Use of a detection reagent for a marker of mixed connective tissue disease

Abstract

This invention belongs to the field of biomedicine and relates to the application of a detection reagent for a marker of mixed connective tissue diseases. This invention discloses... MEF2C Genes can serve as biomarkers for mixed connective tissue disease; they are found in the peripheral blood of patients with mixed connective tissue disease. MEF2C Gene expression levels were significantly higher in the group than in the healthy group. This invention provides a detection reagent for a mixed connective tissue disease marker, comprising an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4, which can detect... MEF2C Gene expression levels, which can then be used to develop products for the early diagnosis of mixed connective tissue diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of a detection reagent for a marker of mixed connective tissue diseases. Background Technology

[0002] Mixed connective tissue disease (MCTD) is a rare systemic autoimmune disease characterized serologically by high titers of antinuclear antibodies and highly specific anti-U1 ribonucleoprotein antibodies. First proposed by Sharp et al. in 1972, it is considered an overlapping syndrome exhibiting clinical features of multiple connective tissue diseases, including systemic lupus erythematosus, systemic sclerosis, and polymyositis / dermatomyositis.

[0003] In terms of clinical manifestations, MCTD often begins with symptoms such as low-grade fever, fatigue, Raynaud's phenomenon, swelling or hardening of the fingers, symmetrical joint pain or arthritis, and anemia, and can gradually affect multiple systems and organs. Involvement of the heart, lungs, kidneys, and central nervous system is a key factor affecting prognosis, with pulmonary hypertension and interstitial lung disease being the leading causes of death. Due to the diverse symptoms and frequent overlap with other rheumatic diseases, patients often present with atypical and nonspecific symptoms in the early stages of the disease, posing a significant challenge to clinical diagnosis.

[0004] Currently, there is a lack of fundamental treatments that can reverse or cure the disease. The main goals of treatment are to alleviate clinical symptoms, prevent organ damage, and improve long-term prognosis. Commonly used drugs include glucocorticoids, immunosuppressants such as methotrexate, cyclophosphamide, azathioprine, and mycophenolate mofetil, as well as targeted therapies for pulmonary hypertension and interstitial lung disease. These drugs control the disease and improve prognosis by inhibiting abnormal immune responses or specific pathological processes, but they still have the drawbacks of not being able to cure the disease, weakening normal immune defenses, and potentially causing direct organ toxicity.

[0005] The pathogenesis of MCTD remains unclear, involving a complex interplay of genetic susceptibility, environmental factors, and immune system abnormalities. Overall, MCTD is a rare rheumatic disease with a prolonged course, significant clinical heterogeneity, and numerous challenges in diagnosis and treatment.

[0006] MEF2C belongs to the MEF2 transcription factor family and encodes a class of transcription factors containing MADS / MEF2 DNA-binding domains, widely involved in embryonic development and tissue differentiation. MEF2C is expressed in cardiac muscle, skeletal muscle, the central nervous system, and hematopoietic / immune cell lineages, regulating key transcriptional networks involved in cell differentiation, proliferation, apoptosis, and immunity. MEF2C-related pathways include the Toll-like receptor 7 / 8 cascade and inflammation-related transcription. It plays important roles in the nervous, cardiovascular, and immune systems, particularly in regulating T cell differentiation, macrophage function, and inflammatory responses. In autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, aberrant MEF2C expression may promote inflammation by affecting T / B cell function and the interferon signaling pathway.

[0007] Currently, the American College of Rheumatology and the European League Against Rheumatism do not have a definitive diagnostic standard for MCTD. Furthermore, the currently developed biomarkers for MCTD have poor sensitivity and specificity and low efficiency, which means that the diagnostic methods for MCTD still face challenges. Therefore, it is urgent to explore the key regulatory factors involved in the pathogenesis of MCTD and develop more specific biomarkers. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an application of a detection reagent for markers of mixed connective tissue diseases, offering a new strategy for the early diagnosis of mixed connective tissue diseases.

