A rapid detection kit for the extracellular vesicle COPD screening marker GLIPR1

Abstract

The present invention relates to the field of biomedical technology, and specifically to a rapid detection kit for the extracellular vesicle COPD screening marker GLIPR1, wherein the kit includes an EVs in situ capture plate or chip, and each well of the EVs in situ capture plate or chip contains a fluorescein-labeled molecular beacon, and the molecular beacon is a specific DNA probe for detecting the COPD screening marker GLIPR1mRNA. The kit of the present invention utilizes the EVs in situ capture plate and chip technology to detect the COPD screening marker GLIPR1mRNA contained in EVs in the body fluid samples of COPD patients. Advantages: Integrating multiple COPD screening marker gene detection channels on a capture plate or chip is fast, convenient, and economical, and can realize rapid detection of the COPD screening marker GLIPR1 in extracellular vesicles, and ensure detection accuracy and specificity, and has strong practicality.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a rapid detection kit for an extracellular vesicle chronic obstructive pulmonary disease screening marker GLIPR1. Background Art

[0002] COPD is a common chronic disease characterized by chronic bronchitis and / or emphysema with airflow obstruction, which can further develop into cor pulmonale and respiratory failure. It is related to abnormal inflammatory reactions to harmful gases and particles, with high disability and mortality rates. The global incidence rate of people over 40 years old has reached 9% to 10%. The exact cause of COPD is unclear. It is generally believed that factors related to the occurrence of chronic bronchitis and obstructive emphysema may be involved in the onset of COPD. The risk factors that have been discovered can be roughly divided into two categories: external factors (i.e. environmental factors) and internal factors (i.e. individual susceptibility factors). External factors include smoking, inhalation of dust and chemicals, air pollution, respiratory infections, and people with low socioeconomic status (which may be related to indoor and outdoor air pollution, crowded living quarters, poor nutrition, and other factors associated with low socioeconomic status). Internal factors include genetic factors, increased airway reactivity, and individuals with poor lung development or growth due to various reasons during pregnancy, neonatal period, infancy or childhood. Its clinical symptoms include chronic cough, sputum, shortness of breath or difficulty breathing, wheezing and chest tightness. Existing examination methods include pulmonary function tests, chest X-rays, chest CT scans, blood gas tests or other methods.

[0003] Extracellular vesicles (EVs) are vesicles with a lipid bilayer membrane structure that are secreted by cells into the extracellular environment and contain host cell-specific nucleic acids and proteins. EVs contain proteins, DNA, mRNA, and non-coding RNA (such as miRNAs and lncRNA). The latest research results show that EVs are highly correlated with the occurrence and development of diseases, making them potential biomarkers for disease detection. As a liquid biopsy target, EVs have many advantages, such as high content in body fluids, lipid bilayer membranes that protect proteins and nucleic acids from degradation, and high marker specificity. Pulmonary function is the gold standard for detecting COPD, but pulmonary function testing requires professional technicians and cannot be widely carried out in primary hospitals. We have more than 100 million COPD patients, and most patients do not know that they are COPD patients. Therefore, it is crucial to develop a COPD screening kit.

[0004] EVs in situ capture plate or chip technology is a new EVs in situ capture and detection technology, especially for detecting mRNA and microRNA in EVs. It uses activated glass coated with a biomolecular film as a carrier, and is coated with cationic lipid nanoparticles that are molecular beacons (self-designed) that can specifically identify disease-related microRNA or mRNA targets. After fusing with negatively charged EVs, the molecular beacons bind to the targets to produce fluorescent signals, which are detected by a total internal reflection fluorescence (TIRF, total internal reflection fluorescence) microscope. The signal intensity is proportional to the corresponding target content, thereby judging the course of the disease or locking the pathogen. Due to the ultra-micro and ultra-sensitive characteristics of TIRF imaging to fluorescent signals, the EVs capture plate or chip combined with TIRF imaging technology can achieve direct imaging of nanoscale vesicles such as EVs and semi-quantitative detection of their contents. Schematic diagram of EVs in situ capture and detection, as shown in the figure Figure 1A-1B As shown. EVs capture well plates or chips can be made into 24, 48, 96, 384 wells and various specifications. Each well can be coated with a single or three fluorescently labeled molecular beacons. Multiple COPD screening marker gene detection channels are integrated on a capture well plate or chip, which is fast, convenient, economical and has obvious advantages.

