing: HPLC analysis showed a purity ≥95% ( Figure 2 The SDS-PAGE results show a single, non-spurious band at 14 kDa. Western Blot identification: Incubation with anti-His-tagged antibody, ECL staining showed a single 14kDa band ( Figure 2 This demonstrates protein specificity; Activity assay: ThT fluorescence method was used. The recombinant protein was co-incubated with positive control PFFs. The fluorescence intensity reached 2 × 10⁻⁶ after 8 hours. 5 RFU, specific activity ≥2×107 RFU / mg demonstrates that aggregation can be induced by misfolded α-Syn.

[0048] 2. Preparation and Validation of α-Syn Prefabricated Fibers (PFFs) 2.1 Preparation process Take the purified α-Syn recombinant protein and dissolve it in PBS (pH 7.4) to a concentration of 5-7 mg / mL. Aliquot the protein into sterile EP tubes (1 mL / tube) and incubate them in a constant temperature shaker at 37°C for 7 days. Take samples daily to detect fiber formation. After incubation, centrifuge and discard the supernatant. Resuspend the precipitate in PBS to a concentration of 0.5 mg / mL and aliquot at -80°C (0.1 mL / tube) for storage.

[0049] 2.2 Validation Indicators ThT fluorescence detection: 2 μL of PFFs solution was added to 198 μL of PBS containing 20 μM ThT, and the fluorescence intensity was measured (excitation 450 nm, emission 480 nm). The results showed that the fluorescence intensity of PFFs (RFU 201416) was 10 times that of the initial protein (RFU 6120). Figure 3 ); Induction activity verification: PFFs were added to recombinant α-Syn solution (0.1 mg / mL) at a ratio of 1:100. RT-QuIC detection showed that the fluorescence signal reached a plateau phase (intensity 2.1 × 10⁻⁶) at 8 h. 5 RFU (recombinant protein oxidase) has been shown to effectively induce recombinant protein aggregation. Figure 4 ).

[0050] 3. Reagent kit assembly 3.1 Reagent Kit Assembly Assemble the kit according to the specifications in Table 1. The kit includes reaction buffer, recombinant protein, controls, and related consumables. Table 1 Reagent Kit Information

[0051] 3.2 Detection Method Step 1: Skin sample collection and preprocessing (saves at least 90% of the time compared to traditional extraction methods) Sampling site: preferably the skin behind the ear on the neck or the inner side of the ankle (the sample used in the following text is the skin behind the ear on the neck, C7 position). 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 (the following are the optimal conditions obtained by the present invention. Compared with the pretreatment methods reported in the prior art, the pretreatment operation of the present invention is simpler, requires fewer reagents, and has a shorter processing time. It can be completed in as little as 10 minutes, while general processing methods require the preparation of multiple reagents and a special cryo-tissue homogenizer, and the entire processing process takes more than 5 hours): Rinse three times with physiological saline (1 mL each time, shake for 10 s) to remove surface contaminants; Add 1 mL of a rapid skin tissue protein extraction reagent containing 1×PMSF, whose main components 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 every 2 min). Manual grinding with a disposable grinding stick for 1-5 minutes is sufficient to completely break down the tissue; Centrifuge at 4℃ and 10000g for 1 min, and collect the supernatant; Dilute the supernatant 10 times with sterile water and store in aliquots at -80℃.

[0052] Step 2: Preparation of RT-QuIC reaction solution Dilute 2× reaction buffer: Mix 2× RT-QuIC reaction buffer with sterile water at a 1:1 volume ratio to prepare 1× reaction buffer (store at 4℃ protected from light, use within 2 hours); Add recombinant protein: Add α-Syn recombinant protein lyophilized powder to 1× reaction buffer, gently invert to mix (avoid generating air bubbles), to make the final concentration 0.08 mg / mL (optimal conditions).

[0053] Step 3: Sample addition and reaction in a 96-well plate Well plate preparation: Take out the black 96-well plate pre-loaded with silicon beads and add 98 μL of the above reaction solution to each well; Grouped sampling: Experimental group: Add 2 μL of pretreated sample; Negative control group: Add 2 μL of negative control; Positive control group: Add 2 μL of positive control; Blank control: Add 2 μL of sterile water; Each group has 3 replicates (to ensure the reliability of the results, 2 or more positive replicates are considered as final positive); Sealing and incubation: Seal the edges of the well plate with optical sealing film, place it in a fluorescent microplate reader, and set the detection conditions (optimal conditions): temperature 49℃, rotation speed 600 rpm / min (pause for 1 min every 4 min of shaking), and incubate continuously for 8-12 h.