[0009] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of this invention provides an application of a detection reagent for biomarkers of mixed connective tissue diseases, wherein the biomarker is... MEF2C Gene; the detection reagent is for detecting MEF2C Reagents for gene expression levels.

[0010] Furthermore, the detection reagent is the same reagent used in nucleic acid amplification technology.

[0011] Furthermore, the detection reagent is for detecting... MEF2C A primer set for a gene; the primer set comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer being shown in SEQ ID NO.4.

[0012] Furthermore, the application is for the preparation of products for the early diagnosis of mixed connective tissue diseases.

[0013] Furthermore, in the test samples of the subjects MEF2C Upregulation of gene expression indicates that the subjects are susceptible to mixed connective tissue disease.

[0014] Furthermore, the test sample is blood.

[0015] Furthermore, the test sample is peripheral blood.

[0016] Furthermore, the product is a reagent kit.

[0017] A second aspect of the present invention provides a kit comprising the detection reagents for the mixed connective tissue disease markers described above.

[0018] Furthermore, the kit also contains: 110 mL Trizol reagent, 22 mL chloroform, 55 mL isopropanol, 80 mL anhydrous ethanol, 30 mL DEPC-treated water, 300 μL 5x gDNA Clean Reaction Mix, 420 μL 5x Evo M-MLV RT Reaction Mix, 1500 μL 2x SYBR Green Pro Taq HS Premix, and an amplification internal reference gene. GAPDH Primer set.

[0019] Furthermore, the amplified internal reference gene GAPDH The primer set includes an upstream primer and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.2.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovers MEF2C Genes can serve as biomarkers for mixed connective tissue disease; they are found in the peripheral blood of patients with mixed connective tissue disease. MEF2C The gene expression level was significantly higher than that of the healthy group, and the AUC value reached 0.9921 as verified by ROC curve, indicating high diagnostic value. Therefore, this study provides an application of a detection reagent for a marker of mixed connective tissue diseases. This reagent contains an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4. The detection... MEF2C Gene expression levels are used to develop products for the early diagnosis of mixed connective tissue diseases, providing a new strategy for the early diagnosis of these diseases. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 To analyze the peripheral immune cell atlas and differentially expressed genes in MCTD patients. Figure 1 Figure A in the diagram shows the distribution of peripheral immune cells in MCTD patients. Figure 1 Figure B in the figure shows the differentially expressed genes of different immune cell types in MCTD patients.

[0023] Figure 2 for MEF2C Differences in gene expression between MCTD patients and CTRL healthy controls, *** indicates p <0.001.

[0024] Figure 3 This study aimed to reveal heatmaps of transcription factor activity specific to each single-cell subset in MCTD patients and CTRL healthy controls using SCENIC analysis. The SCENIC algorithm was employed to infer the activity of transcription factor regulators in each single-cell subset. AUC scores quantified the activation level of each regulator within the cell, with higher scores indicating stronger activation. The annotation "extended" after the transcription factor name, along with the number of target genes, indicates the regulatory network comprised of that transcription factor and all its target genes. The annotation of only the number of target genes after the transcription factor name indicates a core regulatory network containing only high-confidence target genes, indicated by a black dashed box. MEF2C Gene.

[0025] Figure 4 qRT-PCR detection of MCTD and CTRL in healthy control groups MEF2C mRNA level of genes.

[0026] Figure 5 for MEF2C Differences in expression between MCTD and CTRL healthy control groups and ROC curve analysis. Figure 5 A in the text is MEF2C Differences in expression levels between MCTD patients and healthy CTRL controls, *** indicates p <0.001. Figure 5 B in MEF2C ROC curve analysis of the diagnostic value of MCTD showed an AUC of 0.9921. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1: MEF2C Differential expression analysis between MCTD patients and healthy controls 1. Patient enrollment and sample collection All enrolled cases were patients from the Department of Rheumatology and Immunology, Tongji Hospital, affiliated with Tongji University, Shanghai. Patient cases were collected according to the physician's inclusion and exclusion criteria. The skin of patients and healthy volunteers was routinely disinfected, and 10 mL of peripheral blood was collected into EDTA anticoagulant tubes for subsequent experiments. This study has been approved by the Ethics Committee of Tongji Hospital, Shanghai. Patients provided informed consent and signed informed consent forms, voluntarily providing specimens and clinical data for relevant medical research.

