A parkinson's disease in vitro diagnosis kit based on DNA hexahedron and application thereof

Abstract

The application discloses an isolated microRNA molecule, a DNA hexagon-based Parkinson's disease in-vitro diagnosis kit and application thereof. The microRNA molecule miR26690 provided by the application is highly expressed in exosomes in peripheral blood of Parkinson's disease patients, and can be used for in-vitro diagnosis and / or screening of Parkinson's disease. In addition, the kit and method for detecting miR26690 in peripheral blood provided by the application can detect miR26690 in peripheral blood with high sensitivity and high specificity, and compared with a traditional Parkinson's disease marker detection method, the method is simple, easy to prepare, has a small sample amount, is higher in sensitivity, has a low detection limit and is low in detection cost.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological agents, and particularly relates to a DNA hexagonal-based Parkinson's disease in vitro diagnosis kit and application thereof. BACKGROUND

[0002] At present, the diagnosis and differential diagnosis of Parkinson's disease and other neurodegenerative diseases mainly rely on the clinical manifestations and imaging indicators of patients, which requires clinicians to have rich experience, resulting in a high misdiagnosis rate. When patients have obvious clinical symptoms, a large number of neurons have already died irreversibly, resulting in limited treatment effect. There is an urgent need for a high-sensitivity and specificity biomarker to assist in diagnosis. At present, the better biomarker is mainly based on the detection of cerebrospinal fluid, but the collection of cerebrospinal fluid is traumatic to patients and has poor patient compliance. Therefore, it is urgent to develop a high-accuracy diagnostic biomarker based on blood (plasma, serum, red blood cells, etc.). Due to the existence of the blood-brain barrier, biomarkers that can reflect the state of brain diseases in the traditional sense are difficult to be detected in peripheral blood. In recent years, it has been found that extracellular vesicles such as exosomes from the central nervous system can penetrate the blood-brain barrier into peripheral blood (Min et al., Acta Neuropathologica, 128(5): 639-650, 2014), which provides a theoretical basis for detecting exosomes in peripheral blood to reflect neurodegenerative diseases in the central nervous system.

[0003] In recent years, it has been found that exosomes carry a large amount of nucleic acids, among which microRNA (microRNA or miRNA) is an important category. An article published in 2022 introduces an experimental method for detecting exosome microRNA based on DNA hexagonal molecular beacon (Dongsheng et al., Biosensors and Bioelectronics, 202211 4077). It suggests that we can use DNA hexagonal molecular beacon to detect exosomes carrying certain specific miRNAs in blood to diagnose Parkinson's disease and other neurodegenerative diseases and develop biomarkers. SUMMARY

[0004] The inventors have accidentally found a microRNA whose expression level in the peripheral blood exosomes of Parkinson's disease patients is much higher than that of non-Parkinson's disease patients during long-term research on biomarkers for Parkinson's disease. Therefore, it can be used as a biomarker for diagnosing and / or screening Parkinson's disease, and based on this, the present application is completed.

[0005] The first aspect of the present application provides an isolated microRNA molecule having a nucleotide sequence as set forth in SEQ ID NO. 9, or a nucleotide sequence having at least 90%, at least 95%, or at least 99% sequence identity to the nucleotide sequence as set forth in SEQ ID NO. 9.

[0006] The second aspect of the present application provides use of the microRNA molecule of the first aspect of the present application or a reagent for detecting the microRNA molecule of the first aspect of the present application in the preparation of a Parkinson's disease diagnosis and / or screening reagent or kit.

[0007] The third aspect of the present application provides a composition for detecting the microRNA molecule of the first aspect of the present application, comprising nucleic acid molecules as set forth in SEQ ID NO. 1 to SEQ ID NO. 8.

[0008] The fourth aspect of the present application provides a Parkinson's disease diagnosis and / or screening kit comprising a reagent for detecting the microRNA molecule of the first aspect of the present application.

[0009] The fifth aspect of the present application provides use of the microRNA molecule of the first aspect of the present application and / or an exosome comprising the microRNA molecule as a biomarker for Parkinson's disease diagnosis and / or screening.

[0010] The sixth aspect of the present application provides a method for detecting the microRNA molecule of the first aspect of the present application or an exosome comprising the microRNA molecule in vitro, comprising:

[0011] a) mixing nucleic acid molecules as set forth in SEQ ID NO. 1 to SEQ ID NO. 6, SEQ ID NO. 8, and reacting under suitable conditions to form a DNA hexagon structure; wherein the 5' end of the nucleic acid molecule as set forth in SEQ ID NO. 8 is connected with a quenching group; preferably, the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB, or TAMARA;

[0012] b) adding a nucleic acid molecule as set forth in SEQ ID NO. 7 to the reaction system of step a), and reacting at 36-38°C to generate a detection hexagon, wherein the 3' end of the nucleic acid molecule as set forth in SEQ ID NO. 7 is connected with a fluorescent group; preferably, the fluorescent group is selected from PE, FITC, FAM, TAMRA, Alexa Fluor, VIC, JOE, NED, TET, HEX, ROX, TEXASRED, CY3, CY5, CY5.5, or CY7;

[0013] c) contacting the sample to be tested with the detection hexagon; preferably, the sample to be tested is a peripheral blood sample of a subject; preferably, the sample to be tested is peripheral blood plasma;

[0014] d) determining the microRNA molecule in the sample to be tested by detecting the fluorescent signal; preferably, the determination is a quantitative determination.

