Molecular marker for diagnosing severity of parkinson's disease and use thereof

Abstract

The application relates to a marker for predicting the severity or stage of Parkinson's disease and the use thereof, and specifically the exosome miRNA is two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421. The molecular marker provided by the application has good diagnostic efficiency when applied to predicting the severity or stage of Parkinson's disease, and the application provides a new way for diagnosing or assisting in diagnosing the severity or stage of Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, more particularly, to the field of RNA technology, and in particular to a molecular marker for diagnosing the severity of Parkinson's disease and its application. BACKGROUND

[0002] Parkinson's disease (PD) is a chronic and progressive neurological disease that usually occurs in the elderly. The disease is mainly characterized by the loss of dopaminergic neurons and motor dysfunction. According to the severity of the disease and the manifestation of symptoms, Parkinson's disease can be divided into five stages, with the severity of symptoms increasing step by step. The diagnosis of Parkinson's disease is usually made by a doctor's clinical evaluation and related examinations, such as neurological examination, brain imaging examination, etc.

[0003] Since the symptoms of PD in the early stage are not typical, and it is easy to be confused with diseases such as multiple system atrophy and progressive supranuclear palsy, the current early diagnosis of PD is still a difficult problem. For PD patients, they often fall into the dilemma of entering the middle and late stages once diagnosed. Many PD patients miss the opportunity of early diagnosis and early treatment. Parkinson's disease has a serious impact on the quality of life and daily life function of patients, so timely diagnosis and treatment are crucial.

[0004] Biomarkers are beneficial for early diagnosis and prediction of disease progression, which will be beneficial for treatment and more accurate disease monitoring, ultimately improving the prognosis of PD patients. Although some progress has been made in the study of PD blood markers, there are still some challenges: (1) low specificity and sensitivity, which makes it difficult to distinguish different stages of the disease; (2) some potential biomarkers, such as alpha-synuclein oligomers, are difficult to detect, requiring more sensitive detection techniques; (3) PD is a heterogeneous disease, and the pathophysiological mechanisms of different patients may differ, which poses a challenge to finding a universal blood marker.

[0005] With the development of molecular biology technology, miRNAs, as a class of small non-coding RNAs, have attracted attention due to their regulatory role in various diseases. Studies have shown that the expression levels of specific miRNAs change in PD patients, which may be closely related to the occurrence and development of the disease. However, there is currently no widely recognized miRNA marker for PD staging prediction. SUMMARY

[0006] The purpose of the present application is to provide a set of molecular markers with high sensitivity and good specificity for diagnosing or assisting in the diagnosis of the severity of Parkinson's disease, a kit and its application.

[0007] In order to solve the above problems, the technical scheme adopted by the present application is as follows:

[0008] In one aspect, the application provides use of a reagent for detecting a molecular marker, wherein the molecular marker is two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, in the preparation of a product for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease.

[0009] Preferably, the molecular marker comprises at least hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0010] Preferably, the molecular marker further comprises one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0011] Preferably, the reagent comprises an miRNA primer and / or a probe, wherein the miRNA primer comprises a reverse transcription primer, an RT-PCR upstream primer and an RT-PCR downstream primer.

[0012] Preferably, the reverse transcription primer sequence of hsa-miR-106b-5p is shown as SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 9;

[0013] the reverse transcription primer sequence of hsa-miR-142-5p is shown as SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 12;

[0014] the reverse transcription primer sequence of hsa-miR-421 is shown as SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 30.

[0015] Further preferably, the reagent further comprises a reagent for detecting the internal reference hsa-miR-423-5p.

[0016] More preferably, the reverse transcription primer sequence of the hsa-miR-423-5p is shown as SEQ ID NO: 31, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 32, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 33.

[0017] Preferably, the molecular marker is derived from an exosome, and the exosome is derived from one or more of blood, saliva, urine, and sputum.

[0018] In another aspect, the present application provides a kit for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease, the kit comprising reagents for detecting the expression level of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p, and hsa-miR-421.

[0019] Preferably, the kit further comprises reagents for detecting the expression level of one or more of the molecular markers hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, and hsa-miR-27b.