[0029] 2. Preparation and storage of PBMC suspension (1) Take 5 mL of whole blood and transfer it to a 15 mL centrifuge tube. Add 5 mL of PBS solution to dilute the blood and gently pipette to mix. (2) Take 5 mL of Ficoll solution and add it to two new 15 mL centrifuge tubes respectively. Then take 5 mL of diluted blood and slowly add it to the top layer of Ficoll solution in the centrifuge tubes. (3) Centrifuge at room temperature, 2000 rpm, for 30 min; (4) After centrifugation, the blood in the tube will be divided into four layers: plasma, PBMC, separation fluid and red blood cell layer. The cell layer containing PBMC is white. (5) Use a 1mL sterile pipette tip to aspirate the upper layer of liquid from both tubes; (6) Take a new 1mL sterile pipette tip and transfer PBMC into two new 15mL centrifuge tubes respectively; (7) Add PBS to 10-15 mL and gently mix by pipetting. (8) Centrifuge at 1500 rpm at room temperature for 10 min, then slowly remove the supernatant. Repeat twice;

[0030] (9) Take 1 mL of RPMI 1640 complete culture medium to resuspend the cells, and take 10 μL of the cell suspension for cell counting and viability detection; (10) Add complete culture medium according to the number of cells, and adjust the cell concentration of the cell suspension to 2×10⁻⁶. 7 cells / mL; (11) Add an equal volume of cell cryopreservation solution to the cell suspension, mix gently, transfer to a cell cryopreservation tube and place in a programmed cooling box. (12) After placing the programmable cooling box in a -80°C refrigerator overnight, transfer it to liquid nitrogen for long-term storage.

[0031] 3. PBMC resuscitation and single-cell suspension preparation (1) Heat the water bath to 37°C, take the cryovial out of the liquid nitrogen tank, and thaw it quickly in the 37°C water bath; (2) In a clean bench, transfer the cell suspension to a 15 mL centrifuge tube; slowly add complete culture medium to the centrifuge tube to dilute the cells at a ratio of 1:1; (3) Centrifuge at 300g at room temperature for 5 min, then use a pipette to remove most of the supernatant, leaving 1 mL of liquid; (4) Add 9 mL of complete growth medium to the centrifuge tube to reach a total volume of 10 mL, and count the cells using a hemocytometer. (5) 2×10 6 Transfer the cells to a new 15mL centrifuge tube, centrifuge at 300g at room temperature for 5min, and gently aspirate the supernatant without disturbing the cell pellet. (6) Add 1 mL of 1×PBS containing 0.04% BSA, mix gently and transfer the cells to a 2 mL centrifuge tube; (7) Centrifuge at 300g at room temperature for 5 minutes, and slowly aspirate the supernatant without damaging the cell pellet; (8) Add an appropriate volume of 1×PBS containing 0.04% BSA and mix gently with a pipette until a single-cell suspension is formed; (9) Take an appropriate volume of cell suspension, count the cells using a hemocytometer, and calculate the cell viability using 0.4% trypan blue staining. If the cell viability is ≥80%, adjust the cell concentration to 700 cells / μL~1200 cells / μL and place the cell suspension on ice.

