Parkinson's disease diagnostic kit based on peripheral red blood cell alpha-synuclein RT-QuIC technology and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a Parkinson's disease diagnosis kit based on a peripheral red blood cell alpha-synuclein RT-QuIC technology and application of the Parkinson's disease diagnosis kit. The Parkinson's disease diagnostic kit based on the peripheral red blood cell alpha-synuclein RT-QuIC technology comprises a substrate protein, a reaction reagent, a negative reference substance and a positive reference substance, and a sample is selected from red blood cells in blood. The RT-QuIC technology is applied to diagnosis of neurodegenerative diseases, particularly, the sowing activity of pathological alpha-Syn protein is detected in blood red blood cell samples of PD patients of Chinese population for the first time, and the kit has extremely high sensitivity and specificity and provides important reference value for diagnosis and screening of PD. The invention provides a rapid, efficient and accurate diagnostic kit for diagnosis of Parkinson's disease, the kit can be used for accurately detecting Parkinson's disease patients and healthy control, and powerful technical support is provided for clinical diagnosis of the Parkinson's disease patients.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a Parkinson's disease diagnostic kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells and its application. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease occurring in middle-aged and elderly people, second only to Alzheimer's disease. It has an insidious onset and a slow progression. Its core pathological feature is the abnormal deposition of amyloid α-synuclein (α-Syn) in the nervous system.

[0003] The clinical manifestations of Parkinson's disease are mainly bradykinesia, resting tremor, muscle rigidity, and postural balance disorders, etc., and are accompanied by various non-motor symptoms such as olfactory disorders. By the time clinical diagnostic symptoms appear, about 75% of striatal dopaminergic terminals and 50% of substantia nigra cell bodies have been lost. Observing the morphological and quantitative changes of neurons under light microscopy shows the formation of eosinophilic α-Syn aggregates in the cytoplasm. The early formation of aggregates and the sequestration of normal α-Syn from the presynaptic terminal may lead to neuronal dysfunction before neurodegeneration, thus leading to the prodromal stage of the disease.

[0004] Currently, the clinical diagnosis of Parkinson's disease is mainly based on the clinical signs judged by clinicians. Biopsy or autopsy reveals Lewy bodies formed by the aggregation of amyloid α-Syn in brain tissue. Traditional clinical pathological tissue detection is not convenient, difficult to obtain samples, causes greater damage to patients, and is not conducive to the rapid screening and diagnosis of PD. The lack of reliable diagnostic markers for Parkinson's disease has always been a challenge faced by clinical neurology. Therefore, there is an urgent need to provide a new convenient, rapid, minimally invasive, and highly sensitive PD detection kit. Summary of the Invention

[0005] The purpose of the present invention is to provide a Parkinson's disease diagnostic kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells and its application, so as to provide strong technical support for the clinical diagnosis of Parkinson's disease and can be applied to the detection and diagnosis of Parkinson's disease.

[0006] The technical solution of a Parkinson's disease diagnostic kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells and its application of the present invention is as follows: A Parkinson's disease diagnostic kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells, comprising a substrate protein, a reaction reagent, a negative control product, and a positive control product, wherein the sample is selected from red blood cells in blood.

[0007] Preferably, the substrate protein is a 6His-α-Syn recombinant protein, and its amino acid sequence is shown in SEQ ID NO.6.

[0008] Preferably, the reaction reagents include 10× buffer, sterile water, and thioflavin T. The 10× buffer consists of 200 - 800 mM PB buffer, 1.0 - 2.0 M NaCl, and 0.001 - 0.01% SDS, and the pH value of the 10× buffer is 7.0 - 8.5.

[0009] More preferably, the negative control is phosphate buffer; the positive control is 6His-α-Syn preformed fibrils, and the 6His-α-Syn preformed fibrils are fibrillar proteins prepared by in vitro culture of the substrate protein.

[0010] Even more preferably, the kit includes 1 mg of 6His-α-Syn recombinant protein, 1.5 mL of 10× PBS buffer, 10 mL of sterile water, 0.5 mL of thioflavin T, and 0.1 mL of 6His-α-Syn preformed fibrils.

[0011] Preferably, the kit further includes a black 96-well cell plate pre-loaded with silica beads. The diameter of the silica beads is 0.8 mm, and each well contains 4 silica beads.