[0020] Preferably, the reagents comprise miRNA primers and probes, and the miRNA primers comprise reverse transcription primers, RT-PCR upstream primers, and RT-PCR downstream primers.

[0021] Preferably, the kit further comprises RT-PCR detection reagents and / or standards.

[0022] In yet another aspect, the present application provides a judging device for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease, the device comprising an information acquisition module, a calculation module, and a diagnosis module, wherein,

[0023] The information acquisition module is configured to acquire detection information of a subject, the detection information comprising Ct values of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p, and hsa-miR-421, the Ct values being normalized by a reference miRNA;

[0024] The calculation module is configured to input the Ct values of the molecular markers, the age and gender of the subject into a diagnosis model, and acquire a model value.

[0025] The diagnosis module is configured to judge the severity or stage of Parkinson's disease suffered by the subject according to the model value.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] The application provides application of a reagent for detecting a molecular marker in diagnosis or auxiliary diagnosis of Parkinson's severity or staging, which has high sensitivity and high specificity, and provides a more accurate prediction for PD staging. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is the identification result of the exosomes extracted in the embodiment 1 of the present application, wherein A is an electron microscope image of brain-derived exosomes, and typical tea tray-like vesicle structures can be seen; B is a particle size distribution diagram of brain-derived exosomes, 90% of which are between 30-150 nm, meeting the exosome standard; and C is a small RNA quality inspection result of brain-derived exosomes, and there is a peak value in the range of 15-40 nt.

[0029] Figure 2 It is a sequencing result heat map of part of the differentially expressed miRNAs in the embodiment 2 of the present application.

[0030] Figure 3 It is a ROC curve diagram of a multivariate model constructed according to a random forest algorithm in combination with gender and age factors.

[0031] Figure 4 It is a scatter plot of target relative expression amounts of different queue samples.

[0032] Figure 5 It is a ROC curve diagram of an auxiliary staging diagnosis model provided by the present application. DETAILED DESCRIPTION

[0033] In order to make the technical solutions and beneficial effects of the present application more obvious and easy to understand, the following will be described in detail by means of listing specific embodiments. The drawings are not necessarily drawn to scale, and local features can be enlarged or reduced to more clearly show the details of local features; unless otherwise defined, the technical and scientific terms used herein have the same meaning as the technical and scientific terms in the technical field to which the present application belongs.

[0034] The present inventors have made extensive and in-depth research, and unexpectedly screened out molecular markers closely related to Parkinson staging through high-throughput sequencing and bioinformatics analysis. The expression patterns of the markers in PD patients are significantly different from those in healthy controls. The markers are miRNAs derived from plasma-derived exosomes, including 10 exosome miRNAs and corresponding combinations, wherein the 10 exosome miRNAs are hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421. In particular, when a combination of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421 is selected, it has high sensitivity and high specificity, and a diagnostic model combined with age and gender improves the stability and accuracy of detection. Experiments further show that the markers of the present application can be used to predict the severity or stage of Parkinson's disease. On this basis, the present application is completed.

[0035] As used herein, "a plurality" means any integer. Preferably, "a plurality" in "one or more" can be any integer in a range, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0036] The term "about" as used herein encompasses a range of values ±25% of a given numerical value. In other embodiments, the term "about" encompasses a range of values ±20%, ±15%, ±10%, or ±5% of a given numerical value. For example, in one embodiment, "about 3 grams" means a value of 2.7-3.3 grams (i.e., 3 grams ±10%) or the like.

[0037] As used herein, the term "exosome" refers to a small vesicle with an intact membrane structure, with a diameter of 30-150 nm, which is secreted by a variety of cells and widely exists and distributes in body fluids including blood, saliva, urine, and pleural and peritoneal fluid, and is an important mediator of intercellular information transmission. Exosomes contain a large amount of miRNAs, mRNAs, and proteins secreted by their source cells, and therefore exosomes can be a component of liquid biopsy.

