WO2024098369A1 - 一种基于dna六面体的帕金森病体外诊断试剂盒及其应用 - Google Patents

一种基于dna六面体的帕金森病体外诊断试剂盒及其应用 Download PDF

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WO2024098369A1
WO2024098369A1 PCT/CN2022/131321 CN2022131321W WO2024098369A1 WO 2024098369 A1 WO2024098369 A1 WO 2024098369A1 CN 2022131321 W CN2022131321 W CN 2022131321W WO 2024098369 A1 WO2024098369 A1 WO 2024098369A1
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seq
sample
disease
parkinson
nucleic acid
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PCT/CN2022/131321
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English (en)
French (fr)
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于震维
郑元初
冯涛
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北京市神经外科研究所
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Publication of WO2024098369A1 publication Critical patent/WO2024098369A1/zh

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  • the present application belongs to the field of biological preparations, and specifically relates to a DNA hexahedron-based in vitro diagnostic kit for Parkinson's disease and its application.
  • the diagnosis and differential diagnosis of neurodegenerative diseases such as Parkinson's disease mainly rely on the patient's clinical manifestations and imaging indicators, requiring clinicians to have rich experience, resulting in a high misdiagnosis rate.
  • a large number of neurons have already died irreversibly, resulting in limited treatment effects.
  • fluid biomarkers with high sensitivity and specificity are mainly based on cerebrospinal fluid detection, but the collection of cerebrospinal fluid is more traumatic to patients and patient compliance is poor. Therefore, there is an urgent need to develop high-accuracy diagnostic biomarkers based on blood (plasma, serum, red blood cells, etc.).
  • microRNA microRNA or miRNA
  • An article published in 2022 introduced an experimental method for detecting exosomal micro RNA based on DNA hexahedron molecular beacons (Dongsheng et al., Biosensors and Bioelectronics, 2022 11 4077). This suggests that we can use DNA hexahedron molecular beacons to detect exosomes carrying certain specific miRNAs in the blood to diagnose neurodegenerative diseases such as Parkinson's disease and develop biomarkers.
  • microRNA the expression level of which in the peripheral blood exosomes of Parkinson's disease patients is much higher than that in non-Parkinson's disease patients. Therefore, it can be used as a biomarker for diagnosing and/or screening Parkinson's disease, and this application was completed based on this.
  • the first aspect of the present application provides an isolated microRNA molecule having a nucleotide sequence shown in SEQ ID NO.9, or a nucleotide sequence thereof having at least 90%, at least 95%, or at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO.9.
  • 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 reagent or kit for diagnosing and/or screening Parkinson's disease.
  • the third aspect of the present application provides a composition for detecting the microRNA molecules of the first aspect of the present application, which comprises nucleic acid molecules shown in SEQ ID NO.1 to SEQ ID NO.8.
  • the fourth aspect of the present application provides a Parkinson's disease diagnosis and/or screening kit, which comprises a reagent for detecting the microRNA molecules of the first aspect of the present application.
  • the present application provides use of the microRNA molecules and/or exosomes containing the microRNA molecules according to the first aspect of the present application as biomarkers for diagnosis and/or screening of Parkinson's disease.
  • the present application provides a method for in vitro detection of the microRNA molecule or the exosome containing the microRNA molecule according to the first aspect of the present application, comprising:
  • nucleic acid molecules shown in SEQ ID NO.1 to SEQ ID NO.6 and SEQ ID NO.8 are connected to a quenching group; preferably, the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB or TAMARA;
  • step 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 hexahedron, wherein the 3' end of the nucleic acid molecule shown in SEQ ID NO.7 is connected to 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;
  • the sample to be tested is a peripheral blood sample of a subject; preferably, the sample to be tested is peripheral blood plasma;
  • the microRNA molecules in the sample to be tested by detecting the fluorescence signal; preferably, the determination is a quantitative determination.
  • the seventh aspect of the present application provides a method for diagnosing Parkinson's disease, which includes using the method of the sixth aspect of the present application to detect the content of the microRNA molecules or exosomes containing the microRNA molecules in the subject's body fluid sample, and judging whether the subject suffers from Parkinson's disease based on the content of the microRNA molecules or exosomes containing the microRNA molecules.
  • the microRNA molecule miR26690 provided in the present application is highly expressed in the exosomes in the peripheral blood of Parkinson's disease patients, and can therefore 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 in the present application can detect miR26690 in peripheral blood with high sensitivity and high specificity, and its use in diagnosing Parkinson's disease is simple, easy to prepare, requires a small amount of sample for detection, has higher sensitivity, a low detection limit, and low detection cost compared to traditional Parkinson's disease marker detection methods.
  • FIG1 is a schematic diagram showing the principle of the detection method of the present application.
  • FIG. 2 is a schematic diagram of the results of detecting miR26690 positive exosomes in plasma using the nanoflow detection system of Example 1.
  • Figure 3 shows the detection results of plasma miR26690-positive exosome concentrations in Parkinson's disease patients and healthy controls.
  • Figure 4 shows the results of plasma miR26690-positive exosome concentration detection in early Parkinson's disease patients and healthy controls.
  • Figure 5 is the ROC curve of DNA hexahedron detecting the concentration of plasma miR26690-positive exosomes as a diagnostic biomarker for Parkinson's disease.
  • the terms “about,” “substantially,” and “similar to” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which error range may depend in part on the manner in which the value is measured or determined, or on the limitations of the measurement system.
  • the term “comprising/including/containing” means including the described whole or group of wholes, but not excluding any other wholes or groups of wholes.
  • the term “consisting essentially of” means including the described whole or group of wholes, while excluding other wholes or variations that may substantially affect or change the described whole.
  • the term “consisting of” means including the described whole or group of wholes, and excluding any other wholes or groups of wholes.
  • MicroRNA is a class of non-coding single-stranded RNA molecules with a length of about 20 nucleotides encoded by endogenous genes, which are involved in post-transcriptional gene expression regulation in animals and plants.
  • microRNA microRNA
  • microRNA microRNA
  • miRNA is a class of non-coding single-stranded RNA molecules with a length of about 20 nucleotides encoded by endogenous genes, which are involved in post-transcriptional gene expression regulation in animals and plants.
  • isolated microRNA molecule essentially refers to the separation of the microRNA molecule from its natural environment in this article.
  • the microRNA molecule preparation is substantially free of other substances associated with its natural environment.
  • the isolated microRNA molecule by weight, 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 associated with its natural environment.
  • Parkinson's disease also named idiopathic Parkinson's syndrome (IPS) or Morbus Parkinson
  • PD Parkinson's disease
  • IPS idiopathic Parkinson's syndrome
  • Morbus Parkinson refers to a condition called a motor system disorder, which is the result of a loss of brain cells that produce dopamine.
  • the main symptoms of PD are tremors or shaking of the hands, arms, legs, jaw, and face; rigidity or stiffness of the limbs and trunk; bradykinesia or slow movement; and postural instability or impaired balance and coordination. As these symptoms become more pronounced, patients may have difficulty walking, talking, or completing other simple tasks.
  • PD usually affects people over 50 years old. The early symptoms of PD are subtle and occur gradually. In some people, the disease progresses faster than others.
  • PD Parkinson's disease
  • determining the course of a subject with PD refers to determining the progression of PD over time, such as whether PD worsens in the subject, does not worsen/stable in the subject, or improves in the subject over time.
  • diagnosis refers to the process of determining a possible disease or disorder, and is therefore a process that attempts to define the (clinical) condition of an individual.
  • the determination of the level of miRNA according to the present application is related to the (clinical) condition of an individual.
  • diagnosis includes/encompasses: (i) determining the occurrence/presence of PD; (ii) monitoring the course of PD; (iii) staging of PD; (iv) measuring the response of individuals with PD to therapeutic interventions; and/or (v) segmentation of individuals with PD.
  • the term "individual” refers to any subject for whom it is desired to know whether she or he suffers from/has PD.
  • the term "subject" refers to a subject suspected of being or affected by PD.
  • the subject may be diagnosed as being affected by PD, i.e., being ill; or may be diagnosed as not being affected by PD, i.e., being healthy with respect to these diseases.
  • the term "subject” also refers to a subject affected by (i.e., ill with) PD.
  • the subject may be retested for PD and may be diagnosed as still affected by, i.e., ill with, PD; or no longer (less affected by) PD, i.e., healthy with respect to PD (e.g., after therapeutic intervention).
