Alzheimer disease auxiliary detection kit based on cerebrospinal fluid sample alpha-synuclein seed amplification technology and application of Alzheimer disease auxiliary detection kit

Abstract

The invention relates to the technical field of biology, in particular to an Alzheimer disease auxiliary detection kit based on a cerebrospinal fluid sample alpha-synuclein seed amplification technology and application of the Alzheimer disease auxiliary detection kit. The Alzheimer disease auxiliary detection kit based on the cerebrospinal fluid sample alpha-synuclein seed amplification technology comprises a substrate protein, a reaction reagent, a negative reference substance and a positive reference substance, and the sample is a to-be-detected cerebrospinal fluid sample. Cerebrospinal fluid of a subject is used as a detection sample for the first time, and clinical diagnosis research on the Alzheimer's disease by the RT-QuIC technology is carried out. The kit has obvious advantages in the aspects of high sensitivity, specificity, repeatability, practicability, rapidness, low cost and the like, the kit is simple in clinical sampling, the detection kit has the characteristics of simple operation process, low cost and the like, the detection time is short, the accuracy and the specificity are high, clinical popularization is easy, and the application prospect is wide. The kit can be clinically used for helping clinicians to diagnose the Alzheimer's disease in an auxiliary manner.

Description

Technical Field

[0002] The present disclosure relates to the field of biotechnology, and particularly relates to an Alzheimer's disease auxiliary detection kit based on α-synuclein seed amplification technology for cerebrospinal fluid samples and its application. Background Art

[0003] Alzheimer's disease (AD) is an age-related neurodegenerative disease of the central nervous system. The typical pathological manifestations are the formation of abnormally aggregated Aβ senile plaques and tau protein fibril deposition. In addition, studies have shown that in autopsy-confirmed familial or sporadic AD patients, in addition to the formation of Aβ senile plaques and tau protein deposition, there is also deposition of α-synuclein (α-Syn), suggesting a common pathology of AD and synucleinopathy, that is, the coexistence of non-AD pathological markers. The final diagnosis of AD requires brain tissue biopsy or autopsy after the patient's death to detect abnormally aggregated misfolded proteins in the brain tissue. Currently, AD lacks specific biological markers, and clinical diagnosis can only be made when obvious cognitive impairment appears. Due to the heterogeneity of clinical manifestations and the overlap of clinical symptoms between different diseases, the clinical diagnosis of AD faces severe challenges, especially in the early stage of the disease, where there are no characteristic clinical manifestations, and it is extremely easy to miss or misdiagnose. Therefore, finding specific non-invasive biological markers is crucial for the accurate diagnosis and intervention of AD.

[0004] In 2018, the National Institute on Aging - Alzheimer's Association (NIA-AA) in the United States proposed a research framework for AD based on the AT(N) system, regarding AD as a disease continuum composed of different pathological stages, thus transforming AD from a syndrome oriented by clinical symptoms to a biological definition guided by biomarkers. Recently, the latest draft of the NIA-AA revised Alzheimer's disease guidelines (2023-NIA-AA) was released, adding three new biomarker classifications: I for describing inflammatory / immune mechanisms, and non-AD pathological biomarkers, namely vascular brain injury (V) and synucleinopathy S.

[0005] Both AD and synucleinopathy belong to neurodegenerative diseases, and their core pathological changes are the misfolding and abnormal aggregation of related proteins. Recent studies have found that misfolded α-Syn has prion-like seeding properties, using its own misfolded protein as a template to convert the corresponding native conformation α-Syn protein into the same conformation of misfolded α-Syn. The seed amplification technology (SAA) is based on this principle and is used to amplify and detect pathogen seeds, capable of detecting trace amounts of misfolded proteins in samples. The SAA method has the characteristics of high throughput, high speed, high sensitivity, and high specificity. Recent studies have shown that SAA has high diagnostic accuracy in detecting misfolded α-Syn in the cerebrospinal fluid of patients with Parkinson's disease and dementia with Lewy bodies. Summary of the Invention

[0006] The object of the present invention is to provide an auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples and its application. The present invention provides a convenient, rapid, minimally invasive and highly sensitive AD detection kit based on CSF α-Syn-SAA to meet the clinical need for auxiliary diagnosis of AD.