[0038] As used herein, microRNA (miRNA) is a class of endogenous, short chain RNAs of about 20-24 nucleotides in length, which are non-coding RNAs involved in the expression regulation of various target genes at the post-transcriptional stage. Almost all types of cells release miRNAs to the circulatory system passively (e.g. through apoptotic bodies) or actively (e.g. present in exosomes or microvesicles), and these molecules can affect the homeostatic balance of other tissues through mechanisms similar to paracrine signals, or trigger some pathogenic mechanisms, including normal cell transformation into tumor cells and promotion of tumor cell proliferation, etc. A large number of miRNAs play a role after being packaged and transported in exosomes.

[0039] As used herein, "miRNA", "microRNA", "miRNA markers of the present application" and "exosomal miRNA" are used interchangeably to refer to the exosomal miRNAs of the present application for use in the diagnosis or auxiliary diagnosis of the severity of Parkinson's disease.

[0040] It should be noted that "hsa" before miRNA represents the corresponding species, and "hsa" represents human, and "5p" or "3p" after miR is used to distinguish which arm the miRNA is formed from.

[0041] The exosomal miRNAs of the present application include one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421. The expression patterns of these markers in PD patients are significantly different from those in healthy controls, and can be used to determine or predict the stage of Parkinson's disease suffered by the patient.

[0042] The sequences of the 10 markers of the present application are shown in Table 1:

[0043] Table 1

[0044]

[0045]

[0046] In one aspect, the present application provides the use of a reagent for detecting a molecular marker, which is two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, in the preparation of a product for the diagnosis or auxiliary diagnosis of the severity or stage of Parkinson's disease.

[0047] In some embodiments, the molecular markers comprise at least hsa-miR-106b-5p, hsa-miR-142-5p, and hsa-miR-421.

[0048] In some embodiments, the molecular markers further comprise one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, and hsa-miR-27b.

[0049] In some embodiments, the reagents comprise miRNA primers and / or probes, the miRNA primers comprise reverse transcription primers, RT-PCR upstream primers, and RT-PCR downstream primers.

[0050] As used herein, "primer" refers to a nucleic acid molecule with a specific nucleotide sequence that directs synthesis at the initiation of nucleotide polymerization. Primers are usually two oligonucleotide sequences synthesized artificially, one primer is complementary to one DNA template strand at one end of the target region, and the other primer is complementary to the other DNA template strand at the other end of the target region, and its function is to serve as the starting point of nucleotide polymerization. In vitro artificially designed primers are widely used in polymerase chain reaction (PCR), qPCR, sequencing, and probe synthesis, etc. Generally, primers are designed to be 50-150 bp, 60-140 bp, 70-130 bp, 80-120 bp in length of the amplified product. The design method of PCR primers for templates is known in the art. Preferably, the product length is 50-100 bp.

[0051] As used herein, "probe" mainly refers to a DNA or RNA sequence that hybridizes to a target sequence under stringent conditions. Usually, the 5' end of the probe sequence is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. When discussing primers or probes, "recognition" or "hybridization" as described herein refers to the hybridization of primers or probes to template sequences under stringent or highly stringent conditions, which are well known in the art, for example, the highly stringent conditions can be hybridization at 65°C in a solution of 0.1x SSPE (or 0.1x SSC), 0.1% SDS and washing the membrane.

[0052] In some embodiments, the reverse transcription primer sequence of hsa-miR-106b-5p is shown in SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 9;

[0053] The reverse transcription primer sequence of the hsa-miR-142-5p is shown as SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 12;

[0054] The reverse transcription primer sequence of the hsa-miR-421 is shown as SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 30.

[0055] Further preferably, the reagent further comprises a reagent for detecting the internal reference hsa-miR-423-5p.

[0056] More preferably, the reverse transcription primer sequence of the hsa-miR-423-5p is shown as SEQ ID NO: 31, the RT-PCR upstream primer sequence is shown as SEQ ID NO: 32, the RT-PCR downstream primer sequence is shown as SEQ ID NO: 34, and the probe sequence is shown as SEQ ID NO: 33.

[0057] The reverse transcription primer, the RT-PCR upstream primer and the RT-PCR downstream primer involved in the present application are shown in Table 2:

[0058] Table 2

[0059]

[0060]

[0061] In the present application, the reverse universal primer of the markers hsa-let-7g-5p and hsa-miR-26b-5p is the universal primer-R2, shown as SEQ ID NO: 35; and the reverse universal primer of other markers is the universal primer-R1, shown as SEQ ID NO: 34.