  • the subject may be further retested for PD and may be diagnosed as having developed an advanced or severe form of PD.
  • the subject may be any mammal, including a human and another mammal, for example, an animal such as a rabbit, mouse, rat or monkey.
  • a human subject is particularly preferred as the subject.
  • the term "subject” may refer to a subject known to be unaffected by PD (negative control) (such subjects are also referred to as non-Parkinson's disease patients), i.e., healthy with respect to PD.
  • the term “subject” may also refer to a subject known to be affected by PD (i.e., ill). The subject may have developed an advanced form of PD.
  • Non-Parkinson's disease patients who are known not to have PD (i.e., healthy with respect to PD) may also have another untested/unknown disease.
  • the subject may be any mammal, including a human and another mammal, for example, an animal such as a rabbit, a mouse, a rat or a monkey.
  • a human subject is particularly preferred as an individual.
  • the term "treatment” refers to any therapy that improves the health status of an individual and/or prolongs (increases) the life span of an individual.
  • the therapy can eliminate the disease of the individual, prevent or slow the development of the disease of the individual, inhibit or slow the development of the disease of the individual, reduce the frequency or severity of the symptoms of the individual, and/or reduce the recurrence of the individual who currently or previously had the disease.
  • the disease can be PD. (Therapeutic) treatment of PD includes, but is not limited to, drug administration, speech therapy, exercise training, psychological training and/or physical rehabilitation.
  • 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 (ear wax) sample, a gastric juice sample, a mucus sample, an endolymph sample, a perilymph 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 vomitus sample, including their components (components) or components (fractions).
  • CSF cerebrospinal fluid
  • cerumen ear wax
  • gastric juice sample a mucus sample
  • an endolymph sample a perilymph sample
  • a peritoneal fluid sample a pleural fluid sample
  • saliva sample a sebum (skin oil) sample
  • body fluid sample also encompasses body fluid components, such as blood components, urine components or sputum components.
  • Body fluid samples can be mixed or merged. Therefore, a body fluid sample can be a mixture of blood and urine samples, or a mixture of blood and cerebrospinal fluid samples.
  • the body fluid sample can be provided by taking out body fluid from an individual or a subject, but can also be provided by using previously separated body fluid sample materials. Body fluid samples enable non-invasive analysis to be performed on an 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.
  • peripheral blood means blood that circulates or has circulated systemically in a mammal and is not obtained from blood sequestered from, for example, the bone marrow (BM), lymphocytes (eg, lymph nodes), spleen, or liver.
  • blood sample encompasses a whole blood sample or a blood component sample, such as a blood cell/cellular component, serum or plasma sample.
  • plasma is as conventionally defined.
  • human plasma may be, or the source of human plasma may be fresh plasma, freeze-dried 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 mixture of two or more thereof.
  • Plasma is usually obtained from a sample obtained by apheresis or from a whole blood sample, which is provided with or in contact with an anticoagulant (e.g., heparin, citric acid, oxalic acid or EDTA). Subsequently, the cellular components of the blood sample are separated from the liquid component (plasma) by appropriate techniques, usually by centrifugation.
  • an anticoagulant e.g., heparin, citric acid, oxalic acid or EDTA
  • the blood sample in order to obtain plasma suitable for use in the present application, can 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).
  • a vacuum blood collection tube e.g., BD Vacutainer plastic EDTA tube, 10 ml, 1.8 mg/mL
  • the sample is gently shaken and then centrifuged at room temperature for 10 minutes at 1,000-2,000 g to separate plasma from red blood cells.
  • the supernatant (plasma) is collected, optionally pooled (if multiple blood samples are used) and aliquoted into cryovials, which are stored at -80°C until use.
  • plasma refers to a composition of the cell-free components of a blood sample that does not form part of the human or animal body.
  • plasma as contemplated herein is cell-free plasma, e.g., plasma comprising less than about 1.0% w/w, preferably less than about 0.5% w/w or less than 0.1% w/w of whole cellular material, or substantially no whole cellular material.
  • the plasma may preferably be unprocessed plasma, i.e. plasma obtained by separation from whole blood and not subjected to subsequent processing steps that alter its chemical, biochemical or cellular composition, other than optional heat inactivation, "storage” (cryogenic or non-cryogenic), sterilization, filtration, freeze drying and/or solvent/detergent treatment.
  • unprocessed plasma i.e. plasma obtained by separation from whole blood and not subjected to subsequent processing steps that alter its chemical, biochemical or cellular composition, other than optional heat inactivation, "storage” (cryogenic or non-cryogenic), sterilization, filtration, freeze drying and/or solvent/detergent treatment.
  • the term "plasma” may specifically exclude processed plasma, i.e. plasma that has been subjected to one or more processing steps that alter its composition, in particular its chemical, biochemical or cellular composition, after its separation from whole blood.
  • the term "plasma” as contemplated herein specifically excludes platelet-rich plasma (PRP), i.e. plasma that has been enriched with platelets.
  • PRP platelet-rich plasma
  • PRP may contain about 1.0 ⁇ 10 6 platelets/ ⁇ l, although the platelet concentration in whole blood may be about 1.5 ⁇ 10 5 to 3.5 ⁇ 10 5 / ⁇ l.
  • plasma as contemplated herein may contain less than about 8.0 ⁇ 10 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 ⁇ 10 5 , such as less than about 4.0 ⁇ 10 5 platelets/ ⁇ l.
  • Plasma as contemplated herein is human plasma, ie, obtained from a single human subject or from multiple human subjects (eg, a mixture of plasma).
  • Plasma can be used directly in the methods of the present application. Plasma can also be appropriately stored for subsequent use (e.g., for a shorter period of time, e.g., up to about 1-2 weeks, at a temperature above the freezing point of plasma but below ambient temperature, which will typically be about 4°C to 5°C; or for a longer period of time, typically about -70°C to about -80°C, by freezing).
  • a shorter period of time e.g., up to about 1-2 weeks, at a temperature above the freezing point of plasma but below ambient temperature, which will typically be about 4°C to 5°C; or for a longer period of time, typically about -70°C to about -80°C, by freezing.
  • the methods of the present application can use plasma that is autologous to the subject to be diagnosed or screened, and thus the plasma is autologous to the cells stored in the composition comprising the plasma.
  • autologous with respect to plasma means that the plasma is obtained from the same subject that is contacted with the plasma or diagnosed or screened with the plasma.
  • plasma may be heat inactivated, specifically to remove complement.
  • heat inactivation of the plasma may not be necessary.
  • level refers to the amount (eg, measured in grams, moles, or counts such as ion or fluorescence counts) or concentration (eg, absolute or relative) of a miRNA described 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.
  • the level determined herein is an expression level.
  • Sensitivity refers to the number of true positive patients (%) relative to the total number of patients (100%).
  • the individual may be a subject with PD.
  • the term "accuracy” refers to a statistical measure of the correctness of the classification or identification of a sample type. Accuracy is the proportion of true results (both true positives and true negatives).
  • exosome refers to a tiny membrane vesicle with a diameter of about 30-150 nm, which is secreted by a variety of cells and contains specific proteins (for example, the exosome membrane is rich in transmembrane protein families CD63, CD81 and CD9 involved in exosome transport), lipids, cytokines or genetic material.
  • specific proteins for example, the exosome membrane is rich in transmembrane protein families CD63, CD81 and CD9 involved in exosome transport
  • lipids for example, the exosome membrane is rich in transmembrane protein families CD63, CD81 and CD9 involved in exosome transport
  • lipids for example, the exosome membrane is rich in transmembrane protein families CD63, CD81 and CD9 involved in exosome transport
  • lipids lipids
  • cytokines cytokines
  • a variety of cells can secrete exosomes under normal and pathological conditions. They are widely present in body fluids such as blood, saliva, urine, cerebro
  • biomarker refers to a biochemical index that can mark changes or possible changes in the structure or function of a system, organ, tissue, cell, or subcellular structure, and has a very wide range of uses. Biomarkers can be used to diagnose diseases, determine disease stages, or evaluate the safety and effectiveness of new drugs or new therapies in target populations.
  • label refers to a component that can be detected by spectroscopy, photochemical, biochemical, immunochemical, chemical or other physical means.
  • useful labels include 32P, fluorescent dyes, fluorescent groups, electron-dense reagents, enzymes (e.g., enzymes commonly used in ELISA), biotin, digoxigenin or haptens, as well as other entities that can be detected.