[0007] The technical solution of the auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples of the present invention is as follows: An auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples, comprising a substrate protein, a reaction reagent, a negative control and a positive control, and the sample is a cerebrospinal fluid sample to be tested.

[0008] Preferably, the substrate protein is 6His-α-Syn recombinant protein, and the amino acid sequence of the 6His-α-Syn recombinant protein is shown in SEQ ID NO.6: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH.

[0009] Further preferably, the reaction reagent includes 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 of the 10× buffer is 7.0-8.5.

[0010] Even more preferably, the negative control is phosphate buffer.

[0011] Still more preferably, the positive control is 6His-α-Syn preformed fibrils, and the 6His-α-Syn preformed fibrils are fibril proteins prepared by in vitro culture of the substrate protein.

[0012] Still 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, 0.1 mL of phosphate buffer and 0.1 mL of 6His-α-Syn preformed fibrils.

[0013] Preferably, the kit further includes a black 96-well cell plate pre-loaded with silicon beads. The diameter of the silicon beads is 0.8 mm, and 4 silicon beads are loaded in each well.

[0014] The technical solution of the application of the Alzheimer's disease auxiliary detection kit based on the cerebrospinal fluid sample α-synuclein seed amplification technology of the present invention is as follows: Application of a diagnostic kit based on the above-mentioned cerebrospinal fluid sample α-synuclein seed amplification technology in Alzheimer's disease.

[0015] Preferably, the detection method of the kit is as follows: (1) Collect the cerebrospinal fluid to be tested, centrifuge and separate the supernatant, and store it 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 a 100 μL reaction solution per portion; (3) Add 95 μL / well of the reaction solution to the black 96-well cell plate, and set up negative control group, positive control group and experimental group respectively, and set up duplicate wells. Add 5 μL of PBS to each well in the negative control group, add 5 μL of 6His-α-Syn pre-prepared fibrils to the positive control group, and add 5 μL of pretreated cerebrospinal fluid sample to the experimental group; (4) Set the reaction program of the RT-QuIC instrument, stop the detection after running for 36 - 60 h; read the data and analyze the detected data.

[0016] More 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.

[0017] Beneficial effects: (1) In this application, the abnormal conformation α-Syn protein derived from the patient's cerebrospinal fluid is co-incubated with the in vitro recombinant substrate protein 6His-α-Syn with normal conformation. Through oscillation cycling, α-Syn is amplified in large quantities, causing the recombinant protein to undergo a conformational change to form new misfolded proteins. This cycle continues, causing the misfolded α-Syn protein to increase exponentially, and is monitored in real time by the amyloid-specific dye thioflavin T (Thioflavin T, ThT). If there is an abnormal conformation of α-Syn protein in the patient sample, according to the prion-like seeding characteristics, theoretically it can cause the recombinant protein to undergo a conformational change to form new misfolded proteins. This cycle continues, causing the misfolded α-Syn protein to increase exponentially. Therefore, after the cycle amplification is completed, detecting its fluorescence intensity can indicate whether there is an abnormal conformation of α-Syn protein in the patient sample, so as to achieve the auxiliary diagnosis of Alzheimer's disease.

[0018] (2) This application is the first in China and abroad to use the cerebrospinal fluid of subjects as the detection sample for the clinical diagnostic study of RT-QuIC in Alzheimer's disease. The kit of this application has obvious advantages in terms of high sensitivity, specificity, repeatability, practicability, rapidity, low cost, etc. The clinical sampling of this kit is simple, and the detection kit has the characteristics of simple operation process and low cost, short detection time, high accuracy and specificity, and is easy to be clinically promoted. It can be applied clinically to help clinicians accurately diagnose and monitor the treatment progress of Alzheimer's disease.