[0062] In the present application, the specific probe in the present application is a fluorescent probe, and the fluorescent group is a conventional fluorescent group in the art. The present application does not limit the fluorescent group, as long as it can achieve real-time fluorescent quantitative detection of RT-PCR.

[0063] In some embodiments, the molecular marker is derived from an exosome, and the exosome is derived from one or more of blood, saliva, urine and sputum.

[0064] In another aspect, the present application provides a kit for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease, the kit comprising reagents for detecting the expression level of two or more of the molecular markers hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421.

[0065] In certain embodiments, the kit further comprises reagents for detecting the expression level of one or more of the molecular markers hsa-let-7b, hsa-let-7g-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p and hsa-miR-27b.

[0066] In certain embodiments, the reagents comprise miRNA primers and probes, the miRNA primers comprising reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers.

[0067] In certain embodiments, the kit further comprises RT-PCR detection reagents and / or standards.

[0068] The present application also provides a method for detecting exosome-derived miRNA markers for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease, the method comprising reverse transcription and real-time fluorescent quantitative polymerase chain reaction (RT-PCR).

[0069] In addition, for the detection method of the above-mentioned miRNAs, methods other than the RT-PCR method provided by the present application, such as sequencing, microarray, Northern blotting, bioluminescence and probe methods, etc., can also be used to detect the molecular markers.

[0070] In another aspect, the present application provides a judging device for diagnosing or aiding in the diagnosis of the severity or stage of Parkinson's disease, the device comprising an information acquisition module, a calculation module and a diagnosis module, wherein,

[0071] The information acquisition module is configured to perform the operation of acquiring subject detection information, the detection information comprising the Ct value of two or more of hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421, the Ct value being normalized by a reference miRNA;

[0072] The calculation module is configured to perform the operation of inputting the Ct value of the molecular marker, the age and gender of the subject into a diagnosis model to obtain a model value;

[0073] The diagnosis module is configured to perform the operation of judging the severity or stage of Parkinson's disease suffered by the subject according to the model value.

[0074] In the present application, for the detection sample, when the score is less than the threshold value, then the result is negative, which represents that the patient is an early-stage PD patient, showing H+Y staging as 1 / 2, and vice versa, which is positive, representing that the patient is a middle / late-stage PD patient, showing H+Y staging as 3 / 4 / 5.

[0075] When the exosome miRNA is selected from the above-mentioned 3 miRNAs, the detection model is Y = 0.163 ╳ Age -0.867 ╳ Gender +1.785 ╳ hsa-miR-106b-5p -1.596 ╳ hsa-miR-142-5p -1.462 ╳ hsa-miR-421 -6.007, and the threshold value is 0.353.

[0076] In the present application, the level or concentration of the exosome miRNA in the sample is represented by the Ct value, and the patient gender is converted into a numerical value, with male being assigned a value of 1 and female being assigned a value of -1.

[0077] As used in the present application, "Ct value" or "Cycle Threshold" refers to the number of amplification cycles corresponding to the fluorescence signal of the amplification product reaching the set fluorescence threshold value in quantitative PCR. Simply put, the Ct value represents the number of cycles when the starting template amplification reaches a certain product amount. The higher the concentration of the starting template amount, the smaller the Ct value; the lower the concentration of the starting template amount, the larger the Ct value.

[0078] The specific operation of screening the key differential miRNAs for preventing PD staging in the present application is as follows:

[0079] (1) Brain-derived exosome extraction: using magnetic beads coated with ATP1A3 antibody to specifically capture brain-derived exosomes in plasma. Enriching brain-derived exosomes can better detect information of brain neuron lesions, reduce interference of exosome background signals from other sources, and improve the accuracy of PD diagnosis. (2) Exosome nucleic acid extraction: using an miRNA extraction kit to extract small nucleic acids of brain-derived exosomes.

