Method for detecting alpha-synuclein aggregates in saliva, kit and use thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a method for detecting alpha-synuclein aggregates in saliva, a kit and application thereof. The method uses real-time vibration-induced transformation technology to detect alpha-synuclein aggregates in saliva for the first time. On the one hand, the saliva sample is simple to process and has few interference factors, which is conducive to simplifying the detection process, reducing the detection cost, and improving the accuracy of the detection result. On the other hand, the saliva sample is non-invasively obtained, realizing the non-invasive detection of alpha-synuclein aggregates in vivo, and laying a foundation for the non-invasive diagnosis, treatment, progress research, pathogenesis research of Parkinson's disease (PD) or multiple system atrophy (MSA), or the non-invasive identification of PD and MSA.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method, kit, and application for detecting α-synuclein aggregates in saliva. Background Technology

[0002] α-synuclein is a soluble protein composed of 140 amino acids, expressed presynaptally and perinuclearly in the central nervous system. With a molecular weight of approximately 19 kDa, it is closely related to the pathogenesis and related functional impairments of Parkinson's disease and is a major component of Lewy bodies. α-synuclein can be abnormally expressed and aggregated under the influence of various factors. Biochemical reactions such as oxidative stress and the formation of oligomeric intermediates produced during this process all play important roles in the pathogenesis of Parkinson's disease. Physiologically, α-synuclein exists as disordered monomers, while pathologically, it exists as aggregates. In the brains of Parkinson's patients, α-synuclein aggregates into α-synuclein aggregates, which, along with other proteins, form Lewy bodies, precipitating in the brain and leading to brain cell death.

[0003] In related technologies, α-synuclein aggregates in biological materials such as saliva, brain tissue, cerebrospinal fluid, skin, or olfactory mucosa are detected, and the results are used as a diagnostic criterion for Parkinson's disease. Common methods for detecting α-synuclein aggregates in saliva include electrochemical immunofluorescence assay and ELISA.

[0004] However, in the process of proposing this invention, the inventors discovered that existing methods for detecting α-synuclein aggregates in saliva require large sample volumes and have poor accuracy. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the shortcomings of existing methods for detecting α-synuclein aggregates in saliva, such as large sample volume and poor accuracy, and to provide a method, kit and application for detecting α-synuclein aggregates in saliva.

[0006] Therefore, the present invention provides a method for detecting α-synuclein aggregates in saliva for non-disease diagnostic purposes. The method utilizes a real-time vibration-induced transformation method, wherein the reaction buffer used in the real-time vibration-induced transformation method comprises: 10-100 mM phosphate buffer, 300-500 mM sodium chloride, 5-10 μM fluorescent dye, sodium dodecyl sulfate with a mass fraction of 0.00125%-0.0015%, and 0.5-1 mg / ml wild-type human α-synuclein.

[0007] Optionally, the reaction buffer comprises: 10 mM phosphate buffer, 500 mM sodium chloride, 10 μM fluorescent dye, 0.0015% sodium dodecyl sulfate, and 1 mg / ml wild-type human α-synuclein;

[0008] Optionally, the pH of the phosphate buffer solution is 7.0–8.0;

[0009] Optionally, the fluorescent dye is thioflavin T.

[0010] Optionally, the wild-type human α-synuclein is a recombinant wild-type human α-synuclein, and the steps for preparing the recombinant wild-type human α-synuclein include:

[0011] The host strain was transformed using an expression vector expressing wild-type human α-synuclein, induced to culture, and then separated into solid and liquid components. The solid was collected to obtain the first cell pellet.

[0012] The first cell pellet was resuspended in sucrose solution, incubated, and then separated into solid and liquid components. The solid was collected to obtain the second cell pellet.

[0013] The second cell precipitate was resuspended in a salt solution, subjected to thermal precipitation, and solid-liquid separation. The liquid was collected to obtain the cell lysate.

[0014] Take the cell lysate, load it onto an anion exchange column, and elute with a first buffer containing 0-1000 mM NaCl according to a NaCl concentration gradient from low to high. Take the eluent with a NaCl concentration between 300 and 350 mM to obtain the crude extract.

[0015] Take the crude extract, concentrate it, filter it, load it onto a molecular sieve, elute it with a second buffer solution, take the eluent, concentrate it, filter it to obtain the recombinant wild-type human α-synuclein;

[0016] Optionally, the sucrose solution contains 30-40% sucrose, 1-2 mM EDTA, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 20-50 mM Tris by mass, and the pH value of the sucrose solution is 7.0-8.0;

[0017] Optionally, the incubation is performed by shaking at room temperature for 5–10 minutes;

[0018] Optionally, the salt solution contains 1–2 mM EDTA, 1 mM PMSF and 20 mM Tris, and the pH value of the salt solution is 7.0–8.0;

[0019] Optionally, the heat precipitation is performed at 85–100°C for 10–15 minutes.