  • the label can be incorporated into any position of the nucleic acid (e.g., at the 3' end or 5' end or inside).
  • the polynucleotides (polynucleotide probes) used to detect miRNA and/or the miRNA itself can be labeled.
  • the first aspect of the present application provides an isolated microRNA molecule, namely miR26690, whose nucleotide sequence is UGGAUAUGGAGGGAAGGA (SEQ ID NO.9).
  • the present application also relates to variants 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 with miR26690 (nucleotide sequence shown in SEQ ID NO.9).
  • the present application provides the use of miR26690 or its variants or reagents for detecting miR26690 or its variants in the preparation of Parkinson's disease diagnosis and/or screening reagents or kits.
  • miR26690 or a variant thereof as a biomarker for the diagnosis of Parkinson's disease is present in a body fluid sample of a subject, such as, for example, 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 sample, a perilymph 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 vomitus sample, etc.
  • a body fluid sample of a subject such as, for example, a urine sample, a blood sample, a sputum sample, a breast milk sample, a cerebrospinal fluid
  • miR26690 or a variant thereof used as a biomarker for diagnosing Parkinson's disease is present in the peripheral blood of the subject, more specifically, in the exosomes of the peripheral blood.
  • the present application also provides the use of miR26690 or its variants, and/or miR26690-positive exosomes (i.e., exosomes containing miR26690 or its variants) as biomarkers for diagnosis and/or screening of Parkinson's disease.
  • miR26690 or its variants and/or miR26690-positive exosomes are present in the peripheral blood of the subject.
  • the reagent for detecting miR26690 or its variants is a reagent for detecting miR26690 or its variants in peripheral blood or a reagent for detecting miR26690 or its variants in exosomes of peripheral blood, or the reagent for detecting miR26690 or its variants can also be a reagent for detecting miR26690-positive exosomes.
  • the reagent for detecting miR26690 or its variants can specifically bind to miR26690 or its variants.
  • the reagent for detecting miR26690 or its variants contains a detection label, such as a fluorescent group.
  • the detection group therein will change, such as the fluorescent group emitting light, or quenching, or the fluorescence intensity changes, etc., so that the presence or absence or the amount of miR26690 or its variants can be detected by detecting the change in the fluorescent signal.
  • the miR26690 or its variant is present in exosomes, and the reagent for detecting miR26690 or its variant can also be used to detect the presence or absence or amount of exosomes containing miR26690 or its variant.
  • the content of miR26690 or its variants or exosomes comprising miR26690 or its variants in the peripheral blood of Parkinson's disease patients is higher than that in non-Parkinson's disease patients.
  • the Parkinson's disease diagnosis and/or screening also includes diagnosing and/or screening the therapeutic effect of Parkinson's disease.
  • diagnosing and/or screening for treatment effects of Parkinson's disease comprises:
  • the concentration of miR26690 or its variants or exosomes containing miR26690 or its variants in the peripheral blood of the subject shows an increasing trend as the treatment progresses, it indicates that the drug treatment is ineffective; if the concentration of miR26690 or its variants or exosomes containing miR26690 or its variants in the peripheral blood of the subject remains unchanged or shows a decreasing trend as the treatment progresses, it indicates that the drug treatment is effective.
  • the present application provides a composition for detecting miR26690 or its variants, or exosomes containing miR26690 or its variants, which comprises nucleic acid molecules shown in SEQ ID NO.1 to SEQ ID NO.8.
  • the 3' end of the nucleic acid molecule shown in SEQ ID NO.7 is connected to a fluorescent group
  • the 5' end of the nucleic acid molecule shown in SEQ ID NO.8 is connected to a quenching group
  • 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
  • the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB or TAMARA.
  • the present application provides a Parkinson's disease diagnosis and/or screening kit, which comprises a reagent for detecting miR26690 or a variant thereof.
  • 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 radial immunodiffusion kit, a two-dimensional two-phase immunodiffusion kit, a rocket immunoelectrophoresis kit, an immunohistochemistry 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.
  • a Western blot kit an enzyme-linked immunosorbent assay (ELISA) kit, a radioimmunoassay (RIA) kit, a radial immunodiffusion kit, a two-dimensional two-phase immunodiffusion kit, a rocket immunoelectrophoresis kit, an immunohistochemistry staining kit, an immunoprecipitation assay kit, a complement fixation assay kit
  • the reagent for detecting miR26690 or its variants includes nucleic acid molecules shown in SEQ ID NO.1 to SEQ ID NO.8, wherein the 3' end of the nucleic acid molecule shown in SEQ ID NO.7 is connected to a fluorescent group, and the 5' end of the nucleic acid molecule shown in SEQ ID NO.8 is connected to a quenching group; wherein, by rationally designing the sequence of the nucleic acid molecules, it is possible to form a DNA hexahedron structure.
  • sequences SEQ ID NO.1 to SEQ ID NO.6 and SEQ ID NO.8 can be preferentially mixed to form a DNA hexahedron structure, wherein SEQ ID NO.2 contains a complementary sequence of miR26690 and can specifically bind to miR26690 or its variants; the formed DNA hexahedron structure is then incubated with SEQ ID NO.7 to form a detection hexahedron, Among them, the sequence SEQ ID NO.7 contains a region that is partially complementary to the sequence SEQ ID NO.1.
  • the fluorescent group and the quencher group are each independently connected to the 3' end or the 5' end of the nucleic acid molecule through a chemical bond, a linker, or a linker consisting of 1-3 nucleotides.
  • 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; in some embodiments, the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB or TAMARA.
  • the kit also includes a reaction buffer, which contains 40-50 mM Tris-acetate and 12-13 mM magnesium acetate; preferably, the pH of the reaction buffer is 7.5-8.5.
  • the present application also provides a method for detecting miR26690 or its variants or exosomes containing miR26690 or its variants in vitro, comprising:
  • nucleic acid molecules shown in SEQ ID NO.1 to SEQ ID NO.6 and SEQ ID NO.8 are connected to a quenching group; preferably, the quenching group is selected from BHQ1, BHQ2, BHQ3, BHQ-X, Dabcyl, MGB or TAMARA;
  • step 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 hexahedron, wherein the 3' end of the nucleic acid molecule shown in SEQ ID NO.7 is connected to 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;
  • the microRNA molecules in the sample to be tested by detecting the fluorescence signal; preferably, the determination is a quantitative determination.
  • the method can be used directly to detect iR26690-positive exosomes.
  • the amount of miR26690 or its variants can be reflected by the amount of miR26690-positive exosomes.
  • the suitable conditions include incubation at 90-97°C for 3-10 min, followed by incubation at 75-85°C for 2-5 min in a reaction buffer; and then decreasing to about 4°C.
  • the temperature is kept at about 95°C for about 5 minutes, then kept at about 80°C for about 3 minutes; then the temperature is reduced to about 4°C; preferably, the temperature is reduced to 4°C at a uniform rate; preferably, the temperature is reduced to 60°C at a rate of 2°C/min, and then reduced to 4°C at a rate of 3°C/min.
  • the reaction buffer contains 40-50 mM Tris-acetate and 12-13 mM magnesium acetate; preferably, the pH of the reaction buffer is 7.5-8.5.
  • 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): (3.8-4.2): (0.8-1.2): (0.8-1.2): (0.8-1.2); preferably 1:1:1:1:4:1:1:1.
  • the mixing ratio can ensure that most DNA can self-assemble into DNA hexahedron.
  • 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 sample, a perilymph 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 vomitus sample of the subject; preferably, the sample to be tested is a peripheral blood sample of the subject; more preferably, the sample to be tested is peripheral blood plasma.
  • the volume of the sample to be tested is 0.001-20 mL, preferably 0.001-10 mL, more preferably 0.001-1 mL, more preferably 0.001-0.5 mL, more preferably 0.001-0.1 mL, more preferably 0.001-0.05 mL, and most preferably 0.001-0.01 mL.
  • the ratio of the detection hexahedron to the sample to be tested is 0.5-1.5 nmol/mL.
  • contacting the sample to be tested with the detection hexahedron comprises: the ratio of the detection hexahedron to the sample to be tested is 0.5-1.5 nmol/mL, and incubating at 36-38° C. for more than 8 hours.
  • step d) further comprises diluting with PBS buffer, so that the diluted sample is suitable for detection.