[0080] (3) Library construction sequencing: using QiaSeq miRNA library construction kit to construct small RNA library, and Ion Torrent 540 chip for library sequencing. Data mapping to HG19 and miRBase v21 database, each read data Reads is normalized to RPM for data analysis, and the differentially expressed miRNA targets of the queue samples are screened.

[0081] (4) Reverse transcription and real-time fluorescent quantitative PCR: The extracted miRNA is reverse transcribed into cDNA, and then the content of the target miRNA is detected by qPCR to verify the diagnostic performance of the target selected by sequencing.

[0082] (5) Diagnosis model establishment: According to the data of the verification set, a diagnosis model is created in combination with factors such as age and gender, algorithm transformation is performed, and finally an evaluation score is calculated to determine the PD staging condition according to the score.

[0083] As used herein, the term "sample" or "specimen" refers to material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The specimen can consist entirely or in part of biological material from the subject.

[0084] The application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the application and not to limit the scope of the application. The experimental methods in the following examples are not specified, and the methods are generally carried out under conventional conditions, for example, the conditions described in Sam brook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0085] Example 1 Extraction of exosome miRNA

[0086] Clinical cohort sample collection:

[0087] The discovery set cohort is: from Guangzhou Zhongshan No. 1 Hospital, a total of 274 cases (including 89 cases of healthy controls, 26 cases of H&Y stage 1, 59 cases of H&Y stage 2, 63 cases of H&Y stage 3, and 37 cases of H&Y stage 4);

[0088] The verification set cohort is: from Guangzhou Zhongshan No. 1 Hospital, 74 cases (including 22 cases of healthy controls, 11 cases of H&Y stage 1, 22 cases of H&Y stage 2, 6 cases of H&Y stage 3, and 13 cases of H&Y stage 4); from Suzhou Soochow University Second Affiliated Hospital, 140 cases (including 74 cases of healthy controls, 20 cases of H&Y stage 1 / 1.5, 33 cases of H&Y stage 2 / 2.5, 12 cases of H&Y stage 3, and 1 case of H&Y stage 5).

[0089] Inclusion criteria:

[0090] 1. Age > 50 years old;

[0091] 2. Can speak Mandarin (or can communicate effectively with the doctor in dialect);

[0092] 3. The subject and his / her family members sign the informed consent form;

[0093] 4. MMSE ≥ 25;

[0094] 5. Diagnosed as Parkinson's disease according to the Parkinson's Disease Diagnostic Criteria in China;

[0095] 6. Belongs to 0-4 grade according to HOEHN & Yahr classification.

[0096] Exclusion criteria:

[0097] (B) Exclusion criteria for Parkinson's disease group

[0098] 1. Hearing impairment, visual impairment, poor cooperation;

[0099] 2. Has a lifetime history of schizophrenia, schizoaffective disorder or bipolar disorder;

[0100] 3. Subjects currently taking antidepressants: subjects currently suffering from depression or having a history of severe depression in the past two years;

[0101] 4. Has received electroconvulsive therapy in the past;

[0102] 5. Has a history of alcohol abuse or alcohol dependence after two years of screening;

[0103] 6. History of long-term heavy drinking (alcohol content greater than 42 degrees of baijiu, greater than 3 liang / day, drinking more than 12 months);

[0104] 7. History of unstable malignant tumor (including nervous system tumor and tumor outside the nervous system) within one year;

[0105] 8. Any serious systemic disease or unstable condition that makes it difficult to comply with the protocol, including: history of severe lung disease (COPD, pulmonary encephalopathy); history of severe cardiovascular disease (heart failure, severe hypertension);

[0106] 9. Subjects with the following clinically significant signs or history: history of severe nervous system infection of any cause, history of multiple sclerosis, history of immune encephalitis, history of Hashimoto's encephalopathy, etc.; current unstable seizures of any cause; genetic diseases affecting cognitive function (such as Huntington's disease, Down syndrome, CADASIL syndrome, adrenoleukodystrophy, mitochondrial encephalopathy, etc.); infection and immune-related diseases affecting the central nervous system (systemic lupus erythematosus, insufficiently treated HIV infection or history of CNS spirochete infection, etc.); metabolic diseases and endocrine disorders affecting the central nervous system (thyroid function abnormalities requiring new treatment or adjustment of current treatment, folate or vitamin B12 deficiency); current unstable seizures of any cause;