[0032] 4. Single-cell capture, library construction, and sequencing 4.1 Preparation of emulsion gel beads (1) Prepare the premixed buffer according to the formula of the premixed buffer, and prepare one well for each sample; each 319.3 μL of premixed buffer contains: 165.4 μL of RT Reagent B, 64.2 μL of PolyT RT Primer, 16.7 μL of Reducing Agent B and 73.0 μL of RT Enzyme C; (2) The chip has three rows of holes from top to bottom. Use a pipette to vertically add the premixed solution and single-cell suspension in hole ① to prevent air bubbles from forming. (3) Take Gel beads, vortex the eight tubes containing Gel beads in a special vortex adapter for 30 seconds, take them out and centrifuge for 5 seconds to remove air bubbles and keep the liquid level. (4) Take 50 μL of Gel beads and slowly inject them into the Gel beads along the bottom of hole ②; (5) Use a pipette to take 45 μL of isolation oil and add it to well ③ along the well wall; (6) Seal the remaining blank channels with 50% glycerol, seal the chip with a 10X pad, move it to the Chromium Controller machine, and run the single-cell GEM packaging program; (7) After 18 minutes, take out 100µL of the GEM suspension in channel ③ and slowly transfer it along the side wall into the eight-tube set on ice; (8) The GEM-RT incubation system was carried out on a PCR instrument, and the reaction conditions were 53℃ for 55 min.

[0033] 4.2 cDNA formation, amplification, and quality control (1) GEMs are collected in a reservoir containing reverse transcriptase. The gel beads dissolve, cells lyse and mRNA is released. The tagged mRNA is reverse transcribed into barcode cDNA in each GEM by the reverse transcriptase. cDNA reverse transcription conditions: 53℃ for 45 min, 85℃ for 5 min, and store at 4℃.

[0034] (2) Take 125µL of Recovery Agent and mix it with GEM to break the oil droplets. After the oil droplets are broken, a large amount of cDNA with barcode and UMI is released. (3) The cDNA was amplified by PCR and purified and enriched by magnetic beads; (4) The concentration of cDNA was detected and quantified using an Agilent bioanalyzer to meet the requirements for library construction.

[0035] 4.3 Constructing sequencing libraries and performing sequencing Construction of 5' transcriptome library: The quality-tested cDNA was fragmented, end-repaired, A-tailed, adapter-ligated, sample-indexed, PCR-purified with magnetic beads to obtain the cDNA library. Finally, the library was quality-tested and sequenced using the NovaSeq6000 sequencing platform.

[0036] 5. Single-cell sequencing data analysis 5.1 Processing of raw Fastq data After sequencing, the corresponding Fastq files are obtained for each sample. This file contains the base information of all genes. The Cell Ranger software provided by 10x Genomics official is used to process the Fastq files to obtain gene expression profile data for downstream analysis. The Cell Ranger software can perform data quality statistics on the raw data, and then use the alignment tool STAR to align with the reference genome downloaded from the 10x Genomics official website. The reference genome is the human GRCh38 genome, and the version number is Human reference (GRCh38)-2024-A; after filtering and correcting barcodes and UMIs, a single-cell gene expression matrix is finally generated.

[0037] 5.2, Cytoplasmic quality control Import the gene expression profile generated by Cell Ranger into Seurat, and perform cell quality control based on the number of genes, the number of UMIs, the proportion of mitochondrial genes, etc. Too high number of genes and UMIs may indicate the presence of doublets, and too high proportion of mitochondrial genes may be caused by cell damage resulting in partial leakage of mRNA in the cytoplasm. The quality control thresholds are UMI < 15000, 200 < count < 4000, and the proportion of mitochondrial genes < 10%.

[0038] 5.3, Single-cell data analysis (1)Data normalization: Use the "NormalizaData" and "ScaleData" functions in the Seurat package to perform data normalization and normalization under default parameters.

[0039] (2)Data dimensionality reduction: Use "Findmarkers" to calculate 2000 highly variable genes for PCA dimensionality reduction.

[0040] (3)Removing batch effects: Due to different experimental batches, non-biological differences may occur between samples. Use "Runharmony" in the Harmony package for sample integration.