[0005] Since EVs exist in large quantities in various body fluid samples and are rich in specific nucleic acids derived from lung cells, they can be isolated and identified using highly sensitive EVs capture well plate or chip detection technology. The present invention creatively proposes to screen COPD patients by detecting the EVs COPD screening marker GLIPR1, which has the advantages of high efficiency, high accuracy, convenience and high efficiency. There is currently no report on the rapid detection kit for the extracellular vesicle COPD screening marker GLIPR1 of the present invention. Summary of the invention

[0006] The purpose of the present invention is to diagnose the deficiencies of the prior art and to provide a rapid detection kit for the extracellular vesicle COPD screening marker GLIPR1.

[0007] To achieve the above object, the technical solution adopted by the present invention is:

[0008] In a first aspect, the present invention provides the use of the extracellular vesicle COPD screening marker GLIPR1 in the preparation of reagents and / or kits for diagnosing COPD.

[0009] In a second aspect, the present invention provides a COPD diagnostic kit, the kit comprising an extracellular vesicle in situ capture well plate or chip, the extracellular vesicle in situ capture well plate or chip containing a fluorescein-labeled molecular beacon, the molecular beacon being a specific DNA probe for detecting the COPD screening marker GLIPR1 mRNA, the 5' stem and loop of the specific DNA probe being completely complementary to the target gene, the 3' stem being partially complementary to the 5' stem, the 5' and 3' ends being modified with a fluorescent group and a quenching group, respectively, and some bases on the loop being modified with a locked nucleic acid, the kit also comprising an instruction manual, which states that the COPD diagnostic threshold is 97240810, and the corresponding sensitivity is 100%, and the specificity is 80%.

[0010] Preferably, the nucleotide sequence of the specific DNA probe is SEQ ID NO.1 to 3, the base modification mode of sequence SEQ ID NO.1 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th and 22nd bases and BHQ1 modification of the 30th base, the base modification mode of sequence SEQ ID NO.2 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th and 22nd bases and BHQ1 modification of the 30th base, and the base modification mode of sequence SEQ ID NO.3 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th and 19th bases and BHQ1 modification of the 30th base.

[0011] Preferably, the specific DNA probe is encapsulated by cationic lipid complex nanoparticles.

[0012] Furthermore, the kit also includes: an extracellular vesicle separation reagent, an extracellular vesicle purification reagent, an extracellular vesicle purification chromatographic column, a negative control substance, a positive control substance, proteinase K and 1×PBS.

[0013] Furthermore, the test subjects of the above-mentioned kit include healthy people, people at high risk of COPD and COPD patients.

[0014] Preferably, the COPD screening marker GLIPR1 is an RNA binding protein with a gene ID number of 11010, and is involved in the degradation process of gene mRNA.

[0015] Furthermore, the present invention also includes a technical solution: use of a GLIPR1 inhibitor in the preparation of a drug for treating COPD, wherein the inhibitor can inhibit the expression of GLIPR1, and the GLIPR1 inhibitor is selected from a small molecule compound or a biological macromolecule, and the biological macromolecule is a small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid that uses the GLIPR1 protein or its transcript as a target sequence and can inhibit the expression of the GLIPR1 protein or gene transcription; or a construct that can express or form the small interfering RNA, dsRNA, microRNA, antisense nucleic acid.

[0016] The advantages of the present invention are:

[0017] For the first time, a diagnostic kit was creatively proposed. The kit is based on EVs detection of GLIPR1 to screen for chronic obstructive pulmonary disease. The experimental results show that the kit has the advantages of rapidity, high efficiency, specificity and high accuracy. The experimental results show that the diagnostic threshold of the kit (cut-off value is 97240810), and the corresponding sensitivity can be as high as 100%, and the specificity can be as high as 80%. It is more convenient and faster than the detection methods in the prior art, can greatly reduce the workload of medical staff, and has a wide range of subjects. At the same time, it will not cause any side effects to the subjects. It is highly practical and has good application prospects.