  • the detection of fluorescent signals uses nano-flow detection technology.
  • it includes subjecting the above sample diluted with PBS buffer to nano-flow detection, and the instruments used include but are not limited to Apogee, NanoFCM, Cytoflex S and other flow detection platforms that can detect 10-1000 nanometer-level particles.
  • a 405nm laser is used as a scattered light detection light source
  • the CY3 channel is a fluorescence detection parameter (corresponding to the fluorescent group connected to the 3' end of the nucleic acid molecule shown in SEQ ID NO.7 is CY3)
  • the concentration of miR26690-positive nanovesicles (i.e., exosomes) with a diameter of less than 1000nm in plasma is detected, which is further used as a diagnostic biomarker for Parkinson's disease.
  • the present application also provides a method for diagnosing Parkinson's disease, which includes using the in vitro method for detecting miR26690 or its variants or exosomes containing miR26690 or its variants of the present application to detect the content of miR26690 or its variants or miR26690-positive exosomes in the subject's body fluids, especially in the peripheral blood, and judging whether the subject suffers from Parkinson's disease based on the content of miR26690 or its variants or miR26690-positive exosomes.
  • the diagnosis of Parkinson's disease is an early diagnosis of Parkinson's disease.
  • the early diagnosis can be understood as H-Y stage ⁇ 2.
  • the miR26690-positive exosome content is higher than 1808/ ⁇ L, which is judged as Parkinson's disease positive.
  • the miR26690 detection kit and method for exosomes in the blood of patients with Parkinson's disease constructed in this application use DNA hexahedron chimeric fluorescent tags and quenching groups to effectively detect the concentration of miR26690 positive exosomes in plasma.
  • a high-sensitivity and high-specificity diagnosis of Parkinson's disease is achieved.
  • the method of this application is simple, easy to prepare, has a small amount of detection sample, higher sensitivity, low detection limit, and low detection cost.
  • ncMB-cy3 was CY3
  • quenching group connected to the 5' end of ncMB-BHQ2 was BHQ2.
  • H-Y Hoehn-Yahr
  • MDS-UPDRS III Movement Disorder Society Unified Parkinson's Disease Rating Scale Part III
  • Sample processing and preparation of plasma to be tested Blood was collected using EDTA anticoagulation tubes and centrifuged within 2 hours. The centrifugation conditions were: 1500 ⁇ g, 4°C centrifugation for 10 min. The upper plasma was drawn into a cryopreservation tube and stored in a -80 refrigerator. Before testing, the frozen plasma was taken out and centrifuged at 12000 ⁇ g, 4°C for 10 min, and the upper plasma was retained.
  • the prepared samples were subjected to nanoscale flow cytometry, using the Cytoflex S platform, a 405nm laser as the scattered light detection light source, and the Cy3 channel as the fluorescence detection parameter to detect the content of miR26690-positive nanovesicles with a diameter of less than 1000nm in plasma.
  • the flow cytometry results are shown in Figure 2.
  • the number of miR26690-positive nanovesicles with a diameter of less than 1000nm and Cy3 fluorescence positive can be obtained 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.
  • the number of miR26690 positive exosomes in the plasma samples of the Parkinson's disease group and the healthy control group was analyzed using the receiver operating characteristic curve (ROC), and the Youden Index was calculated.
  • the calculation formula was sensitivity (Sensitivity, Sen) + specificity (Spe) – 1.
  • the number of miR26690 positive exosomes in the plasma sample corresponding to the maximum value of the Youden Index was the optimal critical value between the groups. After calculation, the optimal critical value was 1808, that is, the number of miR26690 positive exosomes ⁇ 1808/ ⁇ L was judged as Parkinson's disease positive, and the number of miR26690 positive exosomes was less than 1808/ ⁇ L, which was judged as Parkinson's disease negative.