[0041] (4)Cell clustering and visualization: Use the "FindNeighbors" function to analyze based on the first 50 principal components of the PCA results, and use the "FindClusters" function for cell clustering.

[0042] (5)Clustering visualization: Use the "RunTSNE" function to visualize single-cell clustering using the t-Stochastic Neighbor Embedding algorithm.

[0043] (6)Removing doublets or multiplets: Use the scDblFinder package to predict and remove doublets.

[0044] (7) Calculation of differentially expressed genes: The “FindAllMarkers” function is used to calculate the differentially expressed genes of each cluster, which are then used as the high-expression genes of that cluster.

[0045] (8) Cell type annotation: Cell type annotation is performed based on the marker genes of different cell types in the Cellmarker gene databases such as Cellmarker and PanglaoDB, as well as the marker genes of PBMC immune cell subsets in the cell type identification tools SingleR and SingleCellNet.

[0046] (9) Gene Network Regulation Analysis: SCENIC is a method for reconstructing gene regulatory networks and identifying cell states based on co-expression and DNA motif analysis of single-cell transcriptome data. It introduces transcription factor motif sequence validation statistical methods to infer gene co-expression networks, which can identify highly reliable transcription factor-dominated GRNs. GRNs include transcription factors, co-regulatory factors, and their regulated target genes, determining the transcriptional state of cells. SCENIC uses GENIE3 combined with RcisTarget cis-regulatory motif analysis to obtain upstream TFs and their potential direct target genes, referred to as regulators.

[0047] To clarify the peripheral blood immune cell types in MCTD, this invention first analyzed single-cell sequencing data from MCTD and healthy controls, clarifying the immune cell atlas of MCTD. For example... Figure 1 As shown in A, 15 cell types were mainly obtained: monocytes, NK cells, CD8 effector T cells, CD8 naive T cells, CD8 memory T cells, B cells, CD4 naive T cells, CD4 memory T cells, γδ T cells, megakaryocytes, dendritic cells (DCs), regulatory T cells, mucosa-associated invariant T cells, proliferating NK cells, and plasmablasts. This invention further analyzed differentially expressed genes in different PBMC cell subpopulations from MCTD patients and healthy controls, such as... Figure 1 As shown in B in the diagram.

[0048] This invention extracts single-cell sequencing data from MCTD and healthy control groups after Pseudobulk analysis. MEF2C Gene expression data, such as Figure 2 and Figure 3 As shown, the analysis revealed that, compared to healthy controls, MEF2C Transcription factor activity was enhanced in MCTD patients, and expression levels were significantly upregulated, with statistically significant differences.

[0049] SCENIC is a method based on co-expression and DNA motif analysis to reconstruct gene regulatory networks and identify cell states from single-cell transcriptome data. The SCENIC algorithm is used to predict transcription factors potentially involved in MCTD pathogenesis in PBMC subsets, and the AUCell algorithm is used to calculate regulator-specific scores to identify specific regulators in each group, resulting in... Figure 3 The heatmaps showing transcription factor activity specific to each single-cell subset indicate that regulators MEF2C The activity of B cells, plasma cells, dendritic cells and monocytes in MCTD patients was enhanced, with a more significant effect on B cells.

[0050] Example 2: MEF2C Elevated expression in peripheral blood of MCTD patients Peripheral blood was collected from MCTD patients at the Department of Rheumatology and Immunology, Tongji Hospital, affiliated with Tongji University, Shanghai. After routine disinfection of the skin of patients and healthy volunteers, 2 mL of peripheral blood was collected into EDTA anticoagulant tubes for subsequent experiments.

[0051] 1. RNA extraction and qRT-PCR 1.1 Total RNA extraction and purity detection (1) Take 1 mL of whole blood into a new EP tube, add 200 μL of Trizol lysis buffer, mix thoroughly by pipetting, and let stand at room temperature for 5 min.

[0052] (2) Add 200 μL of chloroform to the EP tube, shake vigorously for 30 seconds, and let stand at room temperature for 10 minutes until the liquid separates into layers.