[0020] Optionally, the first buffer solution also contains 20 mM Tris, and the pH value of the first buffer solution is 7.0 to 8.0;

[0021] Optionally, the second buffer solution is a mixture containing 40 mM phosphate and 170 mM NaCl, and the pH value of the second buffer solution is 7.0 to 8.0.

[0022] Optionally, the detection method includes: taking a saliva sample and mixing it with the reaction buffer, adding glass beads, incubating it with intermittent vibration cycles, and detecting the fluorescence value at the excitation wavelength and emission wavelength.

[0023] Optionally, the detection method satisfies at least one of the following conditions:

[0024] 1) The volume ratio of the saliva sample to the reaction buffer is (5-10):(95-90);

[0025] 2) The diameter of the glass beads is 0.8-3mm, and the number of beads added is 1-6;

[0026] 3) The conditions for intermittent vibration cycle incubation include: the vibration mode is dual-track vibration, the vibration frequency is 400-600 rpm; in a single cycle, after each vibration of 1-2 min, the incubation is paused for 1-29 min, and the cycle is repeated 60-96 times; the incubation temperature is 37-70℃.

[0027] 4) The excitation light wavelength is 450nm and the emission light wavelength is 480nm.

[0028] Optionally, the detection method further includes the step of preparing the saliva sample using the saliva to be tested: taking the saliva to be tested, adding a protease inhibitor, separating the solid and liquid, taking the supernatant, and obtaining the saliva sample;

[0029] Optionally, the protease inhibitor is benzyl sulfonyl fluoride;

[0030] Optionally, the solid-liquid separation is performed by centrifugation at a temperature of 4°C, a rotation speed of 10,000–12,000 g, and a time of 10–20 min.

[0031] The present invention also provides a kit for detecting α-synuclein aggregates in saliva, the kit comprising a reaction buffer for a real-time vibration-induced transformation assay, the reaction buffer comprising:

[0032] 10–100 mM phosphate buffer, 300–500 mM sodium chloride, 5–10 μM fluorescent dye, 0.00125%–0.0015% sodium dodecyl sulfate, 0.5–1 mg / ml wild-type human α-synuclein.

[0033] Optionally, the reaction buffer comprises: 10 mM phosphate buffer, 500 mM sodium chloride, 10 μM fluorescent dye, 0.0015% sodium dodecyl sulfate, and 1 mg / ml wild-type human α-synuclein;

[0034] Optionally, the pH of the phosphate buffer solution is 7.0–8.0;

[0035] Optionally, the fluorescent dye is thioflavin T;

[0036] Optionally, the kit may also include glass beads with a diameter of 0.8 to 3 mm, wherein the number of glass beads is 1 to 6.

[0037] Optionally, the wild-type human α-synuclein is a recombinant wild-type human α-synuclein, prepared by the following method:

[0038] The host strain was transformed using an expression vector expressing wild-type human α-synuclein, induced to culture, and then separated into solid and liquid components. The solid was collected to obtain the first cell pellet.

[0039] The first cell pellet was resuspended in sucrose solution, incubated, and then separated into solid and liquid components. The solid was collected to obtain the second cell pellet.

[0040] The second cell precipitate was resuspended in a salt solution, subjected to thermal precipitation, and solid-liquid separation. The liquid was collected to obtain the cell lysate.

[0041] Take the cell lysate, load it onto an anion exchange column, and elute with a first buffer containing 0-1000 mM NaCl according to a NaCl concentration gradient from low to high. Take the eluent with a NaCl concentration between 300 and 350 mM to obtain the crude extract.

[0042] Take the crude extract, concentrate it, filter it, load it onto a molecular sieve, elute it with a second buffer solution, take the eluent, concentrate it, filter it to obtain the recombinant wild-type human α-synuclein;

[0043] Optionally, the sucrose solution contains 30-40% sucrose, 1-2 mM EDTA, 1 mM MPMSF and 20-50 mM Tris by mass, and the pH value of the sucrose solution is 7.0-8.0;

[0044] Optionally, the incubation is performed by shaking at room temperature for 5–10 minutes;

[0045] Optionally, the salt solution contains 1–2 mM EDTA, 1 mM PMSF and 20 mM Tris, and the pH value of the salt solution is 7.0–8.0;

[0046] Optionally, the heat precipitation is performed at 85–100°C for 10–15 minutes.