[0107] 10. Subjects with the following diseases or clinical symptoms: atypical features including Parkinsonism (progressive supranuclear palsy (PSP), multiple system atrophy (MSA), corticobasal degeneration (CBD)), including cerebellar symptoms, supranuclear gaze palsy, apraxia and other cortical symptoms, or prominent autonomic failure;

[0108] 11. History of clinically significant and unstable gastrointestinal disease within the last two years, including ulcer, severe or occult gastrointestinal bleeding;

[0109] 12. Systemic illness affecting the function of the nervous system, abnormal liver and kidney function related exclusion criteria (undergoing abdominal dialysis or hemodialysis during the screening period; AST or ALT > 3 x upper limit of normal (ULN) or total bilirubin > 2 x ULN during the screening period)

[0110] 13. Uncontrolled diabetes mellitus;

[0111] 14. History of unstable malignancy (including tumors of the nervous system and outside the nervous system) within 1 year;

[0112] 15. Exclusion of drugs required for participation in the study, including:

[0113] 1) Use of narcotic drugs, methyldopa and clonidine within 4 weeks prior to screening;

[0114] 2) Use of short-term anxiolytic or sedative-hypnotic drugs more than 2 times per week within 4 weeks prior to screening (Note: sedative drugs should not be used within 72 hours after screening);

[0115] 3) Use of systemic corticosteroids within 3 months prior to screening;

[0116] 4) Use of anticonvulsant drugs (such as phenytoin, phenobarbital, carbamazepine) within 2 months prior to screening;

[0117] 5) Use of warfarin (coumarin) within 4 weeks prior to screening;

[0118] 6) Use of phosphodiesterase inhibitors within 4 weeks prior to screening.

[0119] Extraction of brain-derived exosomal miRNA

[0120] The method for making the immunocapture microspheres in this example is: using Dynabeads TM ATP1A3 antibody (Thermo, MA3-915) was coupled to Dynabeads M-270 epoxy resin microspheres using the antibody coupling kit (Thermo, 14311D) according to the manufacturer's instructions, with a coupling amount of 5 μg antibody / mg microspheres.

[0121] Collect 1 mL of clinical plasma samples from the cohort, centrifuge at 3000 g for 10 min, and centrifuge at 10000 g for 20 min to remove impurities. Take the supernatant and use the Exoquick kit (SBI Company, EXOQ5TM-1) to extract total exosomes according to the manufacturer's instructions. A total of 500 uL of total exosomes is obtained.

[0122] Specific enrichment of exosomes from brain neurons in total exosomes using immunocapture microspheres

[0123] Add 1 mg of microspheres to the above 500 uL of total exosomes and rotate overnight at 4°C. Use a magnetic stand for solid-liquid separation, remove the supernatant, and wash the microspheres with 500 uL of PBST (0.01 M phosphate buffer containing 0.05% Tween 20) three times. Magnetically separate and remove the washing solution, resuspend the microspheres with 200 uL of PBS, and enrich the brain-derived exosomes on the microspheres. Use the miRcute serum and plasma miRNA extraction and separation kit (Tiangen Biochemical, DP503) to extract the brain-derived exosome miRNA according to the instructions. Perform electron microscopy and particle size detection on the enriched brain-derived exosomes as follows:

[0124] Electron microscopy: 1. Take the exosome sample from the -80°C freezer and place it in an ice box. After dissolving, centrifuge slightly, and use a pipette to take 15 uL of the exosome sample on a copper mesh and stand for 1 min.

[0125] 2. Use filter paper to absorb the exosome sample on the copper mesh, then use a pipette to take 15 uL of 2% uranyl acetate staining solution and stain at room temperature for 1 min.

[0126] 3. Use filter paper to absorb the exosome sample on the copper mesh, place the stained sample under a lamp for 10 min, observe and take pictures, and save the pictures.

[0127] The electron microscopy results are shown in Part A of Figure 1 Under the microscope, the tea tray-like vesicle structure is visible, and the size is within the standard range of exosomes.