[0019] (3) The 6His-α-Syn recombinant protein prepared in this application has the advantages of high activity and high stability. When the kit disclosed in this application is used for sample detection, the required time is relatively short, and it can be completed in only 36-60 h. Description of the Drawings

[0020] Figure 1 It is the prokaryotic induction expression identification result of the substrate protein in Example 1. Among them, the abscissas 1, 3, 5, 7, and 9 are the protein expression results of monoclonal colonies A, B, C, D, and E before induction respectively, the abscissas 2, 4, 6, 8, and 10 are the protein expression results of monoclonal colonies A, B, C, D, and E after induction respectively, and the position pointed by the right arrow is the expression result of the substrate protein; Figure 2 It is the Ni-NTA purification identification result of the substrate protein in Example 1. Among them, the abscissas 1-9 represent the purification results of the experimental groups of FT, 10 mM, 20 mM, 50 mM, 100 mM, 150 mM, 200 mM, 300 mM, and 500 mM imidazole buffer respectively; Figure 3 It is the anion exchange column purification identification result of the substrate protein in Example 1. Among them, the abscissas 1-4 represent the purification results of the experimental groups of 100 mM imidazole buffer, IEX-FT, IEX-A, and IEX-B respectively. IEX-FT, IEX-A, and IEX-B represent the flow-through liquid that is not bound to the packing and the elution liquids collected at different salt concentrations during the ion exchange chromatography purification process; Figure 4 It is the molecular sieve purification identification result of the substrate protein in Example 1. Among them, the abscissas 1-8 represent the groups of IEX-A, IEX-A SEC-A, IEX-A SEC-B1, IEX-A SEC-B2, IEX-B, IEX-BSEC-A, IEX-B SEC-B, and IEX-B SEC-C respectively. IEX-A and IEX-B are the samples of peak A and peak B collected after anion column chromatography respectively. IEX-A SEC-A, B1, and B2 are different eluted proteins in the molecular size exclusion chromatography (SEC) of the IEX-A sample. IEX-B SEC-A, B, and C areFigure 3 Different eluted proteins of the IEX-B fraction in size-exclusion chromatography (SEC); Figure 5 Detection results of cerebrospinal fluid by RT-QuIC instrument for patients with cognitive impairment and control patients in Example 12; Detailed implementation manners

[0021] The embodiments of the present invention will be described in detail below. The examples are shown in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation to the present invention.

[0022] Example 1 Preparation of α-Syn recombinant protein 1. Codon optimization The molecular weight of α-synuclein (SNCA) is 14 kDa and it consists of 140 amino acids. According to the coding gene sequence of SNCA in the NCBI (National Center for Biotechnology Information) database (GenBank: 6622), the human SNCA gene sequence was selected and codon-optimized. Gene synthesis was performed by Anhui General Biotechnology Co., Ltd. The nucleotide sequence of SNCA after codon optimization is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0023] The nucleotide sequence of SEQ ID NO.1 is as follows: ATGGACGTGTTCATGAAAGGTCTGTCTAAAGCGAAAGAGGGTGTTGTTGCTGCGGCTGAGAAGACCAAACAGGGTGTTGCTGAAGCTGCTGGTAAGACTAAAGAAGGCGTTCTGTACGTTGGTTCTAAGACCAAAGAAGGTGTTGTTCACGGTGTTGCGACTGTTGCGGAAAAAACCAAAGAACAGGTTACCAACGTTGGTGGTGCGGTTGTTACTGGTGTTACCGCAGTTGCGCAGAAGACCGTTGAAGGTGCTGGTTCCATCGCAGCAGCTACCGGTTTCGTTAAGAAAGACCAGCTGGGTAAGAACGAAGAAGGTGCTCCGCAAGAAGGTATCTTGGAAGATATGCCGGTAGATCCGGACAACGAAGCATACGAAATGCCGTCTGAAGAAGGTTACCAAGACTACGAACCGGAAGCA The amino acid sequence of SEQ ID NO.2 is as follows: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA 2. Expression vector construction Select the pET32a(+) prokaryotic expression vector, select the Nde1 and Xho1 restriction enzyme sites, and perform homologous recombination ligation on the synthesized SNCA gene and the empty plasmid pET32a(+). The reaction system is 10 μL, including 100 ng of pET32a(+), 16 ng of the SNCA gene, and 5 μL of 2x cloning recombinant mix. The plasmid pET32a(+) has 6 His tags at the C-terminus. After transformation into Escherichia coli competent cells DH5α (Solarbio, C1100), it is spread on an ampicillin-resistant LB (Luria-Bertani) solid medium and cultured overnight at 37°C. Single colonies are picked for PCR positive identification and sequencing identification. The correctly identified positive clone is named pET32-α-Syn, and its nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0024] 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 Transform 10 ng of the above-constructed pET32-α-Syn plasmid into 100 μL of Escherichia coli BL21 (DE3) competent cells (Solarbio, C1400) with a cell density of 10 7 CFU / mL, and culture it overnight at 37 °C in an inverted incubator on an LB solid medium with ampicillin resistance for 12 - 16 h. Pick 5 single colonies (named A, B, C, D, and E respectively) and culture them in 3 - 5 mL of LB liquid medium supplemented with ampicillin antibiotic. Culture them in a shaking incubator at 37 °C and 220 rpm until the OD600 reaches 0.6 - 1. Inoculate the culture into 5 mL of LB auto-induction medium containing ampicillin antibiotic at a ratio of 1:100, and continue to culture it overnight at 37 °C and 200 rpm for 12 - 18 h to induce the expression of recombinant protein. Identify the protein expression by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining. 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.