[0012] A detection method for a Parkinson's disease diagnosis kit based on the α-synuclein RT-QuIC technology of peripheral blood red blood cells includes the following steps: (1) Collect the peripheral blood to be tested, centrifuge and separate the red blood cells, and store them in aliquots at -80 °C for later use; (2) Dilute the 10× buffer with sterile water, and add thioflavin T and 6His-α-Syn recombinant protein to prepare 100 μL of reaction solution per portion; (3) Add 90 μL / well of the reaction solution to the black 96-well cell plate, set up negative control group, positive control group, and experimental group respectively, and set up duplicate wells. Add 10 μL of PBS to each well in the negative control group, add 10 μL of 6His-α-Syn preformed fibrils to the positive control group, and add 10 μL of pretreated red blood cell lysate to the experimental group; (4) Set the reaction program of the RT-QuIC instrument, stop the detection after running for 48 - 96 h; read the data and analyze the detected data.

[0013] Preferably, the final concentration of thioflavin T in the reaction solution is 10 - 30 μM, and the concentration of the substrate protein 6His-α-Syn is 0.05 - 0.5 mg / mL.

[0014] More preferably, the method for pre-treating red blood cells is as follows: 1) Collect peripheral blood, centrifuge at 1500 g for 10 min at 4°C, and take the red blood cells. 2) Wash the red blood cells three times with PBS. 3) Collect the supernatant by repeated freezing and thawing, which is the red blood cell lysate for loading onto the machine.

[0015] Beneficial effects: The present invention applies the RT-QuIC technology to the diagnosis of neurodegenerative diseases. In particular, for the first time in China, the seeding activity of pathological α-Syn protein is detected in the blood red blood cell lysate samples of PD patients, which has extremely high sensitivity and specificity, providing important reference value for the diagnosis and screening of PD. The present invention provides a rapid, efficient and accurate kit for the diagnosis of Parkinson's disease. Using this kit, PD patients and healthy controls can be accurately detected, providing strong technical support for its clinical diagnosis.

[0016] For the first time in China, the present invention uses blood red blood cell lysate as the detection sample for the clinical diagnosis research of RT-QuIC in PD. By detecting the expression of pathological protein α-Syn in the blood red blood cells of patients, PD patients and healthy controls can be distinguished, realizing the diagnosis of PD patients. The sample required by the present invention is the patient's blood, which is simple to sample and has little trauma. The operation process of this kit is simple, and the cost is low, which is easy to promote clinically. As a diagnostic tool for PD, the RT-QuIC technology has obvious advantages in terms of high sensitivity, specificity, repeatability, practicability, rapidity, low cost, etc. This kit can detect α-synuclein in different conformations of PD, which helps clinicians accurately diagnose and monitor the treatment progress of PD. Description of the drawings

[0017] Figure 1 It is the identification result of the prokaryotic induced expression of substrate protein α-Syn identified by 4-20% SDS-PAGE; Figure 2 It is the identification result of Ni-NTA purified substrate protein α-Syn identified by 4-20% SDS-PAGE; Figure 3 It is the identification result of anion exchange column purified substrate protein α-Syn identified by 4-20% SDS-PAGE; Figure 4 It is the identification result of molecular sieve purified substrate protein α-Syn identified by 4-20% SDS-PAGE; Figure 5 It is the detection result of RT-QuIC of blood red blood cell lysate of PD patients and healthy controls. Detailed implementation manners

[0018] Embodiments of the present invention will be described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation of the present invention.