[0015] The seventh aspect of the present application provides a method for diagnosing Parkinson's disease, which comprises the method of the sixth aspect of the present application, detecting the content of the microRNA molecule or the exosome containing the microRNA molecule in the sample of the body fluid of the subject, and determining whether the subject has Parkinson's disease according to the content of the microRNA molecule or the exosome containing the microRNA molecule.

[0016] The microRNA molecule miR26690 provided by the present application is highly expressed in the exosome in the peripheral blood of Parkinson's disease patients, and therefore can be used for in vitro diagnosis and / or screening of Parkinson's disease; further, the kit and method for detecting miR26690 in peripheral blood provided by the present application can detect miR26690 in peripheral blood with high sensitivity and high specificity, and compared with the traditional method for detecting Parkinson's disease markers, the method is simple, easy to prepare, has less sample amount, higher sensitivity, lower detection limit and lower detection cost. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a schematic diagram of the detection method principle of the present application.

[0018] Figure 2 It is a schematic diagram of the results of detecting miR26690 positive exosomes in plasma by the nano-flow detection system of Example 1.

[0019] Figure 3 It is the detection results of plasma miR26690 positive exosome concentration of Parkinson's disease patients and healthy controls.

[0020] Figure 4 It is the detection results of plasma miR26690 positive exosome concentration of early Parkinson's disease patients and healthy controls.

[0021] Figure 5 It is the ROC curve of DNA hexagon detecting plasma miR26690 positive exosome concentration as a biomarker for diagnosing Parkinson's disease. DETAILED DESCRIPTION

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only represent one embodiment of the present application, and for those skilled in the art, other embodiments can also be obtained from these drawings.

[0023] Definitions

[0024] As used herein, the terms "a" and "an" and "the" and similar referents are intended to indicate the singular as well as the plural, unless the context clearly indicates otherwise.

[0025] As used herein, the terms "about", "substantially", and "similarly" refer to an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, or on the limitation of the measurement system.

[0026] According to the present application, the term "comprising" means including, containing or comprising, but not excluding any further integers or steps. According to the present application, the term "consisting essentially of means including the integers or groups of integers specified but excluding other integers or groups of integers that alter, perhaps materially, the basic and novel activity or use of the invention. According to the present application, the term "consisting of" means including, containing or comprising the specified integers or groups of integers but excluding any other integers or groups of integers.

[0027] As used herein, the term "microRNA (miRNA)" is a class of non-coding single-stranded RNA molecules of about 20 nucleotides in length encoded by endogenous genes that are involved in the regulation of post-transcriptional gene expression in animals and plants. The terms "microRNA", "miRNA" and "miRNA" are used interchangeably in the present application.

[0028] The term "isolated microRNA molecule" essentially relates herein to a microRNA molecule that is separated from its natural environment. In other words, the microRNA molecule preparation is essentially free of other substances with which it is naturally associated. In some embodiments, the isolated microRNA molecule contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% of other substances with which it is naturally associated, by weight.

[0029] As used herein, the term "Parkinson's disease (PD)" (also named idiopathic Parkinson's syndrome (IPS) or Morbus Parkinson) refers to a disorder of the motor system that is the result of the loss of dopamine-producing brain cells. The main symptoms of PD are a tremor or shaking in hands, arms, legs, jaw, and face; stiffness or rigidity of the limbs and trunk; slowness of movement or bradykinesia; and postural instability or impaired balance and coordination. As these symptoms become more pronounced, patients can have difficulty walking, talking, or completing other simple tasks. PD usually affects people over the age of 50. The early symptoms of PD are subtle and occur gradually. In some people, the disease progresses more rapidly than in others.

[0030] As used herein, the term "diagnosing whether an individual has Parkinson's disease (PD)" refers to determining whether an individual shows signs of PD or has PD. Thus, an individual can be diagnosed as having PD or not having PD.

[0031] As used herein, the term "determining the course of an individual having Parkinson's disease" refers to determining the development of PD over time, e.g. whether PD worsens in the individual over time, does not worsen / stabilizes in the individual over time, or improves in the individual over time.

[0032] As used herein, the term "diagnosing" refers to the process of determining a possible disease or disorder, thus a process that attempts to define the (clinical) status of an individual. The determination of the level of a miRNA according to the present application is correlated with the (clinical) status of the individual. Preferably, diagnosing comprises / embraces: (i) determining the occurrence / presence of PD; (ii) monitoring the course of PD; (iii) staging of PD; (iv) measuring the response of an individual having PD to a therapeutic intervention; and / or (v) segmentation of an individual having PD.

[0033] As used herein, the term "individual" refers to any subject in which it is desired to know whether she or he is affected by / having PD.