[0047] Optionally, the first buffer solution also contains 20 mM Tris, and the pH value of the first buffer solution is 7.0 to 8.0;

[0048] Optionally, the second buffer solution is a mixture containing 40 mM phosphate and 170 mM NaCl, and the pH value of the second buffer solution is 7.0 to 8.0.

[0049] The present invention also provides the use of the kit described in any one of the present invention in the detection of α-synuclein aggregates in saliva for non-disease diagnostic purposes.

[0050] The present invention also provides the use of the kit described in any one of the present invention in the preparation of products for diagnosing Parkinson's disease and / or multiple system atrophy;

[0051] Optionally, the use may be in the preparation of products that utilize saliva to diagnose Parkinson's disease and / or multiple system atrophy.

[0052] The technical solution of this invention has the following advantages:

[0053] 1. The detection method provided by this invention is the first to utilize real-time vibration-induced transformation technology to detect α-synuclein aggregates in saliva. On the one hand, the processing of saliva samples is simple and has few interfering factors, which helps to simplify the detection process, reduce detection costs, and improve the accuracy of detection results. On the other hand, saliva samples are obtained non-invasively, realizing non-invasive detection of α-synuclein aggregates in vivo, laying the foundation for non-invasive diagnosis, treatment, progression research, pathogenesis research of Parkinson's disease (PD) or multiple system atrophy (MSA), or non-invasive differentiation between PD and MSA.

[0054] 2. The detection method provided by this invention, for the saliva sample, increases the concentration of sodium chloride and substrate protein (recombinant wild-type human α-synuclein) in the reaction buffer, so that accurate detection results can be obtained with the addition of a small amount of saliva sample. At the same time, the small amount of saliva sample added can further reduce the interference of other factors in saliva on the detection results, thereby improving the accuracy of the detection results of the method of this invention.

[0055] 3. The detection method provided by the present invention significantly shortens the time for fluorescence to appear in the detection results by optimizing the pH of the reaction buffer and the vibration frequency, standing time, and incubation temperature of the "intermittent vibration cycle incubation". Specifically, the maximum fluorescence value can be detected in most samples after 35 hours of incubation, and a significant inoculation reaction can be detected within 48 hours.

[0056] 4. The detection method provided by the present invention uses a specific method to prepare recombinant wild-type human α-synuclein: (1) The method uses a combination of sucrose solution resuspension and heat precipitation to break cells, which can improve the cell disruption effect, fully release the recombinant protein, and lay the foundation for subsequent purification operations; (2) Due to the sucrose solution resuspension operation, the heat precipitation part can achieve a good disruption effect with a lower concentration of salt solution. On the one hand, the lower concentration of salt solution causes less damage to the structure of the recombinant protein, which is conducive to improving the purity and yield of the recombinant protein. On the other hand, the lower concentration of salt solution does not require dialysis, which can simplify the purification operation and shorten the purification time; (3) The method first uses anion exchange column for gradient elution, and then uses molecular sieve for further purification. Compared with the conventional purification method of first performing molecular sieve purification, then performing anion exchange column elution, and then replacing the high-salt buffer in the elution solution, in the method of this application, the molecular sieve purification step can replace the high-salt buffer in the anion exchange column elution solution while purifying the protein. Therefore, the method simplifies the purification operation of recombinant protein and shortens the purification time.

[0057] In other words, this method can simplify the purification process of recombinant proteins and shorten the purification time while ensuring the yield and purity of recombinant proteins.

[0058] 5. The detection method provided by this invention involves solutions with the same pH value. Compared with conventional methods, the pH adjustment operation is omitted, which can avoid the introduction of external interference factors and simplify the process. Attached Figure Description

[0059] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0060] Figure 1 This is a 15% SDS-PAGE image of the α-synuclein in the anion exchange column eluent of Example 1 of the present invention.

[0061] Figure 2 This is a 15% SDS-PAGE image of the α-synuclein in the molecular sieve eluent of Example 1 of the present invention.