[0025] After that, expand the culture of the successfully transformed cells and induce the expression overnight in 1 L of the culture medium.

[0026] 4. Protein collection and purification Collect and purify the protein 6His-α-Syn from the above overnight-induced culture according to the following steps: (1) Centrifuge at 4000 rpm for 20 min at 4 °C to collect the bacterial cells; 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 homogenizer with a pressure of 850 pa to disrupt the bacterial cells for 15 min. Collect the bacterial lysate for later use.

[0027] (2) Centrifuge the bacterial solution collected in step (1) at 12,000 rpm for 45 min at 4°C, collect the supernatant, boil it in boiling water for 15 min, and place it on ice for cooling and incubation for 5 min; centrifuge the supernatant after the above cooling and incubation at 12,000 rpm for 45 min at 4°C, collect the supernatant, add streptomycin sulfate with a final concentration of 10 mg / mL to the supernatant, stir at 4°C for 30 min, and set aside.

[0028] (3) Centrifuge the supernatant after the above stirring and mixing at 12,000 rpm for 45 min at 4°C, collect the supernatant, adjust the pH of the supernatant to 3.5 with 1 M hydrochloric acid, stir at 4°C for 30 min, then centrifuge at 12,000 rpm for 45 min at 4°C, collect the supernatant and adjust the supernatant to pH = 7.5 with 1 M NaOH, and set aside.

[0029] (4) Purify by Ni-NTA column chromatography. Pre-equilibrate the purification column with a buffer of 20 mM Tris, 100 mM NaCl, pH = 7.5. Wash the miscellaneous proteins and elute the target protein in FT (Flow Through), 10, 20, 50, 100, 150, 200, 300, and 500 mM imidazole buffers (20 mM Tris, 100 mM NaCl, pH = 7.5, 0 - 500 mM imidazole) respectively, collect the elution peaks at each stage, and finally identify the target protein by 4 - 20% SDS-PAGE gradient gel electrophoresis and Coomassie brilliant blue staining. The results are as Figure 2 shown. It can be seen from Figure 2 that a large amount of the target protein can be eluted at 100 mM imidazole, and a crude 6His-α-Syn protein solution is obtained.

[0030] (5) Centrifuge the above protein solution at 12,000 rpm for 45 min at 4°C, collect the supernatant, and dialyze it overnight for 16 h in a buffer of 20 mM Tris, 100 mM NaCl, pH = 7.5.

