Application of reagents for detecting biomarkers in the preparation of kits for diagnosing asthma.

Abstract

This invention proposes the application of a reagent for detecting biomarkers in the preparation of a kit for diagnosing asthma. The biomarkers of this invention are any one or more of PDK4, TM4SF1, WIF1, and WNT5A. The reagent for detecting biomarkers of this invention is used to detect the expression level and / or methylation level of the biomarker in a sample. It can be used in a kit for diagnosing asthma, enabling early and accurate diagnosis of asthma with excellent diagnostic efficacy. This allows for the screening of drugs for the prevention or treatment of asthma, solving the problems of inaccurate judgment and inconvenience in traditional techniques that rely on clinical manifestations, pulmonary function tests, or peak flow rate measurements for asthma diagnosis. This fills a gap in asthma diagnostic kits and provides a fast and convenient method for asthma diagnosis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of reagents for detecting biomarkers in the preparation of kits for diagnosing asthma. Background Technology

[0002] Asthma is a chronic inflammatory airway disease involving various cells and cellular components, including airway epithelial cells, eosinophils, and mast cells. It is characterized by airway inflammation, airway hyperresponsiveness, and airway remodeling. In recent years, the prevalence of asthma has been increasing annually, becoming one of the most serious chronic airway inflammatory diseases threatening human health. Its recurrent attacks can lead to chronic obstructive pulmonary disease, bronchiectasis, and pulmonary heart disease, severely impacting patients' quality of life and creating a heavy social burden. For most asthma patients, traditional therapies such as corticosteroids and bronchodilators are effective, controlling clinical symptoms. However, some patients still experience persistent symptoms. Therefore, it is essential to improve the diagnosis and monitoring of asthma to identify susceptible individuals for early preventative treatment. Traditional diagnostic techniques rely on clinical manifestations, pulmonary function tests, or peak flow rate measurements. However, the clinical manifestations of asthma are nonspecific, and pulmonary function tests are not sensitive indicators; some asthma patients show normal or minimal changes in pulmonary function tests. Therefore, a diagnostic kit for asthma is needed. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides the application of a reagent for detecting biomarkers in the preparation of a kit for diagnosing asthma. The technical solution of this invention is implemented as follows:

[0004] In one aspect, the present invention relates to the use of a reagent for detecting a biomarker in the preparation of a kit for diagnosing asthma, wherein the biomarker is any one or more of PDK4, TM4SF1, WIF1, and WNT5A.

[0005] The reagent for detecting biomarkers of this invention is used to detect the expression level and / or methylation level of biomarkers in a sample. It can be used in asthma diagnostic kits, enabling early and accurate diagnosis of asthma with excellent diagnostic efficacy. This allows for the screening of drugs for the prevention or treatment of asthma, overcoming the problems of inaccurate judgment and inconvenience in traditional asthma diagnosis techniques that rely on clinical manifestations, pulmonary function tests, or peak flow rate measurements. It fills a gap in asthma diagnostic kits, providing a rapid and convenient method for asthma diagnosis. The diagnostic efficacy of combined biomarkers is superior to that of single biomarkers. This invention describes the use of a reagent for detecting the expression level and / or methylation level of biomarkers in a sample in the preparation of a kit for diagnosing asthma, where the sample includes tissues and body fluids.

[0006] As a preferred embodiment, the kit is any one of a qPCR kit, immunoblotting kit, immunochromatographic assay kit, flow cytometry kit, immunohistochemistry kit, ELISA kit, and electrochemiluminescence assay kit. This is a kit for diagnosing asthma, containing reagents to detect the expression level and / or methylation level of biomarkers in a sample. The biomarkers are any one or more of PDK4, TM4SF1, WIF1, and WNT5A, with combinations of multiple PDK4, TM4SF1, WIF1, and WNT5A showing better efficacy.

[0007] As a preferred embodiment, the reagent is one that detects the expression level of the marker by polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, RNA blotting, or DNA chip.

[0008] As a preferred embodiment, the polymerase chain reaction is any one of real-time quantitative reverse transcription polymerase chain reaction, reverse transcription polymerase chain reaction, and competitive polymerase chain reaction.

[0009] As a preferred embodiment, the reagents further include reagents for detecting the methylation level of the marker by methylation chip, methylation-specific PCR, bisulfite sequencing, restriction endonuclease analysis combined with sodium bisulfite, quantitative fluorescence method, high-throughput sequencing, pyrosequencing quantification, DNA blotting, restriction marker genome scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, and mass spectrometry.