[0034] In particular, as used herein, the term "individual" refers to a subject suspected of being affected by PD or of being affected. An individual can be diagnosed as being affected by PD, i.e. diseased; or can be diagnosed as not being affected by PD, i.e. being healthy with respect to these diseases.

[0035] As used herein, the term "individual" also refers to a subject affected by PD, i.e. diseased. An individual can be retested for PD and can be diagnosed as still being affected by PD, i.e. diseased; or as no longer being affected by (not being affected by as much) PD, i.e. being healthy with respect to PD (e.g. after a therapeutic intervention). An individual can be further retested for PD and can be diagnosed as having developed into a late or severe form of PD.

[0036] It should be noted that an individual diagnosed as not having PD (i.e. healthy with respect to PD) can also have another undetected / unknown disease.

[0037] An individual can be any mammal, including a human and another mammal, for example, an animal such as a rabbit, a mouse, a rat, or a monkey. Human subjects are particularly preferred as individuals.

[0038] As used herein, the term "subject" can refer to a subject known to be unaffected by PD (negative control) (such subject is also referred to as a non-Parkinson's disease patient), i.e. healthy with respect to PD. As used herein, the term "subject" can also refer to a subject known to be affected by PD (i.e. diseased). The subject can have developed into a late stage form of PD.

[0039] It should be noted that a subject known to be unaffected by PD (i.e. healthy with respect to PD) (non-Parkinson's disease patient) can also have another undetected / unknown disease.

[0040] In the present application, a subject can be any mammal, including a human and another mammal, for example, an animal such as a rabbit, a mouse, a rat, or a monkey. Human subjects are particularly preferred as individuals.

[0041] As used herein, the term "treatment" refers to any therapy that improves the health status of an individual and / or prolongs (increases) the life of an individual. The therapy can eliminate the disease in an individual, arrest or slow the progression of the disease in an individual, inhibit or slow the development of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce the recurrence of the disease in an individual who currently or previously has the disease. The disease can be PD. (Therapeutic) treatment of PD includes, but is not limited to, drug administration, speech therapy, motor training, psychological training, and / or physical rehabilitation.

[0042] As used herein, the term "body fluid" is a broad term and is to be given its ordinary and customary meaning to a person of ordinary skill in the art and is not to be limited to a special or customized meaning. The body fluid sample can be a urine sample, a blood sample, a sputum sample, a breast milk sample, a cerebrospinal fluid (CSF) sample, a cerumen (earwax) sample, a gastric fluid sample, a mucus sample, a perilymph fluid sample, an endolymph fluid sample, a peritoneal fluid sample, a pleural fluid sample, a saliva sample, a sebum (skin oil) sample, a semen sample, a sweat sample, a tear sample, a buccal swab, a vaginal secretion sample, a liquid biopsy, or a vomit sample, including components or fractions thereof. The term "body fluid sample" also encompasses body fluid fractions, such as blood fractions, urine fractions, or sputum fractions. Body fluid samples can be mixed or pooled. Thus, a body fluid sample can be a mixture of a blood and a urine sample, or a mixture of a blood and a cerebrospinal fluid sample. The body fluid sample can be provided by withdrawing the body fluid from an individual or subject, but can also be provided by using a previously isolated body fluid sample material. The body fluid sample enables a non-invasive analysis of the individual. It is further preferred that the volume of the body fluid sample is 0.001-20 mL, preferably 0.001-10 mL, more preferably 0.001-1 mL, and most preferably 0.001-0.05 mL.

[0043] In the present application, the term "peripheral blood" means blood that is or was in circulation throughout the body in a mammal and is not obtained from blood sequestered in, for example, bone marrow (BM), lymph nodes (e.g., lymphocytes), spleen, or liver.

[0044] As used herein, the term "blood sample" encompasses a whole blood sample or a blood component sample, such as a blood cell / cellular component, a serum, or a plasma sample.

[0045] The term "plasma" is used as conventionally defined. In some embodiments, human plasma can be, or the source of human plasma can be, fresh plasma, lyophilized plasma, solvent / detergent treated plasma, fresh frozen plasma, thawed plasma or cryoprecipitate, frozen supernatant or plasma concentrate, such as concentrate from frozen plasma, or any two or more of these. Plasma is typically obtained from a sample obtained by blood purification or from a whole blood sample provided with or in contact with an anticoagulant (e.g., heparin, citrate, oxalate, or EDTA). Subsequently, the cellular components of the blood sample are separated from the liquid components (plasma) using appropriate techniques, typically by centrifugation. By way of specific examples, but not limitation, to obtain plasma suitable for use in this application, a blood sample may be aspirated into a vacuum blood collection tube (e.g., BD Vacutainer plastic EDTA tube, 10 ml, 1.8 mg / mL) containing the anticoagulant EDTA (ethylenediaminetetraacetic acid). The sample is gently agitated and then centrifuged at 1,000-2,000 g for 10 minutes at room temperature to separate the plasma from the red blood cells. The supernatant (plasma) is collected, optionally pooled (if multiple blood samples are used), and aliquoted into frozen vials, which are stored at -80°C until use. Therefore, the term "plasma" refers to a composition of cell-free components of a blood sample that does not form part of a human or animal body. Thus, plasma as contemplated herein is cell-free plasma, for example, plasma containing less than about 1.0% w / w, preferably less than about 0.5% w / w or less than 0.1% w / w of whole-cell material, or substantially free of whole-cell material.