[0053] (3) After balancing the EP tube, place it in a centrifuge at 4℃ and centrifuge at 12000rpm for 15min. The liquid surface is divided into three layers: the upper layer is a colorless supernatant, the middle layer is a white layer, and the lower layer is a red layer.

[0054] (4) Slowly transfer the colorless supernatant to a new EP tube, add an equal volume of isopropanol to the supernatant, mix thoroughly, and let stand at room temperature for 10 minutes.

[0055] (5) After balancing the EP tube, place it in a centrifuge at 4°C and centrifuge at 10,000 rpm for 10 minutes. Gently remove the upper liquid and retain the white precipitate.

[0056] (6) Slowly add 1 mL of 75% ethanol along the tube wall with a pipette, centrifuge at 7500 rpm for 5 min at 4℃, and wash twice.

[0057] (7) Add an appropriate amount of RNAase-free H2O to dissolve the RNA precipitate according to the amount of precipitate.

[0058] (8) 2% agarose gel electrophoresis was used to detect whether RNA was degraded or contaminated with impurities.

[0059] (9) The purity and concentration of RNA were determined using NanoDrop2000.

[0060] (10) The RNA integrity value (RIN) was detected by Agilent 2100 and then frozen at -80℃.

[0061] 1.2 cDNA Synthesis (1) gDNA removal: Dissolve the reagents in the reaction system on ice beforehand, and add the following reaction system to the PCR tube.

[0062] Reaction system: 1 μL gDNA Eraser, 2 μL 5×gDNA Eraser Buffer, 1 μg RNA, and RNase FreeH2O to a final volume of 10 μL.

[0063] (2) Gently tap the bottom of the tube to mix the above reagents, centrifuge for 3 seconds, and run the following PCR instrument program: 42℃ for 2 min; store at 4℃.

[0064] (3) Obtain RNA samples with gDNA removed and prepare reverse transcription system according to Table 1.

[0065] Table 1 Reverse transcription system (4) Run the following PCR program on the reverse transcription system prepared according to Table 1 for reverse transcription: 37℃ for 15 min; 85℃ for 5 s; store at 4℃.

[0066] (5) The cDNA obtained by reverse transcription can be stored at -20℃ or used for subsequent qPCR experiments.

[0067] 1.3, according to MEF2C Primers for real-time quantitative PCR (1) According to MEF2C Gene sequence, design amplification MEF2C The primer set for the gene was prepared by dissolving the dry primer powder in NFW and diluting the primer concentration to 10 μM. The primer sequences are shown in Table 2.

[0068] Table 2 Primer Sequences (2) Dilute the reverse transcribed cDNA 10 times with NFW1 and place it on ice for later use.

[0069] (3) Prepare the reaction system for each sample well according to Table 3.

[0070] Table 3 Real-time quantitative PCR reaction system (4) When using a 384-well plate, avoid light when adding samples. After adding samples, seal the plate with the high-transmittance film that comes with the plate, remove it for 10 seconds, and place it on ice in preparation for instrument testing.

[0071] (5) Run the following PCR instrument program: 95℃ 30s; 95℃ 5s; 60℃ 30s; 95℃ 15s; 60℃ 1min; 95℃ 1s.

[0072] 1.4, -2 ΔΔCT Measurement of indicators and plotting of ROC curves The data from this experiment were analyzed using a relatively quantitative method. GAPDH As internal reference genes, the data were analyzed using the software GraphPad Prism 9.

[0073] qRT-PCR was used to detect the presence of [unclear] in blood samples from MCTD patients and healthy controls. MEF2C mRNA expression levels, results as follows Figure 4 As shown, the findings are consistent with MEF2C Compared to expression in healthy controls, MCTD patients showed [significance / expression / expression]. MEF2C The expression of [a specific substance] was significantly upregulated, and the difference was statistically significant. This result also demonstrates the presence of transcription factors [in MCTD]. MEF2C The phenomenon of enhanced activity.