[0010] In a preferred embodiment, the reagent includes an antibody specific to the biomarker, a probe specific to the biomarker, and / or primers specific to the biomarker.

[0011] In another aspect, the present invention provides a kit for diagnosing asthma, the kit comprising reagents for detecting biomarkers, said biomarkers being any one or more of PDK4, TM4SF1, WIF1, and WNT5A.

[0012] This kit contains reagents to detect the expression level and / or methylation level of biomarkers in samples. The biomarkers are any one or more of PDK4, TM4SF1, WIF1, and WNT5A. Using this kit to diagnose asthma enables early and accurate diagnosis of asthma, with excellent diagnostic efficacy. This allows for the screening of drugs for the prevention or treatment of asthma, and solves the problems of inaccurate judgment and inconvenience in traditional techniques that rely on clinical manifestations, pulmonary function tests, or peak flow rate measurements to diagnose asthma. It fills the gap in asthma diagnostic kits and provides a fast and convenient method for diagnosing asthma.

[0013] In another aspect, the present invention provides the application of a biomarker in screening drugs for the prevention or treatment of asthma, wherein the biomarker is any one or more of PDK4, TM4SF1, WIF1, and WNT5A. The present invention also provides the application of a biomarker in screening drugs for the prevention or treatment of asthma, wherein the biomarker is any one or more of PDK4, TM4SF1, WIF1, and WNT5A.

[0014] As a preferred embodiment, the method for screening drugs for the prevention or treatment of asthma includes the following steps: 1) In the test group, administering a test compound to the test subject and detecting the level V1 of the biomarker in samples derived from the test subject in the test group; in the control group, administering a blank control to the test subject and detecting the level V2 of the biomarker in samples derived from the test subject in the control group; 2) Comparing the levels V1 and V2 detected in step 1) to determine whether the test compound is a candidate compound for the prevention or treatment of asthma. The levels include expression levels and / or methylation levels, and the test subject is an in vitro cell system, a sample separated from a living organism.

[0015] In another aspect, the present invention also provides an application of biomarkers in constructing a diagnostic system for asthma, wherein the biomarkers are any one or more of PDK4, TM4SF1, WIF1, and WNT5A, and the diagnostic system includes: (1) a detection unit: including a biomarker detection module; (2) an analysis unit: using the expression level and / or methylation level of the biomarkers detected by the detection unit as input variables, inputting them into an asthma diagnostic model for analysis, wherein the asthma diagnostic model is any one or more of XGBoost, Random Forest, glmnet, cforest, machine learning classification and regression trees, treebag, K-adjacency, neural networks, radial support vector machines, linear support vector machines, Naive Bayes, or multilayer perceptron; (3) an evaluation unit: outputting the risk value of the individual corresponding to the sample for asthma. This is a computer diagnostic system, and the asthma diagnostic model is determined using the above-mentioned algorithms.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The reagent for detecting biomarkers in the present invention is a reagent for detecting the expression level and / or methylation level of biomarkers in samples. It can be used in a kit for diagnosing asthma. By using this kit to diagnose asthma, early and accurate diagnosis of asthma can be achieved, with good diagnostic efficacy. This allows for the screening of drugs for the prevention or treatment of asthma. It solves the problems of inaccurate judgment and inconvenient operation of traditional techniques that rely on clinical manifestations, pulmonary function tests or peak flow rate measurements to diagnose asthma. It fills the gap in kits for diagnosing asthma and provides a fast and convenient method for diagnosing asthma. Attached Figure Description

[0017] Figure 1 Box plot of differential expression of PDK4 in GSE64913 dataset;

[0018] Figure 2 Box plot of differential expression of TM4SF1 in the GSE64913 dataset;

[0019] Figure 3 Box plot of differential expression of WIF1 in the GSE64913 dataset;

[0020] Figure 4 Box plot of differential expression of WNT5A in the GSE64913 dataset;

[0021] Figure 5 The ROC curve analysis results for PDK4 in the GSE64913 dataset are shown in the figure.

[0022] Figure 6 The ROC curve analysis results for TM4SF1 in the GSE64913 dataset are shown in the figure.

[0023] Figure 7 The ROC curve analysis results for WIF1 in the GSE64913 dataset are shown in the figure.

[0024] Figure 8 The ROC curve analysis results for WNT5A in the GSE64913 dataset are shown in the figure.

[0025] Figure 9 The ROC curve analysis results for PDK4+WIF1+WNT5A in the GSE64913 dataset are shown in the figure.