[0046] Plasma may preferably be untreated plasma, i.e. plasma obtained by separation from whole blood without subsequent processing steps that alter its chemical, biochemical, or cellular composition other than selective heat inactivation, storage (low temperature or non-low temperature), sterilization, filtration, freeze drying, and / or solvent / detergent treatment.

[0047] In some embodiments, the term "plasma" may specifically exclude processed plasma, i.e., plasma that has undergone one or more processing steps after separation from whole blood to alter its composition, specifically its chemical, biochemical, or cellular composition. Preferably, the term "plasma," as contemplated herein, specifically excludes platelet-rich plasma (PRP), i.e., plasma enriched with platelets. Typically, PRP may contain about 1.0 × 10⁻⁶ platelets. 6 The platelet count is approximately 1.5 × 10⁻⁶ / μL, although the platelet concentration in whole blood can be approximately 1.5 × 10⁻⁶ / μL. 5 Up to 3.5×10 5 / μl. Therefore, plasma, as envisioned in this paper, can contain less than about 8.0 × 10⁹ / μl. 5 Preferably less than about 7.0 × 10 5 More preferably less than about 6.0 × 10 5, more preferably less than about 5.0 x 10 5 , such as less than about 4.0 x 10 5 platelets per μl.

[0048] Plasma as contemplated herein is human plasma, i.e., obtained from a single human subject or from multiple human subjects (e.g., a plasma pool).

[0049] Plasma can be used directly in the methods of the application. Plasma can also be stored appropriately for later use (e.g., for a short period of time, such as up to about 1-2 weeks, at a temperature above the freezing point of the plasma, but below ambient temperature, which temperature will typically be about 4°C to 5°C; or for a longer period of time by cryopreservation, typically at about -70°C to about -80°C).

[0050] The methods of the application can use plasma that is autologous to the subject to be diagnosed or screened, and thus the plasma is autologous to the cells preserved in the composition comprising the plasma. The term "autologous" with respect to plasma means that the plasma is obtained from the same subject to which the plasma is contacted or diagnosed or screened.

[0051] Plasma can be heat-inactivated, particularly to remove complement, as is known in the art. When the methods of the application use plasma that is autologous to the subject to be diagnosed or screened, it can not be necessary to heat-inactivate the plasma.

[0052] As used herein, the term "level" or "amount" refers to the quantity (e.g., measured in grams, moles, or counts such as ion or fluorescence counts) or concentration (e.g., absolute or relative concentration) of a miRNA as described herein.

[0053] As used herein, the term "level" or "amount" also includes a scaled amount or value, a normalized amount or value, or a scaled and normalized amount or value. Preferably, the levels determined herein are expression levels.

[0054] As used herein, the term "sensitivity" refers to the number of true positive patients (%) relative to the total number of patients (100%). The individuals can be subjects with PD. Sensitivity is calculated by the formula: Sensitivity = TP / (TP + FN) (TP = true positive; FN = false negative).

[0055] As used herein, the term "specificity" relates to the number of true negative individuals (%) relative to the total number of healthy subjects (100%). Specificity is calculated by the formula: Specificity = TN / (TN + FP) (TN = true negative; FP = false positive).

[0056] As used herein, the term "accuracy" refers to a statistical measure of correctness for the classification or identification of a sample type. Accuracy is the proportion of true results (both true positives and true negatives).

[0057] As used herein, the term "exosome" refers to a microvesicular membrane vesicle of approximately 30-150 nm in diameter, secreted by a variety of cells, containing specific proteins (e.g., the exosomal membrane is rich in the transmembrane protein family CD63, CD81 and CD9 involved in exosome transport), lipids, cytokines or genetic material. A variety of cells can secrete exosomes under normal and pathological conditions, which are widely present in body fluids such as blood, saliva, urine, cerebrospinal fluid and milk, and are considered as specific secreted membrane vesicles, which are involved in intercellular communication.

[0058] As used herein, the term "biomarker" refers to a biochemical indicator that can mark changes in systems, organs, tissues, cells and subcellular structures or functions, or changes that can be susceptible to changes, with very wide use. Biomarkers can be used for disease diagnosis, disease staging, or to evaluate the safety and effectiveness of new drugs or new therapies in target populations.

[0059] As used herein, the term "tag" refers to an element that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical or other physical means. For example, useful tags include32P, fluorescent dyes, fluorescent groups, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin or hapten, as well as other entities that can be detected. The tag can be incorporated into a nucleic acid at any position (e.g., at the 3' end or 5' end or internally). The polynucleotide (polynucleotide probe) used to detect the miRNA and / or the miRNA itself can be labeled.

[0060] The first aspect of the present application provides an isolated microRNA molecule, i.e., miR26690, whose nucleotide sequence is UGGAUAUGGAGGGAAGGA (SEQ ID NO. 9).