[0018] Furthermore, the present invention is based on EVs detection. As a liquid biopsy target, EVs have many advantages, and the TIRF imaging used has the characteristics of ultra-micro and ultra-sensitive to fluorescent signals, so that the EVs capture well plate or chip combined with TIRF imaging technology can realize direct imaging of nanoscale vesicles such as EVs and semi-quantitative detection of their contents, greatly improving the detection specificity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Attached Figure 1A-1B Schematic diagram of in situ capture and detection of EVs.

[0020] Attached Figure 2 TIRF was used to detect the expression of GLIPR1 mRNA in plasma EVs from healthy and smoked mice.

[0021] Attached Figure 3 TIRF was used to detect the expression of GLIPR1 mRNA in plasma EVs of healthy subjects and COPD patients. DETAILED DESCRIPTION

[0022] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0023] Example 1 Specific molecular beacon design

[0024] Designing specific molecular beacons for detecting target genes is crucial for detecting specific nucleic acids in EVs capture wells. To this end, the inventors designed a molecular beacon with a special stem-loop structure, combining the characteristics of the target gene. The 5'-end stem and loop are completely complementary to the target gene, the 3'-end stem is partially complementary to the 5'-end stem, the 5'-end and 3'-end are modified with fluorescent groups and quenching groups, respectively, and some bases on the loop are modified with locked nucleic acids. The specific sequence of the specific molecular beacon is shown in Table 1. Among them, the base modification mode of the sequence shown in SEQ ID NO.1 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th and 22nd bases and BHQ1 modification of the 30th base; the base modification mode of the sequence shown in SEQ ID NO.2 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th and 22nd bases and BHQ1 modification of the 30th base; the base modification mode of the sequence shown in SEQ ID NO.3 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th and 19th bases and BHQ1 modification of the 30th base.

[0025] The specific molecular beacon designed by the present invention maximizes the specificity of the molecular beacon binding to the target gene, while reducing the background fluorescence intensity of the reaction. After the molecular beacon is synthesized, in order to verify its specificity of binding to the corresponding target gene and the optimal working temperature, we designed the following Table 2 to select the best molecular beacon and its working temperature according to the highest signal-to-noise ratio.

[0026] GLIPR1-MBs:

[0027] 5-6FAM-CGCGATCATCACTGGCTGTTGGTGATCCGG-BHQ1-3

[0028] 5-6FAM-CGCGATCGGCGGAAGACACAGAAGATCGCG-BHQ1-3

[0029] 5-6FAM-CGCGATCACTTGGTCTCGCTGTCGATCGCG-BHQ1-3

[0030] Table 1

[0031]

[0032]

[0033] Prepare the reaction solution according to the table below, and place the PCR reaction tube directly in the incubator. Incubate at 37, 42, and 50 degrees for 1 hour, respectively. After cooling at room temperature for 10 minutes, add it to the chip sample well and scan its fluorescence intensity with a TIRF microscope to analyze the optimal working temperature of the probe.

[0034] Table 2

[0035]

[0036] GLIPR1-G6MB: GLIPR1 sixth-generation molecular beacon, GLIPR1-G6TP: GLIPR1 positive template, the fluorescence intensity was read by fluorescence plate reader, and the fluorescence intensity was detected by TIRF microscope.

[0037] Example 2 Detection Experiment

[0038] 1 Method

[0039] 1.1 EVs isolation

[0040] (1) Take 100 μl of plasma sample and centrifuge at 12,000 × g for 30 min at room temperature to remove cells and debris;

[0041] (2) Transfer the supernatant to a new EP tube and add 50 μl of EVs precipitation reagent;

[0042] (3) Mix well and incubate at 4°C for 30 min;

[0043] (4) Centrifugation at 10,000 × g for 10 min at room temperature;

[0044] (5) Discard the supernatant, take 50 μl of 1× PBS to resuspend the EVs-rich precipitate, and let it stand at 4°C until use.

[0045] 1.2EVs purification by chromatography column

[0046] (1) Equilibrate the column: add 100 μl of equilibration solution and centrifuge at 9000 × g for 1 min;

[0047] (2) Sample loading: 100 μl of the resuspension solution was loaded onto the column and centrifuged at 9000 × g for 1 min;

[0048] (3) Elution: Add 50 μl of elution buffer and centrifuge at 9000 × g for 3 min.