[0062] Figure 3 This is a fluorescence detection result diagram of Example 2 of the present invention;

[0063] Figure 4 This is a fluorescence detection result diagram of Example 4 of the present invention;

[0064] Figure 5 This is a fluorescence detection result diagram of Example 5 of the present invention;

[0065] Figure 6 This is a fluorescence detection result diagram of Comparative Example 2 of the present invention;

[0066] Figure 7 This is a graph showing the 15% SDS-PAGE detection results of α-synuclein in the molecular sieve eluent of Comparative Example 3 of this invention. Detailed Implementation

[0067] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0068] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0069] The sources of experimental materials and equipment involved in the embodiments of this invention are as follows:

[0070] Experimental materials and reagents:

[0071] pET-3a plasmid (Dr. Lei Wang (Institute of Biophysics, Chinese Academy of Sciences)); Escherichia coli BL21(DE3) (Yisheng Biotechnology, cat#11804ES80).

[0072] LP0042 (Thermo Fisher Scientific OXOID, Lot #1205028), LP0021 (Thermo Fisher Scientific OXOID, Lot #1177579), IPTG (Amresco, Lot #0202C207), Sucrose (Shanghai Test, cat #10021418), EDTA (Amresco, Lot #1926B030), PMSF (Beyotime, cat #ST506), Tris buffer (Amresco, Lot #1973C458), NaCl (Shanghai Test, cat #10019318), PBS (LABLEAD, cat #P7208B), APS (Amresco, Lot #2891C223), TEMED (Amresco, Lot #19H0156625); 30% AB (Acr-Bis) (LABLEAD, cat #A3291), SDS (Solarbio, cat #S1010).

[0073] LB medium: Weigh 10g LP0042, 5g LP0021, and 10g sodium chloride, add 1L ddH2O, mix well, and autoclave.

[0074] SDS-PAGE stacking gel: 1.73ml ddH2O, 0.52ml 30% AB, 0.75ml 4×SDS (pH 6.8), 30μL 10% APS, 4.5μL TEMED.

[0075] SDS-PAGE separating gel: 1.8ml ddH2O, 3.5ml 30% AB, 1.75ml 4×SDS (pH 8.8), 70μL 10% APS, 3.5μL TEMED.

[0076] Experimental equipment and materials:

[0077] Centrifuge (Beckman Coulter Avanit J26XP with JA-25.50 Rotor);

[0078] HiTrap Q FF anion exchange column (GE Healthcare 17-5156-01);

[0079] 3-kDa MWCO centrifugal filter (Merck Millipore Amicon Ultra-15);

[0080] 0.22μm filter membrane (Merk Millipore);

[0081] Molecular sieve HiPrep 16 / 60 Sephacryl S-300HR (GE Healthcare, 17-1167-01);

[0082] 100-kda MWCO ultracentrifugal filter (Amicon Ultra; Millipore);

[0083] 96-well plate (Costar, cat#3792);

[0084] Glass beads (Fisher Scientific, CAT#S80024);

[0085] Greiner EASY seal (Greiner Bio-one) fluorescent sealing film;

[0086] SYNERGY4 fluorescent plate reader (BioTek).

[0087] Example 1

[0088] This example illustrates the preparation of recombinant wild-type human α-synuclein:

[0089] (1) *E. coli* BL21(DE3) cells were transformed using the recombinant plasmid pET-3a expressing wild-type α-synuclein (as shown in SEQ ID NO:1). The transformed *E. coli* were then inoculated into 25 ml of ampicillin-containing... + After incubating overnight at 37°C and 225 rpm in LB medium, the culture was transferred to 1 L of LB medium and continued. When the bacteria grew to the OD... 600 When the concentration was 0.6-0.8, IPTG was added to a final concentration of 0.2 mM to induce expression for 3 h. Then, the cells were centrifuged at 4℃ and 4000g for 15 min, and the solid was collected to obtain the first cell pellet.

[0090] The method for transforming Escherichia coli BL21(DE3) cells using recombinant plasmids is as follows:

[0091] 1) Thaw cloned competent cells (DH5α Chemically Competent Cell, CatNo.11802ES) on ice;

[0092] 2) Take 10 μL of cooled recombinant plasmid, add it to 100 μL of competent cells, gently tap the tube wall a few times to mix, and place on ice for 30 min;

[0093] 3) Heat shock at 42℃ for 90 seconds, followed by incubation in an ice bath for 2 minutes;

[0094] 4) Add 900 μL of LB medium, incubate at 37°C for 10 min to fully recover, and then shake at 37°C and 200 rpm for 45 min.

[0095] 5) Centrifuge at 5000 rpm for 3 min, discard 900 μL of supernatant, resuspend the bacterial culture with the remaining culture medium, and gently spread it evenly on a plate containing the correct antibiotics (ampicillin and chloramphenicol in this experiment) using a sterile spreader. After the bacterial culture is absorbed, invert the plate and incubate overnight at 37°C.