[0061] Another aspect of the present application also relates to a variant of miR26690, whose nucleotide sequence has at least 90%, at least 95%, or at least 99% (i.e., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity to miR26690 (the nucleotide sequence shown in SEQ ID NO. 9).

[0062] In another aspect, the present application provides use of miR26690 or a variant thereof, or an agent for detecting miR26690 or a variant thereof, in the manufacture of a Parkinson's disease diagnostic and / or screening agent or kit.

[0063] In some embodiments, miR26690 or a variant thereof as a biomarker for Parkinson's disease diagnosis is present in a body fluid sample of a subject, exemplarily such as a urine sample, a blood sample, a sputum sample, a breast milk sample, a cerebrospinal fluid (CSF) sample, a cerumen (earwax) sample, a gastric fluid sample, a mucus sample, an endolymph fluid sample, an perilymph fluid sample, a peritoneal fluid sample, a pleural fluid sample, a saliva sample, a sebum (skin oil) sample, a semen sample, a sweat sample, a tear sample, a buccal swab, a vaginal secretion sample, a liquid biopsy or a vomit sample, etc.

[0064] In some preferred embodiments, miR26690 or a variant thereof as a biomarker for Parkinson's disease diagnosis is present in the peripheral blood of a subject, more particularly, in the exosomes of the peripheral blood.

[0065] In another aspect, the present application also provides use of miR26690 or a variant thereof, and / or miR26690 positive exosomes (i.e. exosomes comprising miR26690 or a variant thereof) as a biomarker for Parkinson's disease diagnosis and / or screening. In some embodiments, the miR26690 or a variant thereof and / or miR26690 positive exosomes are present in the peripheral blood of a subject.

[0066] In some embodiments, the agent for detecting miR26690 or a variant thereof is an agent for detecting miR26690 or a variant thereof in the peripheral blood, or an agent for detecting miR26690 or a variant thereof in the exosomes of the peripheral blood, or the agent for detecting miR26690 or a variant thereof can also be an agent for detecting miR26690 positive exosomes.

[0067] In some embodiments, the agent for detecting miR26690 or a variant thereof is capable of specifically binding to miR26690 or a variant thereof.

[0068] In some embodiments, the agent for detecting miR26690 or a variant thereof contains a detection tag, such as a fluorescent group, etc.

[0069] In some embodiments, when the agent for detecting miR26690 or a variant thereof specifically binds to miR26690 or a variant thereof, the detection group therein will change, such as emitting light, or quenching, or changing the fluorescence intensity, etc. of a fluorescent group, so that the presence or absence or the amount of miR26690 or a variant thereof can be detected by detecting the change of the fluorescent signal.

[0070] In some embodiments, the miR26690 or variant thereof is present in an exosome, and the agent for detecting the miR26690 or variant thereof can also be used for detecting the presence or amount of the exosome comprising the miR26690 or variant thereof.

[0071] In some embodiments, the miR26690 or variant thereof or the exosome comprising the miR26690 or variant thereof is present in a higher amount in the peripheral blood of a Parkinson's disease patient than in a non-Parkinson's disease patient.

[0072] In some embodiments, the diagnosis and / or screening of Parkinson's disease further comprises diagnosing and / or screening the therapeutic effect of Parkinson's disease.

[0073] In some embodiments, the diagnosis and / or screening of the therapeutic effect of Parkinson's disease comprises:

[0074] measuring the concentration of the miR26690 or variant thereof or the exosome comprising the miR26690 or variant thereof in the peripheral blood of the subject at different time points of the treatment, and comparing the concentration of the miR26690 or variant thereof or the exosome comprising the miR26690 or variant thereof in the peripheral blood of the subject measured at different time points.

[0075] Preferably, if the concentration of the miR26690 or variant thereof or the exosome comprising the miR26690 or variant thereof in the peripheral blood of the subject presents a trend of increasing as the treatment proceeds, it indicates that the drug treatment is ineffective; if the concentration of the miR26690 or variant thereof or the exosome comprising the miR26690 or variant thereof in the peripheral blood of the subject remains unchanged or presents a trend of decreasing as the treatment proceeds, it indicates that the drug treatment is effective.

[0076] In another aspect, the present application provides a composition for detecting the miR26690 or variant thereof, or the exosome comprising the miR26690 or variant thereof, comprising the nucleic acid molecule shown in SEQ ID NO. 1 to SEQ ID NO. 8.

[0077] In some embodiments, the 3' end of the nucleic acid molecule shown in SEQ ID NO. 7 is connected with a fluorescent group, and the 5' end of the nucleic acid molecule shown in SEQ ID NO. 8 is connected with a quenching group; preferably, the fluorescent group is selected from PE, FITC, FAM, TAMRA, Alexa Fluor, VIC, JOE, NED, TET, HEX, ROX, TEXASRED, CY3, CY5, CY5.5 or CY7; preferably, the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB or TAMARA.

[0078] In another aspect, the present application provides a Parkinson's disease diagnosis and / or screening kit comprising a reagent for detecting miR26690 or a variant thereof.