  • the ROC curve is shown in Figure 5. It can be seen from the figure that the sensitivity of diagnosing Parkinson's disease using the detection method of the present application can reach more than 85%, and the specificity is close to 90%, which has high sensitivity and specificity
  • the method of the present application can obtain accurate results directly by testing peripheral blood samples, and the sample volume is extremely small (5 ⁇ L). Compared with cerebrospinal fluid testing, it greatly reduces the trauma to patients, improves patient compliance, and has extremely high clinical application value.

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Abstract

本申请公开了一种分离的微小RNA分子、一种基于DNA六面体的帕金森病体外诊断试剂盒及其应用。本申请提供的微小RNA分子miR26690在帕金森病患者外周血中的外泌体中高表达，可用于帕金森病的体外诊断和/或筛查；此外，本申请提供的用于检测外周血中miR26690的试剂盒和方法，能够高灵敏度、高特异性地检测外周血中miR26690，其用于诊断帕金森病与传统的帕金森病标志物检测方法相比，方法简单，易制备，检测样本量少，灵敏度更高，检测限低，检测成本低。

Description

一种基于DNA六面体的帕金森病体外诊断试剂盒及其应用 技术领域
本申请属于生物制剂领域，具体涉及一种基于DNA六面体的帕金森病体外诊断试剂盒及其应用。
背景技术
目前，帕金森病等神经退行性疾病的诊断和鉴别诊断主要依靠患者的临床表现及影像学指标，要求临床医生具有丰富的经验，导致较高的误诊率。当患者出现明显的临床症状时，神经元已经出现了大量不可逆的死亡，导致治疗效果有限。现亟需具有高灵敏度和特异性的体液生物标志物辅助诊断。目前，表现较好的生物标志物主要是基于脑脊液的检测，但脑脊液的采集对患者的创伤性较大，患者依从性较差。因此，迫切地需要研发基于血液(血浆、血清、红细胞等)的高准确率的诊断生物标志物。由于血脑屏障的存在，传统意义上能够反应脑内疾病状态的生物标记物难以在外周血中被检测到。近年来，研究发现来自中枢神经系统的细胞外囊泡，如外泌体，能够穿透血脑屏障进入外周血中(Min et al.,Acta Neuropathologica,128(5):639-650,2014)，这项事实为通过检测外周血中的外泌体反应中枢神经系统的神经退行性疾病提供了可行性的理论基础。
近年来，研究发现外泌体中携带有大量的核酸，其中微小RNA(microRNA或miRNA)是其中重要的一大类。2022年发表的一篇文章介绍了一种基于DNA六面体分子信标的检测外泌体micro RNA的实验方法(Dongsheng et al.,Biosensors and Bioelectronics,2022 11 4077)。提示我们可以通过使用DNA六面体分子信标检测血液中携带某些特定miRNA的外泌体的方法，对帕金森病等神经退行性疾病进行诊断，研发生物标志物。
发明概述
发明人在对帕金森病的生物标志物进行长期研究的过程中，意外地发现了一种microRNA，其在帕金森病患者的外周血外泌体中的表达量要远高于非帕金森病患者，因此能够用于作为诊断和/或筛查帕金森病的生物标记物，并基于此完成了本申请。
本申请第一方面提供了一种分离的微小RNA分子，其具有SEQ ID NO.9所示的核苷酸序列，或其核苷酸序列与SEQ ID NO.9所示的核苷酸序列具有至少90％、至少95％、或至少99％序列同一性。
本申请第二方面提供了本申请第一方面的微小RNA分子或检测本申请第一方面的微小RNA分子的试剂在制备帕金森病诊断和/或筛查试剂或试剂盒中的用途。
本申请第三方面提供了一种用于检测本申请第一方面的微小RNA分子的组合物，其包含SEQ ID NO.1至SEQ ID NO.8所示的核酸分子。
本申请第四方面提供了一种帕金森病诊断和/或筛查试剂盒，其包含用于检测本申请第一方面的微小RNA分子的试剂。
本申请第五方面提供了本申请第一方面的微小RNA分子和/或包含所述微小RNA分子的外泌体作为帕金森病诊断和/或筛查的生物标记物的用途。
本申请第六方面提供了一种体外检测本申请第一方面的微小RNA分子或包含所述微小RNA分子的外泌体的方法，其包括：
a)将SEQ ID NO.1-SEQ ID NO.6、SEQ ID NO.8所示的核酸分子混合，在合适的条件下反应形成DNA六面体结构；其中，所述SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA；
b)将SEQ ID NO.7所示的核酸分子加入到步骤a)的反应体系中，在36-38℃下反应生成检测六面体，其中，所述SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；
c)将待测样品与所述检测六面体接触；优选地，所述待测样品为受试者外周血样品；优选地，所述待测样品为外周血血浆；
d)通过检测荧光信号测定待测样品中所述微小RNA分子；优选地，所述测定为定量测定。
本申请第七方面提供了一种帕金森病诊断方法，其包括采用本申请第六方面的方法，检测受试者体液样品中所述微小RNA分子或包含所述微小RNA分子的外泌体的含量，根据所述微小RNA分子或包含所述微小RNA分子的外泌体的含量判断受试者是否患有帕金森病。
本申请提供的微小RNA分子miR26690在帕金森病患者外周血中的外泌体中高表达，因此可用于帕金森病的体外诊断和/或筛查；进一步的，本申请提供的用于检测外周血中miR26690的试剂盒和方法，能够高灵敏度、高特异性地检测外周血中miR26690，其用于诊断帕金森病与传统的帕金森病标志物检测方法相比，方法简单，易制备，检测样本量少，灵敏度更高，检测限低，检测成本低。
附图说明
附图更进一步说明了本说明书所公开的新特性。参照这些附图将能更好地理解本说明书中所公开的特性和优点，但应当理解，这些附图仅用于说明本文所公开原理的具体的实施方案，而非意欲对所附权利要求的范围加以限制。
图1为本申请检测方法原理示意图。
图2为实施例1的纳米流式检测系统检测血浆中miR26690阳性外泌体结果示意图。
图3为帕金森病患者与健康对照的血浆miR26690阳性外泌体浓度检测结果。
图4为早期帕金森病患者与健康对照的血浆miR26690阳性外泌体浓度检测结果。
图5为DNA六面体检测血浆miR26690阳性外泌体浓度作为帕金森病诊断生物标志物的ROC曲线。
具体实施方式
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一种实施方式，对于本领域普通技术人员来讲，还可以根据这些附图获得其他的实施方式。
术语
如本文所用，术语“一个”和“一种”以及“所述”和类似的指代物指示单数和复数，除非本文另外指明或上下文明显矛盾。
如本文所用，术语“约”、“基本上”和“类似于”是指在本领域普通技术人员所确定的特定值的可接受误差范围内，所述误差范围可部分取决于该值的测量或确定方式，或取决于测量系统的局限性。
根据本申请，术语“包含/包括/含有”是指包含所说明的整体或整体的组，但不排除任何其它整体或整体的组。根据本申请，术语“基本上由……组成”是指包含所说明的整体或整体的组，同时排除实质上会影响或改变所说明的整体的其它整体或变型。根据本申请，术语“由……组成”是指包含所说明的整体或整体的组，并排除任何其它整体或整体的组。
如本文使用的，术语“微小RNA(miRNA)”MicroRNA(miRNA)是一类由内源基因编码的长度约为20个核苷酸的非编码单链RNA分子，它们在动植物中参与转录后基因表达调控。本申请中，术语“微小RNA”“microRNA”和“miRNA”可互换地使用。
术语“分离的微小RNA分子”在本文中本质上涉及的是微小RNA分子从其天然环境分离。换言之，该微小RNA分子制备物基本上不含与其天然相关的其它物质。在一些实施方式中，分离的微小RNA分子，以重量计，含有至多10％，优选至多8％，更优选至多6％，更优选至多5％，更优选至多4％，更优选至多3％，甚至更优选至多2％，最优选至多1％，并且甚至最优选至多0.5％的与其天然相关的其它物质。
如本文所用，术语“帕金森病(PD)”(也命名为特发性帕金森综合症(IPS)或Morbus帕金森)是指称为运动系统障碍的病症，是产生多巴胺的脑细胞缺失的结果。PD的主要症状是手、臂、腿、下巴和面部震颤或颤抖；四肢和躯干僵化或僵硬；运动迟缓或运动缓慢；以及姿势不稳或平衡和协调受损。随着这些症状变得更加明显，患者可能难以行走、说话或完成其它简单任务。PD通常影响超过50岁的人。PD的早期症状很微妙并且逐渐发生。在某些人中，疾病进展得比其他人更快。
如本文所用，术语“诊断个体是否具有帕金森病(PD)”是指确定个体是否表现出PD的迹象或患有PD。因此，个体可被诊断为患有PD或未患有PD。
如本文所用，术语“确定具有PD的个体的病程”是指确定PD随着时间推移的发展，例如PD随着时间推移是否在个体中恶化、在个体中未恶化/稳定或在个体中改善。