[0096] (2) Take the first cell pellet above, gently resuspend it in sucrose solution (pH 7.4, containing 40% sucrose, 2 mM EDTA, 1 mM PMSF and 20 mM Tris by mass), and incubate gently with shaking for 10 minutes at room temperature. Then centrifuge at 20°C and 7900g for 20 minutes, discard the supernatant, and take the solid to obtain the second cell pellet.

[0097] (3) Take the second cell pellet above, resuspend it in 25 ml of salt solution (pH 7.4, containing 2 mM EDTA, 1 mM PMSF and 20 mM Tris), heat precipitate at 95 °C for 15 min, then centrifuge at 4 °C and 18000 rpm for 50 min, discard the pellet, take the liquid, and obtain the cell lysate.

[0098] (4) Take the above cell lysis buffer and load it onto a 50 mL HiTrap Q FF anion exchange column. Elute using a first buffer (pH 7.4) containing 0–1000 mM NaCl and 20 mM Tris, following a NaCl concentration gradient from low to high. Collect the eluent with NaCl concentrations between 300 and 350 mM to obtain the crude extract. The purity of α-synuclein was detected by SDS-PAGE. The results are shown below. Figure 1 As shown, seven components, 22-28, were collected.

[0099] (5) The collected crude extract was concentrated to 5 ml using a 3-kDa MWCO centrifugal filter, filtered through a 0.22 μm filter membrane, and loaded onto a HiPrep 16 / 60 Sephacryl S-300HR molecular sieve for further purification. After column pre-equilibration, the column was eluted with a mixture of 40 mM phosphate buffer (pH 7.4) and 170 mM NaCl, and the eluent was collected. The purity of α-synuclein was detected by SDS-PAGE, and the results are as follows: Figure 2 As shown, 13 components were collected from tubes 31 to 43.

[0100] (6) The collected eluent was concentrated using a new 3-kda MWCO centrifugal filter and then filtered through a 100-kda MWCO ultracentrifugal filter to remove any oligomer particles that may have formed during concentration, yielding recombinant wild-type human α-synuclein. The protein concentration was determined using a spectrophotometer, with a theoretical absorption coefficient of ε0.1% and 0.377 at 280 nm. The protein was aliquoted to a final concentration of 1 mg / ml and stored at -80°C for later use.

[0101] Depend on Figure 1 It can be seen that the target protein collected in tubes 22-28 has a high purity. Figure 2 It can be seen that single-component target proteins were collected in tubes 31-43, and the purity was relatively high. Figure 1 Further improvements were made. Measurements showed that, in this example, the protein purification amount was 10 mg compared to the 1 L induction culture medium in step (1).

[0102] Example 2

[0103] A method for detecting α-synuclein aggregates in saliva, comprising:

[0104] (1) Saliva samples were obtained from 75 clinically diagnosed PD patients (source: outpatient and inpatient departments of Peking University First Hospital) and control saliva samples were obtained from 36 non-degenerative disease control subjects of the corresponding age and sex (source: outpatient and inpatient departments of Peking University First Hospital).

[0105] (2) Take 1-3 ml of each saliva sample to be tested or control saliva sample and place them in 5 ml EP tubes. Add 1 mM PMSF and centrifuge at 4℃ and 12000g for 20 min. Dispense the supernatant and freeze at -80℃ to obtain 75 saliva samples to be tested and 36 control saliva samples.

[0106] (3) Take the recombinant wild-type human α-synuclein obtained in Example 1, concentrate it before the experiment, filter it through 100kDaMWCO, dispense it into Ep tubes, measure the concentration, and dilute it to 1mg / ml during the experiment.

[0107] (4) Take a 96-well plate with a black bottom, add 95 μl of RT-QuIC reaction buffer and 5 μl of saliva sample (saliva sample to be tested or control saliva sample) to each well, and the final reaction volume is 100 μl. The RT-QuIC reaction buffer consists of 10 mM PBS (pH 7.4), 500 mM NaCl, 10 μM ThT, 0.0015% SDS and 1 mg / ml recombinant wild-type human α-synuclein.

[0108] (5) Add one 3mm diameter glass bead to each well. Seal the plate with Greiner EASY seal. Place the 96-well plate in a SYNERGY4 fluorescence reader and incubate intermittently at 37°C using a vibration cycle: vibrate on two tracks for 1 min (600 rpm), then let it stand for 29 min. Measure the ThT fluorescence value every 30 min under excitation wavelength of 450 nm and emission wavelength of 480 nm. Perform 96 cycles of the reaction. Each sample is tested in triplicate. Control saliva samples, test saliva samples, and uninoculated reactions can be tested simultaneously on the same plate.