[0031] (6) Add the sample dialyzed overnight 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 perform linear salt concentration elution (20 mM Tris, pH = 7.5, 0 - 1 M NaCl, pH = 7.5). Collect the samples of each elution peak, and identify the target protein by 4 - 20% SDS-PAGE gradient gel electrophoresis and Coomassie brilliant blue staining. The results are as Figure 3 shown. It can be seen from Figure 3 that the purified target protein exists in the samples of peak A and peak B collected, and a further purified 6His-α-Syn protein solution is obtained.

[0032] (7) The identified target protein was purified by size exclusion chromatography. Samples of the elution peaks A and B were collected. The target protein was identified by 4-20% SDS-PAGE gradient gel electrophoresis and Coomassie Brilliant Blue staining. The results are as Figure 4 shown. As can be seen from Figure 4 , after purification by molecular sieve, the miscellaneous proteins have been basically removed, and the target protein with high purity has been obtained. The identified 6His-α-Syn protein sample was obtained.

[0033] (8) The identified target protein sample was dialyzed in a buffer containing 20 mM Tris, 100 mM NaCl, and pH = 7.5 for 16 h. The protein concentration was determined by detecting the absorbance value at 280 nm and setting the extinction coefficient to 0.387, and the protein concentration was adjusted to 1 mg / mL. Each tube of 1 mL was aliquoted and freeze-dried to obtain the substrate protein 6His-α-Syn sample.

[0034] 5. Protein Sequencing The substrate protein 6His-α-Syn was sequenced, and the amino acid sequence was deduced based on the nucleic acid sequence. 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 AD Detection Kit All components, ratios or concentrations, and volumes in the kit of this example are shown in Table 1 below.

[0035] 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 400 mM phosphate buffer (PB), 1.7 M NaCl, 0.005% SDS, pH = 8.0 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 AD detection kit is as follows: (1) Collect cerebrospinal fluid detection samples, centrifuge at 1300 g for 5 min at 4°C, separate the supernatant, aliquot and store at -80°C for later use.

[0036] (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 portion. Among them, the specific components and ratios of the 10× buffer are shown in Table 1.

[0037] (3) Add 4 silicon beads with a diameter of 0.8 mm to each well of a black 96-well plate, and add 95 μL of the reaction solution to each well. Set up negative control groups, positive control groups and experimental groups respectively. Among them, 5 μL of PBS is added to each well in the negative control group, with 4 replicates set; 5 μL of pre-prepared 6His-α-Syn fibrils is added in the positive control group, with 4 replicates set; 5 μL of pretreated cerebrospinal fluid sample is added in the experimental group, and 4 replicates are set for each sample.

[0038] (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 mode to bottom reading by the machine; the black 96-well plate is shaken at 42°C for 1 min each time, paused for 1 min, and the fluorescence data is detected once every 30 min. Stop the detection after running for 36 - 60 h. Read the data and analyze the detected data.

[0039] Example 3 Different from Example 2, the 10× PBS buffer consists of 200 mM PB buffer, 1.5 M NaCl and 0.001% SDS, and the pH of the 10× buffer is 7.0.

[0040] Example 4 Different from Example 2, the 10× PBS buffer is composed of 500 mM PB buffer, 1.0 M NaCl and 0.01% SDS, and the pH of the 10× buffer is 7.5.

[0041] Example 5 Different from Example 2, the 10× PBS buffer is composed of 800 mM PB buffer, 2.0 M NaCl and 0.007% SDS, and the pH of the 10× buffer is 8.5.

[0042] Example 6 Different from Example 2, the final concentration of thioflavin T in the reaction solution is 10 μM.

[0043] Example 7 Different from Example 2, the final concentration of thioflavin T in the reaction solution is 20 μM.

[0044] Example 8 Different from Example 2, the final concentration of thioflavin T in the reaction solution is 30 μM.

[0045] Example 9 Different from Example 2, the concentration of the substrate protein 6His-α-Syn is 0.05 mg / mL.

[0046] Example 10 Different from Example 2, the concentration of the substrate protein 6His-α-Syn is 0.25 mg / mL.