[0019] Example 1 Preparation of α-Syn recombinant protein 1. Codon optimization α-Synuclein (SNCA) is one of the earliest discovered genes associated with PD and is also one of the most promising targets for revealing the pathogenesis of PD. The molecular weight of SNCA is 14 kDa and it consists of 140 amino acids. According to the coding gene sequence of SNCA in NCBI (GenBank: 6622), the human SNCA gene sequence was selected and codon-optimized, and gene synthesis was carried out by Anhui General Biotechnology Co., Ltd. The nucleotide sequence and amino acid sequence of the SNCA gene after codon optimization are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0020] The nucleotide sequence of SEQ ID NO.1 is as follows: ATGGACGTGTTCATGAAAGGTCTGTCTAAAGCGAAAGAGGGTGTTGTTGCTGCGGCTGAGAAGACCAAACAGGGTGTTGCTGAAGCTGCTGGTAAGACTAAAGAAGGCGTTCTGTACGTTGGTTCTAAGACCAAAGAAGGTGTTGTTCACGGTGTTGCGACTGTTGCGGAAAAAACCAAAGAACAGGTTACCAACGTTGGTGGTGCGGTTGTTACTGGTGTTACCGCAGTTGCGCAGAAGACCGTTGAAGGTGCTGGTTCCATCGCAGCAGCTACCGGTTTCGTTAAGAAAGACCAGCTGGGTAAGAACGAAGAAGGTGCTCCGCAAGAAGGTATCTTGGAAGATATGCCGGTAGATCCGGACAACGAAGCATACGAAATGCCGTCTGAAGAAGGTTACCAAGACTACGAACCGGAAGCA The amino acid sequence of SEQ ID NO.2 is as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKKQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEKGAPQEGILENMPVNPDNEAYEMPSEKGYQDYEPEA 2. Construction of expression vector The vector pET32a(+) prokaryotic expression vector was selected, and the restriction enzyme sites Nde1 and Xho1 were selected. The optimized SNCA gene (nucleotide sequence SEQ ID NO.1) was subjected to homologous recombination reaction with the above-mentioned empty plasmid pET32a(+) vector. Among them, the volume of the reaction system was 10 μL, the dosage of the pET32a(+) vector was 100 ng, and the N-terminus of the pET32a(+) vector carried a thioredoxin (Trx) tag, and the C-terminus of the pET32a(+) vector carried 6 His tags.

[0021] After transformation of Escherichia coli competent cells DH5α (Solarbio, C1100) at 37°C, it was cultured overnight on LB solid medium coated with ampicillin resistance, and monoclonal colonies were picked for PCR positive identification and sequencing. The correctly identified positive clone was named pET32-α-Syn, and its nucleotide sequence and amino acid sequence are shown in SEQ ID NO.3 and SEQ ID NO.4 respectively.

[0022] The nucleotide sequence of SEQ ID NO.3 is as follows: The amino acid sequence of SEQ ID NO.4 is as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH. 3. Cell transformation and protein expression identification The above positive clone pET32-α-Syn (10 ng) was transformed into 100 μL of Escherichia coli BL21(DE3) competent cells (Solarbio, C1400) with a cell density of 10 7 CFU / mL, and cultured overnight at 37 °C in an inverted manner on an LB solid medium coated with ampicillin resistance for 12 - 16 h. Five single colonies (named A, B, C, D, and E) were separately picked and cultured in an LB liquid medium containing 3 - 5 mL of ampicillin antibiotic, shaken and cultured in an incubator at 220 rpm at 37 °C until the OD600 was between 0.6 - 1. The culture was inoculated into 5 mL of LB auto-induction medium containing ampicillin antibiotic at a ratio of 1:100, and continuously cultured overnight at 200 rpm for 12 - 18 h to induce the expression of the recombinant protein. The protein expression was identified by SDS-PAGE gel electrophoresis and Coomassie brilliant blue staining, and the results are as Figure 1 shown. As can be seen from the bands at the arrows in Figure 1 , the substrate protein 6His-α-Syn was significantly expressed after induction in different monoclonal strains, indicating successful cell transformation.

[0023] After that, the successfully transformed cells were induced for large-scale expression overnight in 1 L of the culture medium.