[0049] 1.3EVs capture plate or chip detection

[0050] (1) taking out a well plate or a chip (each well of the well plate or the chip contains a molecular beacon mixture shown in Table 1, wherein the molecular beacon is encapsulated by cationic lipid composite nanoparticles), and adding the purified EVs eluate into the sample well;

[0051] (2) Add the negative and positive controls in the kit (the negative and positive controls are nematode gene fragments and target gene fragments respectively encapsulated by anionic nanoparticles) to the subsequent sample wells;

[0052] (3) Incubate at 42°C for 1 hour;

[0053] (4) After equilibration at room temperature for 5 min, fluorescence images were collected using a TIRF microscope;

[0054] (5) Use DXimageV1 software to analyze the image, automatically set the cut-off value, and automatically interpret the test sample results.

[0055] 2Test results

[0056] To detect the specificity of the GLIPR1 rapid test kit for COPD screening markers in EVs, we selected plasma from three healthy mice as the control group and plasma from three cigarette smoked mice as the experimental group. According to the test process, the results were obtained as follows: Figure 2 As shown, the fluorescence intensity of the experimental group was significantly higher than that of the control group, indicating that the expression level of GLIPR1 mRNA in the plasma EVs of smoked mice was significantly higher than that of healthy mice.

[0057] We further selected 35 healthy subjects’ plasma as the control group and 40 COPD patients’ plasma as the experimental group. According to the test process, we obtained the following results: Figure 3 As shown, the fluorescence intensity of the experimental group was significantly higher than that of the control group, indicating that the expression level of GLIPR1 mRNA in the plasma EVs of COPD patients was significantly higher than that of healthy subjects.

[0058] The experimental results showed that the diagnostic threshold (cut-off value) for COPD was 97240810, with a corresponding sensitivity of 100% and a specificity of 80%.

[0059] In summary, EVs in COPD patient samples contain abundant COPD screening marker GLIPR1 mRNA-specific nucleic acid fragments.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. SEQUENCE LISTING <110> Zhongshan Hospital Affiliated to Fudan University <120> A rapid detection kit for the extracellular vesicle COPD screening marker GLIPR1 <130> / <160> 3 <170> PatentIn version 3.3 <210> 1 <211> 30 <212> DNA <213> Artificial sequence <400> 1 cgcgatcatc actggctgtt ggtgatcgcg 30 <210> 2 <211> 30 <212> DNA <213> Artificial sequence <400> 2 cgcgatcggc ggaagacaca gaagatcgcg 30 <210> 3 <211> 30 <212> DNA <213> Artificial sequence <400> 3 cgcgatcact tggtctcgct gtcgatcgcg 30

Claims

1. Use of a kit for detecting the extracellular vesicle COPD screening marker GLIPR1 in the preparation of a reagent for diagnosing COPD, characterized in that: The kit comprises an extracellular vesicle in situ capture well plate or an extracellular vesicle in situ capture chip, the extracellular vesicle in situ capture well plate or the extracellular vesicle in situ capture chip contains a fluorescein-labeled molecular beacon, the molecular beacon is a specific DNA probe for detecting GLIPR1 mRNA, a COPD screening marker, the 5' end stem and loop of the specific DNA probe are completely complementary to the target gene, the 3' end stem is partially complementary to the 5' end stem, the 5' end and the 3' end are respectively modified with a fluorescent group and a quenching group, and some bases on the loop are modified with locked nucleic acid. The kit also comprises an instruction manual, which records that the COPD diagnostic threshold is 97240810, the sensitivity is 100%, and the specificity is 80%. The nucleotide sequence of the specific DNA probe is SEQ ID NO.1-3, the base modification mode of the sequence SEQ ID NO.1 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th, and 22nd bases, and BHQ1 modification of the 30th base, and the sequence SEQ ID The base modification method of NO.2 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th, 19th and 22nd bases and BHQ1 modification of the 30th base. The base modification method of sequence SEQ ID NO.3 is: 6FAM modification of the first base, LNA modification of the 10th, 13th, 16th and 19th bases and BHQ1 modification of the 30th base. The specific DNA probe is wrapped by cationic lipid composite nanoparticles. The kit also includes: an extracellular vesicle separation reagent, an extracellular vesicle purification reagent, an extracellular vesicle purification chromatographic column, a negative control, a positive control, proteinase K and 1×PBS.

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