[0079] In some embodiments, the kit is selected from one or more of a Western blot kit, an enzyme-linked immunosorbent assay (ELISA) kit, a radioimmunoassay (RIA) kit, a radioimmunodiffusion kit, a two-dimensional double immunodiffusion kit, a rocket immunoelectrophoresis kit, an immunohistochemical staining kit, an immunoprecipitation assay kit, a complement fixation assay kit, a fluorescence-activated cell sorting (FACS) kit, an aptamer chip kit, a microarray kit, and a protein chip kit.

[0080] In some embodiments, the reagent for detecting miR26690 or a variant thereof comprises a nucleic acid molecule as shown in SEQ ID NO. 1 to SEQ ID NO. 8, wherein the 3' end of the nucleic acid molecule as shown in SEQ ID NO. 7 is connected with a fluorescent group, and the 5' end of the nucleic acid molecule as shown in SEQ ID NO. 8 is connected with a quenching group; wherein the sequence of the nucleic acid molecule is reasonably designed to form a DNA hexagonal structure, for example, as shown in Figure 1 the sequences of SEQ ID NO. 1 to SEQ ID NO. 6 and SEQ ID NO. 8 are mixed in priority to form a DNA hexagonal structure, wherein the sequence of SEQ ID NO. 2 comprises a complementary sequence of miR26690 and can specifically bind to miR26690 or a variant thereof; the formed DNA hexagonal structure is incubated with the sequence of SEQ ID NO. 7 to form a detection hexagon, wherein the sequence of SEQ ID NO. 7 comprises a region partially complementary to the sequence of SEQ ID NO. 1, and after the complementary binding of SEQ ID NO. 7 and SEQ ID NO. 1, the shape of the DNA hexagonal structure changes, and the fluorescent group in the sequence of SEQ ID NO. 7 is close to the quenching group in the sequence of SEQ ID NO. 8, so that the detection hexagon almost does not emit fluorescence (i.e., the background fluorescence intensity is very low); further, when the detection hexagon is contacted with the substrate miR26690, miR26690 or a variant thereof is complementary to the fragment on the sequence of SEQ ID NO. 2 and binds thereto, which again changes the structure of the detection hexagon, so that the fluorescent group is away from the quenching group, thereby generating fluorescence, and thus the presence or absence and the amount of miR26690 or a variant thereof can be determined according to the fluorescence intensity, so that miR26690 or a variant thereof or an exosome comprising miR26690 or a variant thereof can be detected.

[0081] In some embodiments, the fluorescent group and the quencher group are each independently connected to the 3' end or 5' end of the nucleic acid molecule by a chemical bond, a linking group, or a linker consisting of 1-3 nucleotides.

[0082] In some embodiments, the fluorescent group is selected from PE, FITC, FAM, TAMRA, Alexa Fluor, VIC, JOE, NED, TET, HEX, ROX, TEXASRED, CY3, CY5, CY5.5, or CY7; and in some embodiments, the quencher group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB, or TAMARA.

[0083] In some embodiments, the kit further comprises a reaction buffer comprising 40-50 mM Tris-acetate and 12-13 mM magnesium acetate; preferably, the pH of the reaction buffer is 7.5-8.5.

[0084] In another aspect, the present application also provides a method for detecting miR26690 or a variant thereof or an exosome comprising miR26690 or a variant thereof in vitro, which comprises:

[0085] a) mixing the nucleic acid molecules shown in SEQ ID NO. 1-SEQ ID NO. 6, SEQ ID NO. 8, and reacting under suitable conditions to form a DNA hexagonal structure; wherein the 5' end of the nucleic acid molecule shown in SEQ ID NO. 8 is connected with a quencher group; preferably, the quencher group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB, or TAMARA;

[0086] b) adding the nucleic acid molecule shown in SEQ ID NO. 7 to the reaction system of step a), and reacting at 36-38°C to generate a detection hexagon, wherein the 3' end of the nucleic acid molecule shown in SEQ ID NO. 7 is connected with a fluorescent group; preferably, the fluorescent group is selected from PE, FITC, FAM, TAMRA, Alexa Fluor, VIC, JOE, NED, TET, HEX, ROX, TEXASRED, CY3, CY5, CY5.5, or CY7;

[0087] c) contacting the sample to be tested with the detection hexagon;

[0088] d) determining the microRNA molecule in the sample to be tested by detecting the fluorescent signal; preferably, the determination is a quantitative determination.

[0089] In some embodiments, the method can be directly used for detecting iR26690 positive exosomes.

[0090] In some embodiments, the amount of miR26690 or the variant thereof can be reflected by the amount of miR26690 positive exosomes.

[0091] In some embodiments, the suitable conditions comprise incubation in a reaction buffer at 90-97℃ for 3-10 min, followed by 75-85℃ for 2-5 min; and then reduced to about 4℃.

[0092] In some preferred embodiments, incubation at about 95℃ for about 5 min, followed by incubation at about 80℃ for about 3 min; and then reduced to about 4℃; preferably, the temperature is reduced to 4℃ at a constant rate; preferably, the temperature is reduced to 60℃ at a rate of 2℃ / min, and then reduced to 4℃ at a rate of 3℃ / min.