[0054] Step 4: Fluorescence detection and result interpretation Detection parameters: excitation wavelength 450nm, emission wavelength 480nm, fluorescence signal detected once every 30min; Threshold setting: The threshold is set at "mean fluorescence intensity of negative control + 20 standard deviations (SD)". Result determination: The experimental group was identified as PD positive if the fluorescence intensity was ≥ the threshold. The fluorescence intensity in the experimental group was below the threshold, and it was determined to be PD negative.

[0055] 4. Clinical sample validation 4.1 Sample Inclusion Criteria PD group: 52 cases, all of whom met the diagnostic criteria for MDS Parkinson's disease (2015 edition), excluding patients with other neurological diseases (such as Alzheimer's disease); Control group: 38 cases, including patients with essential tremor, multiple system atrophy (MSA), transthyretin cardiac amyloidosis (TTR), Alzheimer's disease (AD), and healthy individuals (with no history of neurological disease).

[0056] 4.2 Testing Process The test was conducted by two lab technicians in a double-blind manner, following the above-mentioned detection method. Each sample was tested in three replicates, and negative, positive, and blank controls were also included. The instrument used for the test was a fluorescent microplate reader.

[0057] 4.3 Verification Results The core performance indicators are shown in Table 2: Table 2 Detection performance of the present invention

[0058] Among them, the detection rate of early-stage PD patients was 93.75% (30 / 32), and the detection rate of mid-to-late-stage PD patients was 100% (20 / 20), proving that the kit can be used for early diagnosis of PD. Figure 5 The fluorescence signal curves for skin samples from PD patients and healthy individuals are shown. The signal in PD patients was significantly higher than the threshold, while the signal in healthy individuals was lower than the threshold. Results showed that the fluorescence signal in PD patient samples increased significantly after 4-5 hours, reaching a plateau after 7-10 hours, with 2 false negatives, resulting in a sensitivity of 96%. The signals in healthy individuals and other disease samples were all below the threshold, with a specificity of 100%. Recorded detection time results show that, based on the aforementioned optimized reaction system and detection conditions, the detection cycle of this kit was significantly shortened, and the detection efficiency was greatly improved, with most samples being tested and results determined within 8 hours.

[0059] As demonstrated by the above embodiments, this invention provides a skin sample-based auxiliary diagnostic kit for Parkinson's disease. This kit can stably and specifically detect pathological protein markers in skin tissue obtained through minimally invasive extraction. Experimental verification shows that this kit has good identification ability for Parkinson's disease patient samples and can effectively distinguish between healthy controls and patients with other similar diseases, demonstrating its potential application value in the clinical auxiliary diagnosis of Parkinson's disease.

[0060] 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 amino acid sequence shown in SEQ ID NO.

4.

2. The reagent kit according to claim 1, characterized in that, The reaction buffer contains phosphate, sodium chloride, surfactant, and thioflavin T.

3. The reagent kit according to claim 2, characterized in that, The phosphate is selected from one or more of Na2HPO4, NaH2PO4, KH2PO4 and KCl.

4. The reagent kit according to claim 2, characterized in that, The surfactant is selected from one or more of SDS, Triton X-100, ammonium sulfate, CHAPS, Tween-20, Tween-80, Ficoll 70 and PEG400.

5. The reagent kit according to claim 2, characterized in that, The concentration of the phosphate is 10 mM to 100 mM; The pH value of the reaction buffer solution is 7.2~8.5; The concentration of sodium chloride is 0.1M~1M; The concentration of the surfactant is 0.001% to 1.0%; The concentration of thioflavone T is 10 μM to 100 μM.

6. The reagent kit according to claim 1, characterized in that, The negative control was sterile physiological saline, and the positive control was an ultrasonically diluted solution of α-synuclein pre-formed fibrous material.

7. 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 samples taken from the skin of the subject's neck, behind the ear, abdomen, or ankle.

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 products for early screening, clinical diagnosis or monitoring of treatment effects of Parkinson's disease.

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