[0109] In this embodiment, as the reaction proceeded, fluorescence was detected in the culture wells of 57 out of 75 saliva samples tested, and the fluorescence value gradually increased with increasing reaction time. Figure 3 As shown in the figure (PD, 57 samples); no obvious fluorescence value was detected in the culture wells containing saliva samples from 36 control samples, as shown. Figure 3 As shown in the figure (NNCs, the percentage of relative fluorescence values ​​of 36 control saliva samples is the average).

[0110] Example 3

[0111] The method for detecting α-synuclein aggregates in saliva was followed as described in Example 2, except that the concentration of NaCl in the RT-QuIC reaction buffer used in this example was 300 mM.

[0112] In this embodiment, as the reaction proceeded, fluorescence was detected in the culture wells containing the saliva sample to be tested, and the fluorescence value gradually increased with the increase of reaction time; no obvious change in fluorescence value was detected in the culture wells containing the control saliva sample.

[0113] Example 4

[0114] The method of Example 2 was used to detect α-synuclein aggregates in saliva, except that the concentration of recombinant wild-type human α-synuclein in the RT-QuIC reaction buffer used in this example was 0.5 mg / ml, and two saliva samples to be tested and one control saliva sample were tested.

[0115] In this embodiment, as the reaction proceeded, fluorescence was detected in the culture wells containing two test saliva samples (PD, 2 samples in the figure), and the fluorescence value gradually increased with increasing reaction time; no significant change in fluorescence value was detected in the culture wells containing the control saliva sample (NNC, 1 sample in the figure). Figure 4 As shown.

[0116] Example 5

[0117] The method of Example 2 was used to detect α-synuclein aggregates in saliva, except that the saliva samples used in this example were collected from 18 clinically diagnosed MSA patients (source: outpatient and inpatient wards of Peking University First Hospital).

[0118] In this embodiment, as the reaction proceeded, fluorescence was detected in the culture wells of 11 out of 18 saliva samples tested, and the fluorescence value gradually increased with increasing reaction time (MSA in the figure, 11 samples); no significant change in fluorescence value was detected in the culture wells containing 36 control saliva samples (NNCs in the figure, the average percentage of relative fluorescence values ​​of the 36 control saliva samples). Figure 5 As shown.

[0119] Comparative Example 1

[0120] The method of Example 2 was used to detect α-synuclein aggregates in saliva, except that the concentration of NaCl in the RT-QuIC reaction buffer used in this comparative example was 170 mM.

[0121] In this comparative example, no significant changes in fluorescence values ​​were detected in either the culture well containing the saliva sample to be tested or the culture well containing the control saliva sample as the reaction proceeded.

[0122] Comparative Example 2

[0123] The method of Example 2 was used to detect α-synuclein aggregates in saliva, except that the concentration of recombinant wild-type human α-synuclein in the RT-QuIC reaction buffer used in this comparative example was 0.2 mg / ml, and two saliva samples to be tested and one control saliva sample were tested.

[0124] In this comparative example, as the reaction proceeded, no significant changes in fluorescence values ​​were detected in the culture wells containing two test saliva samples (PD, 2 samples in the figure) and in the culture wells containing the control saliva sample (NNC, 1 sample in the figure). Figure 6 As shown.

[0125] Comparative Example 3

[0126] Recombinant wild-type human α-synuclein was prepared according to the method of Example 1, except that the sucrose solution resuspension in step (2) was not performed in this comparative example.

[0127] The purity of α-synuclein in the molecular sieve eluent was determined by 15% SDS-PAGE, and the results are as follows: Figure 7 As shown. Figure 7It can be seen that after omitting the sucrose solution resuspension step, two bands appeared near 15 kDa in the molecular sieve eluent collected in tubes 31-42, indicating that the purity of α-synuclein in the molecular sieve eluent decreased.