[0024] 4. Protein collection and purification Collect the above-mentioned overnight-induced culture broth, and collect and purify the protein 6His-α-Syn through the following steps: (1) Centrifuge at 4000 rpm for 20 min at 4 °C to collect the bacterial cells; (2) Resuspend the collected bacterial cells in 50 mL of lysis buffer (20 mM Tris, 100 mM NaCl, 1 mM PMSF, pH = 7.5), and use a high-pressure homogenizer with a pressure of 850 pa to disrupt the bacterial cells for 15 min. Collect the culture broth and set it aside for later use; (3) Centrifuge the collected culture broth at 12000 rpm for 45 min at 4 °C, collect the supernatant, boil it in boiling water for 15 min, and place it on ice for 5 min for cooling and incubation, and set it aside for later use; (4)The supernatant after the above cooling incubation was centrifuged at 12,000 rpm for 45 min at 4°C, the supernatant was collected, streptomycin sulfate with a final concentration of 10 mg / mL was added to the supernatant, and it was stirred at 4°C for 30 min for standby; (5)The supernatant after the above stirring and mixing was centrifuged at 12,000 rpm for 45 min at 4°C, the supernatant was collected, the pH value of the supernatant was adjusted to 3.5 with 1M hydrochloric acid, stirred at 4°C for 30 min, and then centrifuged at 12,000 rpm for 45 min at 4°C. After collecting the supernatant, the pH value of the supernatant was adjusted to 7.5 with 1M NaOH for standby; (6)Purification was performed using a Ni-NTA column chromatography. The purification column was pre-equilibrated with a buffer of 20 mM Tris, 100 mM NaCl, and pH = 7.5. Miscellaneous proteins were washed and the target protein was eluted in FT (Flow Through), 0 mM, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM, and 500 mM imidazole buffers (20 mM Tris, 100 mM NaCl, pH = 7.5, 0 - 500 mM imidazole). The elution peaks at each stage were collected, and finally, the target protein was identified by 4 - 20% SDS-PAGE gradient gel electrophoresis and Coomassie brilliant blue staining. The results were as Figure 2 shown. It can be Figure 2 seen that a large amount of the target protein can be eluted at 100 mM imidazole, and a crude 6His-α-Syn protein solution was obtained; (7)The above protein solution was centrifuged at 12,000 rpm for 45 min at 4°C, the supernatant was collected, and it was dialyzed in a buffer of 20 mM Tris, 100 mM NaCl, and pH = 7.5 for 16 h; (8)The sample dialyzed overnight was added to an anion exchange column pre-equilibrated with 20 mM Tris, pH = 7.5 to further purify the target protein 6His-α-Syn, and then linear salt concentration elution was performed (20 mM Tris, pH = 7.5, 0 - 1M NaCl, pH = 7.5). Samples of each elution peak were collected, and the target protein was identified by 4 - 20% SDS-PAGE gradient gel electrophoresis and Coomassie brilliant blue staining. The results were as Figure 3 shown. It can be Figure 3 seen that the purified target protein exists in the collected samples of peak A and peak B, and a further purified 6His-α-Syn protein solution was obtained; (9)The identified target protein was further purified by size exclusion chromatography. Samples of the A and B elution peaks were collected, and the target protein was identified by 4 - 20% SDS-PAGE gradient gel electrophoresis and Coomassie brilliant blue staining. The results were as Figure 4 shown. It can be Figure 4It can be seen that after purification by molecular sieve, the miscellaneous proteins have been basically removed, and the target protein with high purity has been obtained, and the 6His-α-Syn protein sample after identification has been obtained; (10)Dialyze the identified target protein sample overnight in a buffer containing 20 mM Tris, 100 mM NaCl, and pH = 7.5. The dialysis duration is 16 h. Detect the absorbance value of CD280 and set the extinction coefficient to 0.387 to determine the protein concentration, and adjust the protein concentration to 1 mg / mL. Aliquot 1 mL per tube and freeze-dry to obtain the substrate protein 6His-α-Syn sample.

[0025] 5. Protein Sequencing Perform sequence determination on the substrate protein 6His-α-Syn, and deduce the amino acid sequence based on the nucleic acid sequence.

[0026] The coding nucleotide sequence encoding 6His-α-Syn is shown in SEQ ID NO.5, and its amino acid sequence is shown in SEQ ID NO.6. The nucleotide sequence of SEQ ID NO.5 is as follows: ATGGACGTGTTCATGAAAGGTCTGTCTAAAGCGAAAGAGGGTGTTGTTGCTGCGGCTGAGAAGACCAAACAGGGTGTTGCTGAAGCTGCTGGTAAGACTAAAGAAGGCGTTCTGTACGTTGGTTCTAAGACCAAAGAAGGTGTTGTTCACGGTGTTGCGACTGTTGCGGAAAAAACCAAAGAACAGGTTACCAACGTTGGTGGTGCGGTTGTTACTGGTGTTACCGCAGTTGCGCAGAAGACCGTTGAAGGTGCTGGTTCCATCGCAGCAGCTACCGGTTTCGTTAAGAAAGACCAGCTGGGTAAGAACGAAGAAGGTGCTCCGCAAGAAGGTATCTTGGAAGATATGCCGGTAGATCCGGACAACGAAGCATACGAAATGCCGTCTGAAGAAGGTTACCAAGACTACGAACCGGAAGCACTCGAGCACCACCACCACCACCACTGA. The amino acid sequence of SEQ ID NO.6 is as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH. Example 2 PD Detection Kit All components, ratios or concentrations, and volumes in the kit of this example are shown in Table 1 below.