如本文所用，术语“诊断”是指确定可能的疾病或紊乱的过程，因此是试图定义个体的(临床)状况的过程。根据本申请的miRNA的水平的确定与个体的(临床)状况相关。优选地，诊断包括/涵盖：(i)确定PD的发生/存在；(ii)监测PD的病程；(iii)对PD的分期；(iv)测量具有PD的个体对治疗干预的响应；和/或(v)对患有PD的个体的细分(segmentation)。
如本文所用，术语“个体”是指希望知道她或他是否患有/具有PD的任何受试者。
具体而言，如本文所用，术语“个体”是指疑似受PD或影响的受试者。个体可被诊断为受PD的影响，即患病；或者可被诊断为未受PD的影响，即就这些疾病而言是健康的。
如本文所用，术语“个体”还指受PD影响(即患病)的受试者。个体可以针对PD进行再测试，并可被诊断为仍然受PD的影响，即患病；或不再受(不再那么受)PD的影响，即相对于PD而言是健康的(例如在治疗干预之后)。个体可以针对PD进一步进行再测试，并可被诊断为已发展为PD的晚期形式或严重形式。
应当注意的是，被诊断为未患有PD(即相对于PD而言是健康的)的个体也可能患有另一种未测试/未知的疾病。
个体可以是任何哺乳动物，包括人类和另一哺乳动物，例如，诸如兔、小鼠、大鼠或猴的动物。特别优选人类受试者作为个体。
如本文所用，术语“受试者”可以是指已知未受PD影响的受试者(阴性对照)(此类受试者也称为非帕金森病患者)，即相对于PD而言是健康的。如本文所用，术语“受试者”还可以指已知受PD影响(即患病)的受试者。所述受试者可能已经发展为PD的晚期形式。
应当注意的是，已知未患有PD(即相对于PD而言是健康的)的受试者(非帕金森病患者)也可能患有另一种未 测试/未知的疾病。
本申请中，受试者可以是任何哺乳动物，包括人类和另一哺乳动物，例如，诸如兔、小鼠、大鼠或猴的动物。特别优选人类受试者作为个体。
如本文所用，术语“治疗”是指改善个体健康状况和/或延长(增加)个体寿命的任何疗法。所述疗法可消除个体的疾病，阻止或减慢个体的疾病发展，抑制或减慢个体的疾病发展，降低个体的症状的频率或严重性，和/或降低当前或以前具有疾病的个体的复发。疾病可以是PD。PD的(治疗性)治疗包括但不限于药物施用、言语治疗、运动训练、心理训练和/或身体康复。如本文所用，术语“体液”是广义的术语且被赋予对本领域普通技术人员而言普通且惯常的含义，并且不限于特殊或自定义的含义。所述体液样品可以是尿液样品、血液样品、痰样品、母乳样品、脑脊液(CSF)样品、耵聍(耳垢)样品、胃液样品、粘液样品、内淋巴液样品、外淋巴液样品、腹膜液样品、胸膜液样品、唾液样品、皮脂(皮肤油脂)样品、精液样品、汗液样品、泪液样品、面颊拭子、阴道分泌物样品、液体活检或呕吐物样品，包括它们的成分(components)或组分(fractions)。术语“体液样品”还涵盖体液组分，例如血液组分、尿液组分或痰组分。体液样品可以混合或合并。因此，体液样品可以是血液和尿液样品的混合物，或者血液和脑脊液样品的混合物。所述体液样品可以通过从个体或受试者取出体液来提供，但也可以通过使用先前分离的体液样品材料来提供。体液样品使得能够对个体进行非侵入性分析。进一步优选体液样品的体积为0.001-20mL，优选为0.001-10mL，更优选为0.001-1mL，并且最优选为0.001-0.05mL。
在本申请中，术语“外周血”意思是指在哺乳动物中全身性循环或曾经循环的血液，并且不是从例如骨髓(BM)、淋巴细胞(例如，淋巴结)、脾脏或肝脏中螯合的血液中获得的。
如本文所用，术语“血液样品”涵盖全血样品或血液组分样品，例如血细胞/细胞组分、血清或血浆样品。
术语“血浆”如常规定义。在某些实施方式中，人血浆可以是，或者人血浆的来源可以是新鲜血浆、冻干血浆、溶剂/去污剂处理的血浆、新鲜冷冻血浆、解冻的血浆或冷沉淀物、冷冻上清液或血浆浓缩物，如来自冷冻血浆的浓缩物，或者它们中任何两种或更多种的混合物。血浆通常获得自通过清血法获得的样品或者获得自全血样品，其提供有或接触抗凝血剂(例如，肝素、柠檬酸、草酸或EDTA)。随后，通过适当的技术，通常通过离心，将血液样品的细胞组分与液体组分(血浆)分离。通过具体实例但不限制的方式，为了获得适合在本申请中使用的血浆，可以将血液样品吸入含有抗凝血剂EDTA(乙二胺四乙酸)的真空采血管(例如，BD Vacutainer塑料EDTA管，10ml，1.8mg/mL)。将样品轻轻振荡，然后在室温下以1,000-2,000g离心10分钟以将血浆与红细胞分离。收集上清液(血浆)，可选地汇集(如果使用多个血液样品)并等分至冷冻小瓶，其在-80℃下储存直至使用。因此，术语“血浆”是指血液样品的无细胞组分的组合物，其不形成人或动物体的一部分。因此，如本文所设想的血浆是无细胞血浆，例如，血浆包含小于约1.0％w/w，优选地小于约0.5％w/w或小于0.1％w/w的全细胞物质，或者基本不包括全细胞物质。
血浆可以优选地是未处理的血浆，即通过与全血分离且未经受可选的热失活、”存(低温或非低温)、除菌、过滤、冷冻干燥和/或溶剂/去污剂处理之外的改变其化学、生物化学或细胞组成的后续处理步骤所得的血浆。
在某些实施方式中，术语“血浆”可以具体地排除了处理的血浆，即在其与全血分离后经受了改变其组成，具体地，其化学、生物化学或细胞组成的一个或多个处理步骤的血浆。优选地，如本文所设想的术语“血浆”具体地排除了富血小板血浆(PRP)，即已富集了血小板的血浆。通常，PRP可以含有约1.0×10 6个血小板/μl，尽管全血中血小板浓度可以为约1.5×10 5至3.5×10 5/μl。因此，如本文所设想的血浆可以含有小于约8.0×10 5、优选地小于约7.0×10 5、更优选地小于约6.0×10 5、更优选地小于约5.0×10 5、如小于约4.0×10 5个血小板/μl。
如本文所预期的血浆是人血浆，即获得自单一人类受试者或得自多个人类受试者(例如，血浆混合物)。
血浆可以直接用于本申请的方法。血浆还可以适当储存用于后续使用(例如，储存较短的时间，例如，最长约1-2周，在血浆的冰点以上，但低于环境温度的温度，该温度通常将为约4℃至5℃；或者通过冻藏储存更长的时间，通常为约-70℃至约-80℃)。
本申请的方法可以使用要诊断或筛查的受试者自体的血浆，并因此血浆对包含血浆的组合物中保存的细胞是自体的。关于血浆的术语“自体”表示血浆获得自与血浆接触或用血浆诊断或筛查的相同的受试者。
如本领域中已知的，可以使血浆热失活，具体地以除去补体。当本申请的方法使用要诊断或筛查的受试者自体的血浆时，可以不必需使血浆热失活。
如本文所用，术语“水平”或“含量”是指本文所述的miRNA的量(例如以克、摩尔或诸如离子或荧光计数之类的计数所测量的)或浓度(例如绝对浓度或相对浓度)。
如本文所用，术语“水平”或“含量”还包括缩放的量或值、归一化的量或值、或者缩放且归一化的量或值。优选地，本文确定的水平是表达水平。
如本文所用，术语“灵敏度”是指相对于患者总数量(100％)的真阳性患者数量(％)。个体可以是具有PD的受试者。灵敏度通过以下公式计算：灵敏度＝TP/(TP+FN)(TP＝真阳性；FN＝假阴性)。
如本文所用，术语“特异性”涉及相对于健康受试者总数量(100％)的真阴性个体数量(％)。特异性通过以下公式计算：特异性＝TN/(TN+FP)(TN＝真阴性；FP＝假阳性)。
如本文所用，术语“准确性(accuracy)”指用于样品类型的分类或鉴别的正确性的统计量度。准确性是真实结果(真阳性和真阴性二者)的比例。
如本文中所使用的，术语“外泌体(exosome)”是指直径大约为30-150nm的微小膜泡，由多种细胞分泌，含有 特定的蛋白质(例如，外泌体膜上富含参与外泌体运输的跨膜蛋白家族CD63，CD81和CD9)、脂质、细胞因子或遗传物质。多种细胞在正常及病理状态下均可分泌外泌体，它们广泛存在于血液、唾液、尿液、脑脊液和乳汁等体液中，被视为特异性分泌的膜泡，参与细胞间通讯。
如本文中所使用的，术语“生物标志物(Biomarker)”是指可以标记系统、器官、组织、细胞及亚细胞结构或功能的改变或可能患的改变的生化指标，具有非常广泛的用途。生物标志物可用于疾病诊断、判断疾病分期或者用来评价新药或新疗法在目标人群中的安全性及有效性。
如本文所用，术语“标签”是指可通过光谱学手段、光化学手段、生物化学手段、免疫化学手段、化学手段或其它物理手段检测的成分。例如，有用的标签包括32P、荧光染料、荧光基团、电子致密试剂、酶(例如，如在ELISA中通常使用的酶)、生物素、地高辛或半抗原，以及可被检测到的其它实体。标签可掺入核酸的任何位置(例如在3'端或5'端或内部)。用于检测miRNA的多核苷酸(多核苷酸探针)和/或miRNA本身可被标记。
本申请第一方面提供了一种分离的微小RNA分子，即miR26690，其核苷酸序列为UGGAUAUGGAGGGAAGGA(SEQ ID NO.9)。
本申请另一方面还涉及miR26690的变体，其核苷酸序列与miR26690(SEQ ID NO.9所示的核苷酸序列)具有至少90％、至少95％、或至少99％(即至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％)序列同一性。
在另一个方面，本申请提供了miR26690或其变体或检测miR26690或其变体的试剂在制备帕金森病诊断和/或筛查试剂或试剂盒中的用途。
在一些实施方式中，作为帕金森病诊断的生物标记物的miR26690或其变体存在于受试者的体液样品中，示例性地，如尿液样品、血液样品、痰样品、母乳样品、脑脊液(CSF)样品、耵聍(耳垢)样品、胃液样品、粘液样品、内淋巴液样品、外淋巴液样品、腹膜液样品、胸膜液样品、唾液样品、皮脂(皮肤油脂)样品、精液样品、汗液样品、泪液样品、面颊拭子、阴道分泌物样品、液体活检或呕吐物样品等。