[0128] Use a particle size analyzer (Spectradyne Company, model nCS1) to measure particle size and particle concentration. Use the microfluidic resistance pulse induction (MRPS) method as follows:

[0129] Turn on and load the cleaning cartridge, run the instrument prime program.

[0130] Take an appropriate amount of EVs, dilute with PBST, and take 4 uL into the cartridge well.

[0131] Load the cartridge into the instrument and run the particle size detection program.

[0132] After completion, replace the cleaning cartridge and clean the instrument lines with purified water.

[0133] The particle size distribution chart is shown in Part B of FIG. 13, wherein 90% of the particles (D90) are less than 135 nm in size, and the particle size distribution meets the exosome standard range. Figure 1 The concentration of the extracted miRNA was determined using a Qubit microRNA assay kit (Thermo, Cat. No. Q32881) in combination with a Qubit 4 fluorometer, and the extracted miRNA was quality controlled using a small RNA detection chip in combination with a 2100 Bioanalyzer (Agilent). The detection chart is shown in Part C of FIG. 12.

[0134] Figure 1

[0135] Example 2: miRNA NGS sequencing of discovery cohort samples and screening of differential miRNA targets

[0136] The library was constructed using a small RNA library construction kit (Qiaseq miRNA library kit, KANGJIE), and sequencing was performed on an Ion Torrent (Thermo) platform using 540 Chips. The data was mapped to the HG19 and miRBase v21 databases, and each read data was normalized to RPM for data analysis. The miRNAs with high expression in the cohort samples and significant differences between normal people and PD patients were screened.

[0137] The markers screened in this example are hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b, and hsa-miR-421. The miRNA heat map of the higher expression is shown in FIG. 14, which shows that they have differential expression in different stages of PD. Figure 2 A multivariate model was constructed according to age, gender, and the normalized value RPM of sequencing using a random forest algorithm, the diagnostic performance of the candidate targets was viewed, and support was provided for further screening of targets for subsequent qPCR verification. The ROC curve chart is shown in FIG. 15.

[0138] Figure 3 The area under the curve AUC = 0.873, the sensitivity is 93.51%, the specificity is 54.99%, and the accuracy is 81.02%.

[0139] ​​​Example 3 Validation of molecular markers

[0140] The validation cohort samples were collected and brain-derived exosomal miRNA was extracted according to the method in Example 2.

[0141] TaqMan reverse transcription kit TM The miRNA was reverse transcribed into cDNA using the MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific, 4366597). 15 μL of reverse transcription system was used. The amounts of each component were shown in Table 3. The reverse transcription primers (see Table 2) were included in the TaqMan TM Assays (Thermo Fisher, 4440886) reagents, the reaction conditions were: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and maintained at 4°C.

[0142] Table 3

[0143] Reverse transcription system Volume (μL) dNTPs and dTTP (100 mM) 0.3 Reverse transcriptase 3 10X reverse transcription buffer 1.5 RNase inhibitor (20 U / μL) 0.19 RNase-free water 1.01 Reverse transcription primer pool 6 Sample miRNA 3 Total volume 15

[0144] Using TaqMan preamplification reagents TM The reverse transcription products obtained above were pre-amplified using PreAmp premix (Thermo Fisher Scientific, 4488593). The reaction system was 25 μL. The components and amounts were shown in Table 4. The primers (see Table 2) were included in Assay (Thermo Fisher Scientific, 4440886) reagents, reaction conditions were: 95°C for 10 min, 55°C for 2 min, 72°C for 2 min, 12 cycles of (95°C for 15 s, 60°C for 4 min), 99.9°C for 10 min, and 4°C hold. After the reaction, 175 μL of 0.1× TE, pH 8.0, was added to each reaction well for dilution, to a final volume of 200 μL.

[0145] Table 4

[0146]

[0147] use Fast Advanced premix (2×) (Thermo Fisher, 4444557) was used for RT-qPCR detection of the Ct values ​​of exosomal miRNA markers. A 10 μL system was used. The amounts of each component were shown in Table 5. The primers and probes (see Table 2) were included in Assay (Thermo Fisher, 4440886) reagent, the reaction conditions were: 50°C for 2 min, 95°C for 20 s, (95°C for 1 s, 60°C for 20 s) for 40 cycles.