[0027] Table 1 Components and Contents of the Kit Component Ratio or Concentration Volume or Capacity Substrate Protein (Lyophilized Powder) 6His-α-Syn 1 mg 10×PBS Buffer 600 mM Phosphate Buffer (PB), 1.5 M NaCl, 0.007% SDS, pH = 7.5 1.5 mL Black 96-Well Cell Plate Pre-loaded with Silica Beads The diameter of the silica beads is 0.8 mm 4 silica beads per well Thioflavin T (ThT) 1 mM 0.5 mL Sterile Water - 10 mL Negative Control PBS 0.1 mL Positive Control Pre-prepared Fibrils of 6His-α-Syn 0.1 mL The usage method of the above PD detection kit is as follows: (1) Collect peripheral blood test samples, centrifuge at 1500g for 10 min at 4°C, separate red blood cells, aliquot and store at -80°C for later use.

[0028] (2) Prepare the reaction solution, dilute the 10× buffer with sterile water, and add ThT with a final concentration of 20 μM and the substrate protein 6His-α-Syn at 0.1 mg / mL to prepare 100 μL of reaction solution per sample. Among them, the specific components and ratios of the 10× buffer are shown in Table 1.

[0029] (3) Add 4 silicon beads with a diameter of 0.8 mm to each well of a black 96-well plate, and add 90 μL of the reaction solution to each well. Set up negative control groups, positive control groups and experimental groups respectively. Among them, 10 μL of PBS is added to each well in the negative control group, with 4 replicates, 10 μL of pre-prepared fibrils of 6His-α-Syn is added in the positive control group, with 4 replicates, and 10 μL of pretreated red blood cell lysate is added in the experimental group, with 4 replicates for each sample.

[0030] (4) Set the reaction program of the RT-QuIC instrument, the excitation light wavelength is 450 nm, the emission light wavelength is 480 nm, and set the reading method as bottom reading by the machine; the black 96-well plate is shaken at 42°C for 4 min each time, paused for 1 min, and the fluorescence data is detected once every 45 min, and the detection is stopped after running for 48 - 96 h. Read the data and analyze the detected data.

[0031] Example 3 Differing from Example 2, the 10× buffer consists of 200 mM PB buffer, 1.0 M NaCl and 0.001% SDS, and the pH value of the 10× buffer is 7.0.

[0032] Example 4 Different from Example 2, the 10× buffer consists of 800 mM PB buffer, 2.0 M NaCl, and 0.01% SDS, and the pH value of the 10× buffer is 8.5.

[0033] Example 5 Different from Example 2, the final concentration of thioflavin T in the reaction solution is 10 μM, and the concentration of the substrate protein 6His-α-Syn is 0.05 mg / mL.

[0034] Example 6 Different from Example 2, the final concentration of thioflavin T in the reaction solution is 30 μM, and the concentration of the substrate protein 6His-α-Syn is 0.5 mg / mL.

[0035] The method for preprocessing red blood cells is as follows: 1) Collect peripheral blood, centrifuge at 1500 g for 10 min at 4°C, and take the red blood cells; 2) Wash the red blood cells three times with PBS; 3) Freeze-thaw repeatedly and collect the supernatant, which is the red blood cell lysate for loading.

[0036] Example 7 Clinical Sample Detection and Verification Select 15 red blood cell samples from PD patients who have been clinically diagnosed (named X1 - X15 respectively) and 15 healthy control (CR) biopsy skin samples (named C2 - C16 respectively), and perform detection and verification on the above 30 samples according to the detection steps of Example 2. The results are shown in Figure 5 , and it can be seen from Figure 5 that the fluorescence starts to increase after 10 h of amplification and tends to stabilize after 32 h. Through statistical analysis of the results of PD and control group samples, it is found that the maximum fluorescence value of red blood cell samples from PD patients is significantly higher than that of the non-PD control group (P < 0.001). Among the 15 PD red blood cell lysate samples, 14 showed positive α-Syn seeding activity, while none of the 14 non-PD control samples showed seeding activity, and 1 case showed false positive, indicating that the sensitivity of this detection method is 93.3% and the specificity is as high as 93.3%.