在一些优选的实施方式中，用于作为帕金森病诊断的生物标记物的miR26690或其变体存在于受试者的外周血中，更具体地，其存在于外周血的外泌体中。
在另一个方面，本申请还提供了miR26690或其变体，和/或miR26690阳性外泌体(即包含miR26690或其变体的外泌体)作为帕金森病诊断和/或筛查的生物标记物的用途。在一些实施方式中，所述miR26690或其变体和/或miR26690阳性外泌体存在于受试者外周血中。
在一些实施方式中，所述检测miR26690或其变体的试剂是用于检测外周血中的miR26690或其变体的试剂或用于检测外周血的外泌体中的miR26690或其变体的试剂，或者，所述检测miR26690或其变体的试剂还可以是检测miR26690阳性外泌体的试剂。
在一些实施方式中，所述检测miR26690或其变体的试剂能够特异性地与miR26690或其变体结合。
在一些实施方式中，所述检测miR26690或其变体的试剂中含有检测标签，例如荧光基团等。
在一些实施方式中，当所述检测miR26690或其变体的试剂与miR26690或其变体特异性结合后，其中的检测基团的会发生变化，例如荧光基团发光、或淬灭、或荧光强度改变等，从而可以通过检测荧光信号的变化检测miR26690或其变体的存在与否或量的多少。
在一些实施方式中，所述miR26690或其变体存在于外泌体中，所述检测miR26690或其变体的试剂还可以用于检测包含miR26690或其变体的外泌体的存在与否或量的多少。
在一些实施方式中，所述miR26690或其变体或包含miR26690或其变体的外泌体在帕金森病患者外周血中的含量高于非帕金森病患者。
在一些实施方式中，所述帕金森病诊断和/或筛查还包括诊断和/或筛查帕金森病的治疗效果。
在一些实施方式中，其中，诊断和/或筛查帕金森病的治疗效果包括：
在治疗的不同时间点测定受试者外周血中miR26690或其变体或包含miR26690或其变体的外泌体的浓度，比较在不同时间点测定的受试者外周血中miR26690或其变体或包含miR26690或其变体的外泌体的浓度；
优选地，如果受试者外周血中miR26690或其变体或包含miR26690或其变体的外泌体的浓度随着治疗进行呈现升高的趋势，则提示药物治疗无效；如果受试者外周血中miR26690或其变体或包含miR26690或其变体的外泌体的浓度随着治疗进行不变或呈现降低的趋势，则提示药物治疗有效。
在另一个方面，本申请提供了一种用于检测miR26690或其变体，或包含miR26690或其变体的外泌体的组合物，其包含SEQ ID NO.1至SEQ ID NO.8所示的核酸分子。
在一些实施方式中，SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团，SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA。
在另一个方面，本申请提供了一种帕金森病诊断和/或筛查试剂盒，其包含用于检测miR26690或其变体的试剂。
在一些实施方式中，所述试剂盒选自Western印迹试剂盒、酶联免疫吸附测定(ELISA)试剂盒、放射免疫测定(RIA)试剂盒、放射免疫扩散试剂盒、二维双相免疫扩散试剂盒、火箭免疫电泳试剂盒、免疫组织化学染色试剂盒、 免疫沉淀测定试剂盒、补体结合测定试剂盒、荧光激活细胞分选(FACS)试剂盒、适体芯片试剂盒、微阵列试剂盒和蛋白质芯片试剂盒中的一种或两种以上。
在一些实施方式中，所述用于检测miR26690或其变体的试剂包括SEQ ID NO.1至SEQ ID NO.8所示的核酸分子，其中，所述SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团，所述SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；其中，通过合理地设计所述的核酸分子的序列，使其能够形成DNA六面体结构，示例性地，如图1所示，可以优先混合序列SEQ ID NO.1至SEQ ID NO.6和SEQ ID NO.8，从而形成DNA六面体结构，其中SEQ ID NO.2中包含了miR26690的互补序列，能够与miR26690或其变体特异性结合；形成的DNA六面体结构再与SEQ ID NO.7孵育，形成检测六面体，其中，序列SEQ ID NO.7中包含于与序列SEQ ID NO.1部分互补的区域，SEQ ID NO.7与SEQ ID NO.1互补结合后，DNA六面体形状发生改变，序列SEQ ID NO.7中的荧光基团与序列SEQ ID NO.8中的淬灭基团靠近，因此检测六面体几乎不发荧光(即本底荧光强度很低)；进一步地，当所示检测六面体与底物miR26690接触时，miR26690或其变体与SEQ ID NO.2序列上的片段互补结合，再次改变了检测六面体的结构，使荧光基团远离淬灭基团，从而产生荧光，因此可以根据荧光强度反应miR26690或其变体的存在与否以及量的多少，使得miR26690或其变体或包含miR26690或其变体的外泌体能够被检测到。
在一些实施方式中，所述荧光基团和所述淬灭基团各自独立地通过化学键、连接基团或由1-3个核苷酸构成的接头连接与所述核酸分子的3’末端或5’末端。
在一些实施方式中，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；在一些实施方式中，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA。
在一些实施方式中，所述试剂盒中还包括反应缓冲液，所述反应缓冲液中包含40-50mM Tris-醋酸和12-13mM醋酸镁；优选地，所述反应缓冲液的pH为7.5-8.5。
在另一个方面，本申请还提供了一种体外检测miR26690或其变体或包含miR26690或其变体的外泌体的方法，其包括：
a)将SEQ ID NO.1-SEQ ID NO.6、SEQ ID NO.8所示的核酸分子混合，在合适的条件下反应形成DNA六面体结构；其中，所述SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA；
b)将SEQ ID NO.7所示的核酸分子加入到步骤a)的反应体系中，在36-38℃下反应生成检测六面体，其中，所述SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；
c)将待测样品与所述检测六面体接触；
d)通过检测荧光信号测定待测样品中所述微小RNA分子；优选地，所述测定为定量测定。
在一些实施方式中，所述方法可以直接用于检测iR26690阳性外泌体。
在一些实施方式中，所述miR26690或其变体的量可以以miR26690阳性外泌体的量来反映。
在一些实施方式中，所述合适的条件包括在反应缓冲液中，在90-97℃保温3-10min，随后75-85℃保温2-5min；然后降至约4℃。
在一些优选的实施方式中，在约95℃保温约5min，随后约80℃保温约3min；然后降至约4℃；优选地，匀速降温至4℃；优选地，以2℃/min的速度降至60℃，再以3℃/min的速度降至4℃。
在一些实施方式中，所述反应缓冲液中包含40-50mM Tris-醋酸和12-13mM醋酸镁；优选地，所述反应缓冲液的pH为7.5-8.5。
在一些实施方式中，所述SEQ ID NO.1-SEQ ID NO.8所示的核酸分子混合的摩尔比为(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)；优选为1:1:1:1:4:1:1:1。所示混合比例可保证大部分DNA都能自组装成DNA六面体。
在一些实施方式中，所述待测样品选自受试者的尿液样品、血液样品、痰样品、母乳样品、脑脊液(CSF)样品、耵聍(耳垢)样品、胃液样品、粘液样品、内淋巴液样品、外淋巴液样品、腹膜液样品、胸膜液样品、唾液样品、皮脂(皮肤油脂)样品、精液样品、汗液样品、泪液样品、面颊拭子、阴道分泌物样品、液体活检或呕吐物样品的至少一种；优选地，所述待测样品为受试者外周血样品；更优选地，所述待测样品为外周血血浆。
在一些实施方式中，所述待测样品的体积为0.001-20mL，优选为0.001-10mL，更优选为0.001-1mL，更优选为0.001-0.5mL，更优选为0.001-0.1mL，更有选为0.001-0.05mL，并且最优选为0.001-0.01mL。
在一些实施方式中，所述检测六面体与所述待测样品的比为0.5-1.5nmol/mL。
在一些实施方式中，所述将待测样品与所述检测六面体接触包括：所述检测六面体与所述待测样品的比为0.5-1.5nmol/mL，在36-38℃孵育8小时以上。
在一些实施方式中，步骤d)之前还包括使用PBS缓冲液稀释，从而使稀释后的样品适用于检测。
在一些实施方式中，所述检测荧光信号采用纳米流式检测技术。示例性的，其包括将上述经过PBS缓冲液稀释过的样本进行纳米级流式检测，使用的仪器包括但不限于Apogee，NanoFCM，Cytoflex S等能够检测到10-1000纳米级别颗粒的流式检测平台。以Cytoflex S平台为例，使用405nm激光器作为散射光检测光源，CY3通道为荧光检 测参数(对应于SEQ ID NO.7所示的核酸分子的3’末端连接的荧光基团为CY3)，检测血浆中直径1000nm以下的miR26690阳性的纳米囊泡(即外泌体)浓度，进一步作为帕金森病的诊断生物标志物。