[0128] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention. sequence list <110> Peking University First Hospital <120> Methods, kits, and applications for detecting α-synuclein aggregates in saliva <130> HA202200691 <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1000 <212> DNA <213> Artificial Sequence <400> 1 cctctagaat attttgttta actttaagaa ggagatatac atatggatgt attcatgaaa 60 ggactttcaa aggccaagga gggagttgtg gctgctgctg agaaaaccaa acagggtgtg 120 gcagaagcag caggaaagac aaaagagggt gttctctatg taggctccaa aaccaaggag 180 ggagtggtgc atggtgtggc aacagtggct gagaagacca aagagcaagt gacaaatgtt 240 ggaggagcag tggtgacggg tgtgacagca gtagcccaga agacagtgga gggagcaggg 300 agcattgcag cagccactgg ctttgtcaaa aaaggaccagt tgggcaagaa tgaagaaagga 360 gccccacagg aaaggaattct ggagaatatg cctgtggatc ctgacaatga ggcttatgaa 420 atgccttctg aggaagggta tcaagaactac gaacctgaag cctaatgatc aaatcgaat 480 tcctgcagcc cgatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca 540 ccgctgagca ataattagca taaccccttg gggcccttaa acgggtcttg aggggttttt 600 tgctgaaagg aggaactata tccggatatc cacaggacgg gtgtggtcgc catgatcgcg 660 tagtcgatag tggctccaag tagcgaagcg agcaggactg ggcggcggcc aaagcggtcg 720 gacagtgctc cgagaacggg tgcgcataga aattgcatca acgcatatag cgctagcagc 780 acgccatagt gactggcgat gctgtcggaa tggacgatat cccgcaagag gcccggcagt 840 accggcataa ccaagcctat gcctacagca tccaggtgac gtgccgagga tgacgatgag 900 cgcattgtag attcatacac ggtgcctgac tgcgttagca tttaacctgt gataactacg 960 cataagctta tcgatgatag ctgttcaaac atggagaatt 1000

Claims

1. A method for detecting α-synuclein aggregates in saliva for non-disease diagnostic purposes, characterized in that, The detection was performed using a real-time vibration-induced transformation method, wherein the reaction buffer used in the real-time vibration-induced transformation method comprises: 10 mM phosphate buffer, 500 mM sodium chloride, 10 μM fluorescent dye, 0.0015% sodium dodecyl sulfate, 1 mg / ml wild-type human α-synuclein.

2. The detection method according to claim 1, characterized in that, The pH value of the phosphate buffer solution is 7.0 to 8.

0.

3. The detection method according to claim 1, characterized in that, The fluorescent dye is thioflavin T.

4. The detection method according to claim 1, characterized in that, The wild-type human α-synuclein is a recombinant wild-type human α-synuclein, and the steps for preparing the recombinant wild-type human α-synuclein include: The host strain was transformed using an expression vector expressing wild-type human α-synuclein, induced to culture, and then separated into solid and liquid components. The solid was collected to obtain the first cell pellet. The first cell pellet was resuspended in sucrose solution, incubated, and then separated into solid and liquid phases. The solid was collected to obtain the second cell pellet. The second cell precipitate was resuspended in a salt solution, subjected to thermal precipitation, and solid-liquid separation. The liquid was collected to obtain the cell lysate. Take the cell lysate, load it onto an anion exchange column, and elute with a first buffer containing 0-1000 mM NaCl according to a NaCl concentration gradient from low to high. Take the eluent with a NaCl concentration between 300 and 350 mM to obtain the crude extract. The crude extract was concentrated, filtered, loaded onto a molecular sieve, eluted with a second buffer solution, and the eluent was concentrated and filtered to obtain the recombinant wild-type human α-synuclein.

5. The detection method according to claim 4, characterized in that, The sucrose solution contains 30%–40% sucrose, 1–2 mM EDTA, 1 mM PMSF and 20–50 mM Tris by mass, and the pH value of the sucrose solution is 7.0–8.

0.

6. The detection method according to claim 4, characterized in that, The incubation process involves shaking the incubator at room temperature for 5–10 minutes.

7. The detection method according to claim 4, characterized in that, The salt solution contains 1–2 mM EDTA, 1 mM PMSF and 20 mM Tris, and the pH value of the salt solution is 7.0–8.

0.

8. The detection method according to claim 4, characterized in that, The heat precipitation is performed at 85–100 °C for 10–15 min.

9. The detection method according to claim 4, characterized in that, The first buffer solution also contains 20 mM Tris, and the pH value of the first buffer solution is 7.0 to 8.

0.

10. The detection method according to claim 4, characterized in that, The second buffer solution is a mixture containing 40 mM phosphate and 170 mM NaCl, and the pH value of the second buffer solution is 7.0 to 8.

0.

11. The detection method according to any one of claims 1 to 10, characterized in that, The detection method includes: Take a saliva sample and mix it with the reaction buffer solution. Add glass beads and incubate with intermittent vibration. Detect the fluorescence value at the excitation wavelength and emission wavelength.