[0047] Example 11 Different from Example 2, the concentration of the substrate protein 6His-α-Syn is 0.5 mg / mL.

[0048] Example 12 Clinical sample detection and verification Thirty-four cerebrospinal fluid samples from patients with cognitive impairment diagnosed by clinical signs (named X1 - X34 respectively) and five cerebrospinal fluid samples from healthy controls (CR) (named C1 - C5 respectively) were selected. According to the detection steps of Example 2, the above 39 samples were detected, and the results are as Figure 5 shown in Table 2.

[0049] Table 2 Auxiliary diagnosis of Alzheimer's disease by α-Syn SAA AT α-Syn SAA Number of Cases Clinical Significance A(+) or T(+) α-Syn SAA(+) 4 Co-morbidity of Alzheimer's Disease and Synucleinopathy A(+) or T(+) α-Syn SAA(-) 20 Alzheimer's Disease A(-)T(-) α-Syn SAA(+) 5 Synucleinopathy A(-)T(-) α-Syn SAA(-) 10 Non-Alzheimer's Disease and Non-Synucleinopathy A: Amyloid-β proteinopathy, decreased cerebrospinal fluid (or serum) Ab42 / 40, or positive Amyloid PET; T: Tau proteinopathy, increased cerebrospinal fluid (or serum) P-tau 181 (or 217), or positive Tau PET.

[0050] As can be seen from Figure 5 Table 2, among the 29 patients with cognitive impairment, 24 were diagnosed with AD, 4 of whom had synucleinopathy; 5 patients with cognitive impairment were diagnosed with synucleinopathy (DLB or PDD). 5 patients with cognitive impairment and 5 non-neurodegenerative disease controls were neither Alzheimer's disease nor synucleinopathy.

[0051] 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 may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples.

[0052] 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. An auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples, characterized in that, It includes a substrate protein, reaction reagents, a control, and a positive control. The sample is a cerebrospinal fluid sample to be tested.

2. The Alzheimer's disease auxiliary detection kit based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to claim 1, wherein, The substrate protein is a 6His-α-Syn recombinant protein, and the amino acid sequence of the 6His-α-Syn recombinant protein is as shown in SEQ ID NO.6: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEALEHHHHHH.

3. The auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to claim 2, characterized in that, 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 of the 10× buffer is 7.0 - 8.

5.

4. The Alzheimer's disease auxiliary detection kit based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to claim 3, wherein The negative control is phosphate buffer solution.

5. The Alzheimer's disease auxiliary detection kit based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to claim 4, wherein 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.

6. The Alzheimer's disease auxiliary detection kit based on the cerebrospinal fluid sample α-synuclein seed amplification technology according to claim 5, 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.

7. The auxiliary detection kit for Alzheimer's disease based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to any one of claims 1 to 6, characterized in that, 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 4 silica beads are loaded in each well.

8. Application of the Alzheimer's disease auxiliary detection kit based on the α-synuclein seed amplification technology of cerebrospinal fluid samples in Alzheimer's disease as described in claim 7.

9. Use of the Alzheimer's disease auxiliary detection kit based on the cerebrospinal fluid sample α-synuclein seed amplification technology according to claim 8 in Alzheimer's disease, characterized in that, The detection method of the kit is as follows: (1) Collect the cerebrospinal fluid to be tested, centrifuge and separate the supernatant, and aliquot and store it 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 95 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 respectively, and set up duplicate wells. Add 5 μL of PBS to each well of the negative control group, add 5 μL of 6His-α-Syn preformed fibrils to the positive control group, and add 5 μL of the pretreated cerebrospinal fluid sample to the experimental group; (4) Set the reaction program of the RT-QuIC instrument, stop the detection after running for 36 - 60 h; read the data and analyze the detected data.

10. Use of the Alzheimer's disease auxiliary detection kit based on the α-synuclein seed amplification technology of cerebrospinal fluid samples according to claim 9 in Alzheimer's disease, characterized in that, 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.

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