[0093] In some embodiments, the reaction buffer comprises 40-50mM Tris-acetate and 12-13mM magnesium acetate; preferably, the pH of the reaction buffer is 7.5-8.5.

[0094] In some embodiments, the molar ratio of the nucleic acid molecules shown in SEQ ID NO. 1-SEQ ID NO. 8 is (0.8-1.2):(0.8-1.2):(0.8-1.2):(0.8-1.2):(3.8-4.2):(0.8-1.2):(0.8-1.2):(0.8-1.2); preferably, it is 1:1:1:1:4:1:1:1. The shown mixing ratio can ensure that most of the DNA can self-assemble into a DNA hexagon.

[0095] In some embodiments, the sample to be tested is selected from at least one of a urine sample, a blood sample, a sputum sample, a breast milk sample, a cerebrospinal fluid (CSF) sample, a cerumen (earwax) sample, a gastric juice sample, a mucus sample, an endolymph fluid sample, an perilymph fluid sample, a peritoneal fluid sample, a pleural fluid sample, a saliva sample, a sebum (skin oil) sample, a semen sample, a sweat sample, a tear sample, a cheek swab, a vaginal secretion sample, a liquid biopsy or a vomit sample of a subject; preferably, the sample to be tested is a peripheral blood sample of a subject; more preferably, the sample to be tested is peripheral blood plasma.

[0096] In some embodiments, the volume of the sample to be tested is 0.001-20mL, preferably 0.001-10mL, more preferably 0.001-1mL, more preferably 0.001-0.5mL, more preferably 0.001-0.1mL, more preferably 0.001-0.05mL, and most preferably 0.001-0.01mL.

[0097] In some embodiments, the ratio of the detection hexahedron to the sample to be tested is 0.5-1.5 nmol / mL.

[0098] In some embodiments, the contacting of the sample to be tested with the detection hexahedron comprises that the ratio of the detection hexahedron to the sample to be tested is 0.5-1.5 nmol / mL, and the incubation is performed at 36-38℃ for more than 8 hours.

[0099] In some embodiments, before step d), the method further comprises diluting the sample using PBS buffer, so that the diluted sample is suitable for detection.

[0100] In some embodiments, the detection of the fluorescence signal uses nanoflow detection technology. For example, the above-mentioned sample diluted with PBS buffer is subjected to nanoscale flow detection, and the instrument used includes but is not limited to Apogee, NanoFCM, Cytoflex S, etc. flow detection platform capable of detecting 10-1000 nanometer particles. Taking the Cytoflex S platform as an example, a 405 nm laser is used as a scattering light detection light source, and CY3 channel is used as a fluorescence detection parameter (corresponding to the CY3 of the fluorescent group connected to the 3' end of the nucleic acid molecule shown in SEQ ID NO. 7), the concentration of miR26690 positive nanovesicles (i.e. exosomes) with a diameter of less than 1000 nm in plasma is detected, which is further used as a diagnostic biomarker for Parkinson's disease.

[0101] In another aspect, the present application also provides a method for diagnosing Parkinson's disease, which comprises using the method for detecting miR26690 or its variant or exosome containing miR26690 or its variant in vitro, detecting the content of miR26690 or its variant or miR26690 positive exosome in the body fluid of the subject, especially in the peripheral blood, and determining whether the subject has Parkinson's disease according to the content of miR26690 or its variant or miR26690 positive exosome.

[0102] In some embodiments, the diagnosis of Parkinson's disease is early diagnosis of Parkinson's disease.

[0103] In some embodiments, the early diagnosis can be understood as H-Y staging ≤2.

[0104] In some embodiments, the content of miR26690 positive exosome higher than 1808 / μL is judged as Parkinson's disease positive.

[0105] The Parkinson's disease patient blood exosome miR26690 detection kit and detection method constructed by the application can effectively detect the concentration of miR26690 positive exosomes in blood plasma by using DNA hexagonal chimeric fluorescent tags and quenching groups, and realize high sensitivity and high specificity diagnosis of Parkinson's disease by detecting the blood plasma of Parkinson's disease patients and healthy controls. Compared with the traditional Parkinson's disease marker detection method, the method is simple, easy to prepare, has less detection sample amount, higher sensitivity, lower detection limit and lower detection cost.

[0106] The kit and method of the application will be described below in combination with specific examples.

[0107] Example 1: Detection of miR26690 positive exosomes in peripheral blood samples

[0108] According to the sequences shown in Table 1, the corresponding single-stranded DNA molecules were synthesized by Shanghai Biotechnology, wherein the fluorescent group connected to the 3' end of ncMB-cy3 is CY3; and the quenching group connected to the 5' end of ncMB-BHQ2 is BHQ2.

[0109] Exosome fluorescent labeling reaction buffer: 45mM Tirs-acetic acid and 12.5mM magnesium acetate solution, pH=8.0.