在另一方面，本申请还提供了一种帕金森病诊断方法，其包括采用本申请的体外检测miR26690或其变体或包含miR26690或其变体的外泌体的方法，检测受试者体液中，特别是外周血中，miR26690或其变体或miR26690阳性外泌体的含量，根据所述miR26690或其变体或miR26690阳性外泌体的含量判断受试者是否患有帕金森病。
在一些实施方式中，所述帕金森病诊断为帕金森病早期诊断。
在一些实施方式中，所述早期诊断可以理解为H-Y分期≤2。
在一些实施方式中，miR26690阳性外泌体的含量高于1808/μL判断为帕金森病阳性。
本申请构建的帕金森病患者血液外泌体miR26690检测试剂盒和检测方法，利用DNA六面体嵌合荧光标签和淬灭基团，有效地检测到血浆中miR26690阳性外泌体的浓度，通过对帕金森病患者和健康对照的血浆进行检测，实现了对帕金森病的高灵敏度、高特异性诊断。本申请与传统的帕金森病标志物检测方法相比，方法简单，易制备，检测样本量少，灵敏度更高，检测限低，检测成本低。
以下将结合具体实施例，对本申请的试剂盒和方法进行说明。
实施例
实施例1 外周血样品中miR26690阳性外泌体检测
根据表1所示的序列由上海生工合成相应单链DNA分子，其中，ncMB-cy3的3’末端连接的荧光基团为CY3；ncMB-BHQ2的5’末端连接的淬灭基团为BHQ2。
外泌体荧光标记反应缓冲液：45mM Tirs-醋酸和12.5mM乙酸镁溶液，pH＝8.0。
帕金森病患者血液样本来自首都医科大学附属北京天坛医院运动障碍性疾病科就诊的帕金森病患者(PD)28例。同时从社区招募年龄匹配的健康对照受试者(NC)28例，其中疾病早期(H-Y分期≤2)患者8例；共入组受试者56例。本研究方案由北京天坛医院伦理委员会审查并批准，本研究所有受试者均签署了书面知情同意书。对所有受试者进行一般人口学资料与临床特征资料的问卷调查与评测，包括年龄、性别。对于PD患者，使用病程、“关期”H-Y(Hoehn-Yahr)分期量表和运动障碍协会统一帕金森病评定量表第三部分(Movement Disorder Society Unified Parkinson's Disease Rating Scale PartⅢ,MDS-UPDRSⅢ)来评估疾病的分期及严重程度。
受试者人口学及临床资料如表2所示。样本处理及待测血浆制备：使用EDTA抗凝采血管采血后2小时内进行离心处理，离心条件：1500×g，4℃离心10min。吸取上层的血浆至冻存管内，负80冰箱保存。检测前，将冻存血浆取出，12000×g，4℃离心10min，保留上层血浆。
表1
Figure PCTCN2022131321-appb-000001
表2
Figure PCTCN2022131321-appb-000002
Figure PCTCN2022131321-appb-000003
NA:健康对照无病程以及疾病严重程度相关信息。
将表1中的DNA定量后，以ncMB-1:NcMB26690:ncMB-3:ncMB-4:ncMB-Assist:NcMB-Anchor:ncMB-BHQ2＝1：1：1：1：4：1：1的摩尔比将七条序列在外泌体荧光标记反应缓冲液中混合均匀后，在95℃5min，80℃3min然后降至4℃，形成DNA六面体结构。然后向体系中加入1摩尔比的ncMB-cy3进行37℃1小时孵育，获得包含检测六面体的混合体系。
将上述得到的包含检测六面体的混合体系使用外泌体荧光标记反应缓冲液稀释到检测六面体含量约为100nM，取50uL与5uL血浆混合，在37℃孵育8-24小时，然后添加150uL PBS缓冲液(pH＝7.4)并混匀，完成样本的准备。
将上述制备好的样本进行纳米级流式检测，采用Cytoflex S平台，使用405nm激光器作为散射光检测光源，Cy3通道为荧光检测参数，检测血浆中直径1000nm以下的miR26690阳性的纳米囊泡含量。其中，流式结果如图2所示，圈选出直径1000nm以下且Cy3荧光阳性的纳米囊泡，即可得到每例样本中直径1000nm以下的miR26690阳性的纳米囊泡数量。帕金森病组及健康对照组血浆样品中miR26690阳性外泌体浓度结果如图3所示。从图3的结果中可以看出，帕金森病患者组外周血中miR26690阳性外泌体的浓度要显著高于健康对照组，p＜0.0001(Mann Whitney检验)。此外，与健康对照组相比，处于疾病早期(H-Y分期≤2)的帕金森病患者即出现外周血中miR26690阳性外泌体的浓度显著升高，p＜0.0001(Mann Whitney检验)，结果如图4所示，提示该指标可作为帕金森病早期诊断生物学标志物。
实施例2 方法特异性及灵敏度验证
使用受试者工作特征曲线(Receiver Operating Characteristic Curve,ROC)对帕金森病组及健康对照组血浆样品中miR26690阳性外泌体数量进行分析，并计算约登指数(Youden Index)，计算公式为灵敏度(Sensitivity,Sen)+特异度(Specificity,Spe)–1，约登指数最大值所对应的血浆样品中miR26690阳性外泌体数量即为组间最佳临界值，经计算，最佳临界值为1808，即miR26690阳性外泌体数量≥1808/μL，判断为帕金森病阳性，miR26690阳性外泌体数量小于1808/μL，判断为帕金森病阴性。ROC曲线如图5所示，从图中可以看出，采用本申请的检测方法，对帕金森病进行诊断的灵敏度可达85％以上，特异性接近90％，具有高灵敏度和特异性。
更重要的是，本申请的方法能够直接通过检测外周血样品即可得到准确的结果，且样品量极少(5μL)，相比于脑脊液检测，极大程度地减少了对患者的创伤，提高了患者依从性，具有极高的临床应用价值。
以上所述仅为本申请的较佳实施例，并不用以限制本申请，凡在本申请的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本申请保护的范围之内。

Claims (10)

  1. 一种分离的微小RNA分子，其具有SEQ ID NO.9所示的核苷酸序列，或其核苷酸序列与SEQ ID NO.9所示的核苷酸序列具有至少90％、至少95％、或至少99％序列同一性。
  2. 权利要求1的微小RNA分子或检测权利要求1的微小RNA分子的试剂在制备帕金森病诊断和/或筛查试剂或试剂盒中的用途。
  3. 一种用于检测权利要求1所述的微小RNA分子的组合物，其包含SEQ ID NO.1至SEQ ID NO.8所示的核酸分子。
  4. 根据权利要求3所述的组合物，其中，SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团，SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA。
  5. 一种帕金森病诊断和/或筛查试剂盒，其包含用于检测权利要求1所述的微小RNA分子的试剂；优选地，所述试剂盒包含SEQ ID NO.1至SEQ ID NO.8所示的核酸分子，其中，所述SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团，所述SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA。
  6. 根据权利要求5所述的试剂盒，其还包括反应缓冲液，所述反应缓冲液中包含40-50mM Tris-醋酸和12-13mM醋酸镁；优选地，所述反应缓冲液的pH为7.5-8.5。
  7. 一种体外检测权利要求1的微小RNA分子或包含所述微小RNA分子的外泌体的方法，其包括：
    a)将SEQ ID NO.1-SEQ ID NO.6、SEQ ID NO.8所示的核酸分子混合，在合适的条件下反应形成DNA六面体结构；其中，所述SEQ ID NO.8所示的核酸分子的5’末端连接有淬灭基团；优选地，所述淬灭基团选自BHQ1、BHQ2、BHQ3、BHQ-X、Dabcyl、MGB或TAMARA；
    b)将SEQ ID NO.7所示的核酸分子加入到步骤a)的反应体系中，在36-38℃下反应生成检测六面体，其中，所述SEQ ID NO.7所示的核酸分子的3’末端连接有荧光基团；优选地，所述荧光基团选自PE、FITC、FAM、TAMRA、Alexa Fluor、VIC、JOE、NED、TET、HEX、ROX、TEXASRED、CY3、CY5、CY5.5或CY7；
    c)将待测样品与所述检测六面体接触；优选地，所述待测样品为受试者外周血样品；优选地，所述待测样品为外周血血浆；
    d)通过检测荧光信号测定待测样品中所述微小RNA分子；优选地，所述测定为定量测定。
  8. 根据权利要求7所述的方法，其中，所述合适的条件包括在反应缓冲液中，在90-97℃保温3-10min，随后75-85℃保温2-5min；然后降至4℃；其中所述反应缓冲液中包含40-50mM Tris-醋酸和12-13mM醋酸镁；优选地，所述反应缓冲液的pH为7.5-8.5。
  9. 根据权利要求7所述的方法，其中，所述SEQ ID NO.1-SEQ ID NO.8所示的核酸分子混合的摩尔比为(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)；优选为1:1:1:1:4:1:1:1。
  10. 根据权利要求7-9中任一项所述的方法，其中所述将待测样品与所述检测六面体接触包括：所述检测六面体与所述待测样品的比为0.5-1.5nmol/mL，在36-38℃孵育8小时以上；优选地，步骤d)之前还包括使用PBS缓冲液稀释。
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