12. The detection method according to claim 11, characterized in that, The detection method satisfies at least one of the following conditions: 1) The volume ratio of the saliva sample to the reaction buffer is (5-10):(95-90); 2) The diameter of the glass beads is 0.8–3 mm, and the number of beads added is 1–6; 3) The conditions for intermittent vibration cycle incubation include: the vibration mode is dual-track vibration, the vibration frequency is 400-600 rpm; in a single cycle, after each vibration for 1-2 minutes, the incubation is paused for 1-29 minutes, and the cycle is repeated 60-96 times; the incubation temperature is 37-70 ℃. 4) The excitation light wavelength is 450 nm and the emission light wavelength is 480 nm.

13. The detection method according to claim 11, characterized in that, The detection method further includes the step of preparing the saliva sample using the saliva to be tested: taking the saliva to be tested, adding a protease inhibitor, separating the solid and liquid, taking the supernatant, and obtaining the saliva sample.

14. The detection method according to claim 13, characterized in that, The protease inhibitor is benzyl sulfonyl fluoride.

15. The detection method according to claim 13, characterized in that, The solid-liquid separation step for preparing the saliva sample is centrifugation at a temperature of 4 ℃, a rotation speed of 10000~12000 g, and a time of 10~20 min.

16. A kit for detecting α-synuclein aggregates in saliva, characterized in that, The kit includes a reaction buffer for real-time vibration-induced transformation, the reaction buffer comprising: 10 mM phosphate buffer, 500 mM sodium chloride, 10 μM fluorescent dye, 0.0015% sodium dodecyl sulfate, 1 mg / ml recombinant wild-type human α-synuclein.

17. The detection method according to claim 16, characterized in that, The pH value of the phosphate buffer solution is 7.0 to 8.

0.

18. The detection method according to claim 16, characterized in that, The fluorescent dye is thioflavin T.

19. The detection method according to claim 16, characterized in that, The kit also includes glass beads with a diameter of 0.8 to 3 mm, and the number of glass beads is 1 to 6.

20. The kit according to claim 16, characterized in that, The wild-type human α-synuclein is a recombinant wild-type human α-synuclein, prepared by the following method: The host strain was transformed using an expression vector expressing wild-type human α-synuclein, induced to culture, and then separated into solid and liquid components. The solid was collected to obtain the first cell pellet. The first cell pellet was resuspended in sucrose solution, incubated, and then separated into solid and liquid phases. The solid was collected to obtain the second cell pellet. The second cell precipitate was resuspended in a salt solution, subjected to thermal precipitation, and solid-liquid separation. The liquid was collected to obtain the cell lysate. Take the cell lysate, load it onto an anion exchange column, and elute with a first buffer containing 0-1000 mM NaCl according to a NaCl concentration gradient from low to high. Take the eluent with a NaCl concentration between 300 and 350 mM to obtain the crude extract. The crude extract was concentrated, filtered, loaded onto a molecular sieve, eluted with a second buffer solution, and the eluent was concentrated and filtered to obtain the recombinant wild-type human α-synuclein.

21. The reagent kit according to claim 20, characterized in that, The sucrose solution contains 30-40% sucrose, 1-2 mM EDTA, 1 mM PMSF and 20-50 mM Tris by mass, and the pH value of the sucrose solution is 7.0-8.

0.

22. The reagent kit according to claim 20, characterized in that, The incubation process involves shaking the incubator at room temperature for 5–10 minutes.

23. The reagent kit according to claim 20, characterized in that, The salt solution contains 1–2 mM EDTA, 1 mM PMSF and 20 mM Tris, and the pH value of the salt solution is 7.0–8.

0.

24. The reagent kit according to claim 20, characterized in that, The heat precipitation is performed at 85–100 °C for 10–15 min.

25. The reagent kit according to claim 20, characterized in that, The first buffer solution also contains 20 mM Tris, and the pH value of the first buffer solution is 7.0 to 8.

0.

26. The reagent kit according to claim 20, characterized in that, The second buffer solution is a mixture containing 40 mM phosphate and 170 mM NaCl, and the pH value of the second buffer solution is 7.0 to 8.

0.

27. Use of the kit according to any one of claims 16 to 26 in the detection of α-synuclein aggregates in saliva for non-disease diagnostic purposes.

28. Use of the kit according to any one of claims 16 to 26 in the preparation of a product for diagnosing Parkinson's disease and / or multiple system atrophy using saliva.

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