[0148] Table 5

[0149]

[0150] The NGS sequencing results show that hsa-miR-423-5p is stably expressed in each sample as an internal reference. According to the Ct value after homogenization, the relative expression amounts of hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421 are obtained. The relative expression amounts are grouped according to different queue population normal group, early PD (H&Y stage 1 and 2), and middle and late PD (H&Y stage 3 and above), and the relative expression amount point graph is shown in Figure 4 It can be seen that in the verification of the multi-center queue, hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421 have more obvious expression differences in early and late PD, and the target shows different degrees of differentiation in different stages. Figure 4

[0151] Example 4: Establishment of PD stage diagnosis model

[0152] According to the ΔCt value of the target and the internal reference measured by RT-PCR, a staging model is established, and a binary logistic regression is used to train one or more of hsa-let-7b, hsa-let-7g-5p, hsa-miR-106b-5p, hsa-miR-142-5p, hsa-miR-191-5p, hsa-miR-22-5p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27b and hsa-miR-421 in combination with age and gender to obtain a logistic regression equation (judgment formula). The diagnostic performance of each target is shown in Table 6.

[0153] Table 6

[0154]

[0155]

[0156] Several targets with higher AUC are combined to obtain a fitting function: Y = 0.163 ╳ Age -0.867 ╳ Gender +1.785 ╳ hsa-miR-106b-5p -1.596 ╳ hsa-miR-142-5p -1.462 ╳ hsa-miR-421 -6.007, and the ROC curve is shown in Figure 5 ​, AUC=0.852, wherein the gender of the patient is converted into a numerical value, where male is assigned a value of 1 and female is assigned a value of -1, and each marker in the fitting function is represented as the normalized ΔCt value of each marker. When the score is less than the threshold value, the result is determined to be negative, which represents that the patient is an early stage PD patient, showing H+Y stage 1 / 2, and otherwise, the result is determined to be positive, which represents that the patient is a middle or late stage PD patient, showing H+Y stage 3 / 4 / 5. When the cutoff value is taken as 0.353, the sensitivity is 81.8% and the specificity is 76.3%.

[0157] According to the enrollment criteria in Example 1, 216 samples were collected as a test set, and the model was verified by the above detection method. The results are shown in Table 7.

[0158] Table 7

[0159]

[0160] It should be understood that the above examples are exemplary and are not intended to include all possible embodiments encompassed by the claims. Various modifications and changes can also be made on the basis of the above examples without departing from the scope of the present disclosure. Similarly, any combination of the technical features of the above examples can also be made to form additional embodiments of the present application that can not have been explicitly described. Therefore, the above examples only express several embodiments of the present application and do not limit the protection scope of the present application.

Claims

1. Use of a reagent for detecting the expression level of a molecular marker in the preparation of a product for assisting in the diagnosis of Parkinson's disease stages, characterized in that: The molecular markers are hsa-miR-106b-5p, hsa-miR-142-5p and hsa-miR-421; the staging is to distinguish early-stage patients from middle and late-stage patients, with early-stage patients showing H+Y stage 1 or 2, and middle and late-stage patients showing H+Y stage 3, 4 or 5.

2. The use according to claim 1, characterized in that The reagents include miRNA primers and / or probes, and the miRNA primers include reverse transcription primers, RT-PCR upstream primers and RT-PCR downstream primers.

3. The use according to claim 2, characterized in that The reverse transcription primer sequence of hsa-miR-106b-5p is shown in SEQ ID NO: 7, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 8, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 9; The reverse transcription primer sequence of hsa-miR-142-5p is shown in SEQ ID NO: 10, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 11, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO: 12; The reverse transcription primer sequence of the hsa-miR-421 is shown in SEQ ID NO: 28, the RT-PCR upstream primer sequence is shown in SEQ ID NO: 29, the RT-PCR downstream primer sequence is shown in SEQ ID NO: 34, and the probe sequence is shown in SEQ ID NO:

30.

4. The use according to claim 1, characterized in that The molecular markers are derived from exosomes, and the exosomes are derived from blood.

Images