[0110] The blood samples of Parkinson's disease patients were from 28 Parkinson's disease patients (PD) who visited the Movement Disorder Disease Department of Beijing Tiantan Hospital, Capital Medical University. At the same time, 28 age-matched healthy control subjects (NC) were recruited from the community, including 8 patients in the early stage of the disease (H-Y stage ≤2); a total of 56 subjects were enrolled in the study. The research protocol was reviewed and approved by the Beijing Tiantan Hospital Ethics Committee, and all subjects in this study signed a written informed consent form. All subjects were surveyed and evaluated for general demographic data and clinical characteristics, including age, gender. For PD patients, disease duration, "off period" H-Y (Hoehn-Yahr) staging scale and Movement Disorder Society Unified Parkinson's Disease Rating Scale Part III (MDS-UPDRS III) were used to assess the stage and severity of the disease.

[0111] The demographic and clinical data of the subjects are shown in Table 2. Sample processing and preparation of blood plasma to be tested: within 2 hours after blood collection using EDTA anticoagulation blood collection tube, centrifugation at 1500xg, 4°C for 10 min. The upper layer of blood plasma was aspirated into a cryogenic tube and stored in a -80°C freezer. Before detection, the frozen blood plasma was taken out, centrifuged at 12000xg, 4°C for 10 min, and the upper layer of blood plasma was reserved.

[0112] Table 1

[0113]

[0114] Table 2

[0115]

[0116] NA: Healthy controls without information on disease course and disease severity.

[0117] After quantifying the DNA in Table 1, seven sequences were mixed in the exosome fluorescent labeling reaction buffer with a molar ratio of ncMB-1 : NcMB26690: ncMB-3: ncMB-4: ncMB-Assist: NcMB-Anchor: ncMB-BHQ2 = 1 : 1 : 1 : 1 : 4 : 1 : 1, and then incubated at 95 °C for 5 min, 80 °C for 3 min, and then reduced to 4 °C to form a DNA hexagonal structure. Then 1 molar ratio of ncMB-cy3 was added to the system for 37 °C for 1 hour incubation, and a mixed system containing detection hexagons was obtained.

[0118] The mixed system containing detection hexagons obtained above was diluted with exosome fluorescent labeling reaction buffer to a detection hexagon content of about 100 nM, 50 uL of which was mixed with 5 uL of plasma and incubated at 37 °C for 8-24 hours, then 150 uL of PBS buffer (pH = 7.4) was added and mixed to complete the preparation of the sample.

[0119] The prepared sample was subjected to nanoscale flow detection, using a Cytoflex S platform, using a 405 nm laser as a scattering light detection light source, and Cy3 channel as a fluorescence detection parameter to detect the content of miR26690 positive nanovesicles with a diameter of less than 1000 nm in the plasma. The flow cytometry results are shown in Figure 2 , and the nanovesicles with a diameter of less than 1000 nm and Cy3 fluorescence positive were selected by circle, so as to obtain the number of miR26690 positive nanovesicles with a diameter of less than 1000 nm in each sample. The results of the concentration of miR26690 positive exosomes in the plasma samples of the Parkinson's disease group and the healthy control group are shown in Figure 3 . As can be seen from the results in Figure 3 , the concentration of miR26690 positive exosomes in the peripheral blood of the Parkinson's disease patient group was significantly higher than that of the healthy control group, p < 0.0001 (Mann Whitney test). In addition, compared with the healthy control group, the Parkinson's disease patients in the early stage (H-Y stage ≤ 2) also showed a significant increase in the concentration of miR26690 positive exosomes in the peripheral blood, p < 0.0001 (Mann Whitney test), and the results are shown in Figure 4As shown, it is indicated that the index can be used as a biomarker for early diagnosis of Parkinson's disease.

[0120] Method specificity and sensitivity verification of Example 2

[0121] The number of miR26690 positive exosomes in the plasma samples of the Parkinson's disease group and the healthy control group was analyzed using a Receiver Operating Characteristic Curve (ROC), and the Youden Index was calculated. The calculation formula is Sensitivity (Sen) + Specificity (Spe) - 1. The number of miR26690 positive exosomes in the plasma sample corresponding to the maximum Youden Index is the optimal critical value between groups. After calculation, the optimal critical value is 1808, that is, the number of miR26690 positive exosomes is ≥1808 / μL, which is judged as positive for Parkinson's disease, and the number of miR26690 positive exosomes is less than 1808 / μL, which is judged as negative for Parkinson's disease. The ROC curve is shown in Figure 5 As can be seen from the figure, the detection method of the present application has a sensitivity of more than 85% for the diagnosis of Parkinson's disease, and a specificity close to 90%, with high sensitivity and specificity.

[0122] More importantly, the method of the present application can directly obtain accurate results by detecting peripheral blood samples, and the sample amount is extremely small (5 μL), which greatly reduces the trauma to patients compared to cerebrospinal fluid detection, improves patient compliance, and has extremely high clinical application value.

[0123] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. The use of a reagent for detecting exosomes containing microRNA molecules in peripheral blood in the preparation of reagents or kits for the diagnosis and / or screening of Parkinson's disease; wherein, The nucleotide sequence of the microRNA molecule is shown in SEQ ID NO. 9.

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