[0037] Red blood cells are the main source of α-Syn in the blood. Therefore, red blood cells in the blood can be separately extracted and the correlation between α-Syn in them and Parkinson's disease can be studied. The misfolded α-Syn has prion-like seeding properties and can use its own misfolded protein as a template to convert the corresponding native conformation α-Syn protein into misfolded α-Syn of the same conformation. Based on this principle, the present invention uses the real-time quaking-induced conversion (RT-QuIC) technology, also known as "protein PCR", to detect the misfolded α-Syn protein in the blood red blood cell samples of PD patients. The abnormally conformation α-Syn protein from patients is co-incubated with the in vitro recombinant substrate protein α-Syn of normal conformation. Through the quaking cycle, the recombinant protein undergoes a conformational change to form a new misfolded protein. By successive cycling, the misfolded α-Syn protein increases exponentially and is monitored in real time by the amyloid-specific dye thioflavin T to achieve accurate disease diagnosis. Based on the unique RT-QuIC cyclic amplification detection technology, the sensitivity is extremely high, and the lower limit of detection of misfolded protein can be less than 100 fg. The feasibility of this detection method has been confirmed in prion diseases, and the sensitivity and specificity can be as high as 100% in the cerebrospinal fluid, skin and blood of prion disease patients, and it has been included in the prion disease diagnostic guidelines of the United States and the European Union.

[0038] In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0039] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A Parkinson's disease diagnostic kit based on the RT-QuIC technique for α-synuclein in peripheral blood red blood cells, characterized in that, It includes a substrate protein, reaction reagents, a negative control, and a positive control. Among them, the sample is selected from red blood cells in blood.

2. The Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 1, wherein, The substrate protein is a 6His-α-Syn recombinant protein, and its amino acid sequence is as shown in SEQ ID NO.

6.

3. The Parkinson's disease diagnostic kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 1, wherein, The reaction reagents include a 10× buffer, sterile water, and thioflavin T. The 10× buffer is composed of 200 - 800 mM PB buffer, 1.0 - 2.0 M NaCl, and 0.001 - 0.01% SDS, and the pH value of the 10× buffer is 7.0 - 8.

5.

4. The Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 3, characterized in that, The negative control is phosphate buffer; the positive control is 6His-α-Syn preformed fibrils, and the 6His-α-Syn preformed fibrils are fibrillar proteins prepared by in vitro culture of the substrate protein.

5. The Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 4, wherein, The kit includes 1 mg of 6His-α-Syn recombinant protein, 1.5 mL of 10× PBS buffer, 10 mL of sterile water, 0.5 mL of thioflavin T, and 0.1 mL of 6His-α-Syn preformed fibrils.

6. The Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to any one of claims 1 to 5, characterized in that, The kit also includes a black 96-well cell plate pre-loaded with silica beads. The diameter of the silica beads is 0.8 mm, and 4 silica beads are loaded in each well.

7. The detection method of the Parkinson's disease diagnosis kit based on the RT-QuIC technology of peripheral blood red blood cell α-synuclein according to claim 6, characterized in that, It includes the following steps: (1) Collect the peripheral blood to be tested, centrifuge and separate the red blood cells, aliquot and store them at -80 °C for later use; (2) Dilute the 10× buffer with sterile water, and add thioflavin T and 6His-α-Syn recombinant protein to prepare 100 μL of reaction solution per portion; (3) Add 90 μL / well of the reaction solution to the black 96-well cell plate. Set up a negative control group, a positive control group, and an experimental group, and set up duplicate wells respectively. Add 10 μL of PBS to each well in the negative control group, add 10 μL of 6His-α-Syn preformed fibrils to the positive control group, and add 10 μL of pretreated red blood cell lysate to the experimental group; (4) Set the reaction program of the RT-QuIC instrument, stop the detection after running for 48 - 96 h; read the data and analyze the detected data.

8. The detection method of the Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 7, wherein, The final concentration of thioflavin T in the reaction solution is 10 - 30 μM, and the concentration of the substrate protein 6His-α-Syn is 0.05 - 0.5 mg / mL.

9. The Parkinson's disease diagnosis kit based on the RT-QuIC technology of α-synuclein in peripheral blood red blood cells according to claim 8, characterized in that, The method for pre-treating red blood cells is as follows: 1) Collect peripheral blood, centrifuge at 1500 g for 10 min at 4 °C, and take the red blood cells; 2) Wash the red blood cells three times with PBS; 3) Freeze-thaw repeatedly and collect the supernatant, which is the red blood cell lysate for use in the instrument.

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