[0074] Example 3: Peripheral blood MEF2C Correlation study between expression and pathogenesis of MCTD Peripheral blood was collected from MCTD patients and healthy controls at the Department of Rheumatology and Immunology, Tongji Hospital, affiliated with Tongji University in Shanghai. 2 mL of peripheral blood was taken into EDTA anticoagulant tubes for subsequent experiments.

[0075] The experimental method is the same as in Example 2.

[0076] The results are as follows Figure 5 Analysis of A in the figure revealed that, with MEF2C Compared to expression in healthy controls, MCTD patients showed [significance / expression / expression]. MEF2C The expression was significantly upregulated, with a statistically significant difference. The results were processed, and an ROC curve was plotted, as shown below. Figure 5 As shown in B, the AUC can reach 0.9921. From the results, we can see that... MEF2C It has high diagnostic value. Therefore, genes MEF2C Used for the diagnosis of mixed connective tissue diseases.

[0077] Example 4: A kit for screening and early diagnosis of mixed connective tissue disease. In this embodiment, a kit is provided for screening and early diagnosis of mixed connective tissue diseases in high-risk groups. The kit is used to detect the levels of certain substances in the peripheral blood of subjects. MEF2C The expression level of mRNA was detected using a kit containing the following reagents, with 100 reactions per kit: 1. Trizol reagent 110 mL 2. 22 mL of chloroform 3. Isopropanol 55mL 4. 80 mL of anhydrous ethanol 5. DEPC treated water 30mL 6.5x gDNA Clean Reaction Mix 300μL 7.5x Evo M-MLV RT Reaction Mix420μL 8.2x SYBR Green Pro Taq HS Premix 1500μL 9.10μM MEF2C 150 μL of specific primers for real-time quantitative PCR, the specific sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0078] 10.10μM GAPDH 150 μL of specific primers for real-time quantitative PCR, the specific sequences of which are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0079] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a detection reagent for a mixed connective tissue disease marker, characterized in that, The biomarker is MEF2C Gene; the detection reagent is for detecting MEF2C Reagents for gene expression levels.

2. The application of the detection reagent for mixed connective tissue disease markers according to claim 1, characterized in that, The detection reagent is for detection. MEF2C A primer set for a gene; the primer set comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer being shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer being shown in SEQ ID NO.

4.

3. The application of the detection reagent for mixed connective tissue disease markers according to claim 1, characterized in that, The application is for the preparation of products for the early diagnosis of mixed connective tissue diseases.

4. The application of the detection reagent for mixed connective tissue disease markers according to claim 3, characterized in that, In the test samples of the subjects MEF2C Upregulation of gene expression indicates that the subjects are susceptible to mixed connective tissue disease.

5. The application of the detection reagent for mixed connective tissue disease markers according to claim 4, characterized in that, The test sample was blood.

6. The application of the detection reagent for mixed connective tissue disease markers according to claim 5, characterized in that, The product in question is a reagent kit.

7. A reagent kit, characterized in that, The kit contains the detection reagent for the mixed connective tissue disease marker as described in claim 1.

8. The reagent kit according to claim 8, characterized in that, The kit also contains 110 mL of Trizol reagent, 22 mL of chloroform, 55 mL of isopropanol, 80 mL of anhydrous ethanol, 30 mL of DEPC-treated water, 300 μL of 5x gDNA Clean Reaction Mix, 420 μL of 5x Evo M-MLV RT Reaction Mix, 1500 μL of 2x SYBR Green Pro Taq HS Premix, and an amplification internal control gene. GAPDH Primer set.

9. The reagent kit according to claim 8, characterized in that, The amplified internal reference gene GAPDH The primer set includes an upstream primer and a downstream primer, the nucleotide sequence of which is shown in SEQ ID NO.1 and the nucleotide sequence of which is shown in SEQ ID NO.2.

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