[0026] Figure 10 The ROC curve analysis results for PDK4+TM4SF1+WIF1+WNT5A in the GSE64913 dataset are shown in the figure.

[0027] Figure 11 This is a real-time amplification curve of the internal reference GAPDH gene.

[0028] Figure 12 The melting curve of the internal reference GAPDH gene product;

[0029] Figure 13 This is a real-time amplification curve of the internal reference ACTB gene.

[0030] Figure 14 The melting curve of the internal control ACTB gene product;

[0031] Figure 15 This is a real-time amplification curve of the PDK4 gene.

[0032] Figure 16 Melting curve of PDK4 gene product;

[0033] Figure 17 This is a real-time amplification curve of the WIF1 gene.

[0034] Figure 18 The melting curve of the WIF1 gene product;

[0035] Figure 19 This is a real-time amplification curve of the WNT5A gene.

[0036] Figure 20 Melting curve of WNT5A gene product;

[0037] Figure 21 Scatter plot for Real-time PCR validation of differential PDK4 gene expression;

[0038] Figure 22 Scatter plot for Real-time PCR validation of differential expression of the WIF1 gene;

[0039] Figure 23 Scatter plot for Real-time PCR validation of differential expression of the WNT5A gene;

[0040] Figure 24 ROC curve analysis results for validating the diagnostic efficacy of PDK4 using real-time PCR;

[0041] Figure 25 ROC curve analysis results for validating the diagnostic efficacy of WIF1 using real-time PCR;

[0042] Figure 26 ROC curve analysis results for Real-time PCR validation of the diagnostic efficacy of WNT5A. Detailed Implementation

[0043] definition

[0044] Unless otherwise specified, the following terms as used herein have their own meanings.

[0045] biomarkers, differentially expressed genes

[0046] The term "marker" refers to a molecule that is quantitatively or qualitatively associated with the presence of a biological phenomenon. Examples of "markers" include polynucleotides such as genes or gene fragments, RNA or RNA fragments; or polypeptides such as peptides, oligopeptides, proteins, or protein fragments; or any metabolites, byproducts, or any other identifying molecules such as antibodies or antibody fragments, whether or not they are directly or indirectly related to the mechanism of the phenotype. The markers of this invention include nucleotide sequences (i.e., GenBank sequences) as disclosed herein, particularly full-length sequences, any coding sequences, any fragments, or any complements thereof.

[0047] In this invention, biomarkers, such as PDK4 (gene ID: 5166), TM4SF1 (gene ID: 4071), WIF1 (gene ID: 11197), and WNT5A (gene ID: 7474), include genes and their encoded proteins, as well as their homologs, mutations, and isotypes. This term encompasses full-length, unprocessed biomarkers, as well as biomarkers of any form derived from cell processing. This term also encompasses naturally occurring variants of the biomarker (e.g., splice variants or allelic variants).

[0048] The terms “differentially expressed gene,” “differential gene expression,” and similar phrases refer to genes whose expression is activated to higher or lower levels in a test sample (e.g., a disease), particularly in diseases such as asthma, relative to their expression in a control sample (e.g., a control sample). These terms also include genes whose expression is activated to higher or lower levels in: different stages of the same disease; in relapsing or non-relapsing disease; or in cells with higher or lower levels of proliferation. Differentially expressed genes may be activated or repressed at the polynucleotide or polypeptide level, or may undergo variable splicing to result in different polypeptide products. Such distinctions can be demonstrated, for example, by changes in mRNA levels, surface expression, polypeptide secretion, or other partitioning.

[0049] Differential gene expression can include comparing the expression of two or more genes or their gene products; or comparing the expression ratios of two or more genes or their gene products; or comparing different processed products of the same gene that differ between normal and diseased subjects; or between different stages of the same disease; or between relapsed and non-relapsed diseases; or between cells with higher and lower proliferation levels; or between normal and diseased tissues. Differential expression includes quantitative and qualitative differences in the temporal or cellular expression profiles of genes or their expression products in, for example, normal and diseased cells, or in cells experiencing different disease events or stages, or in cells with different proliferation levels.

[0050] The term "expression" encompasses the production of polynucleotides and polypeptides, particularly the production of RNA (e.g., mRNA) from a gene or a portion of a gene, and includes the production of proteins encoded by RNA or a gene or a portion of a gene, as well as the presence of detectable substances associated with expression. For example, the formation of complexes arising from protein-protein interactions, protein-nucleotide interactions, or similar interactions is also included within the scope of the term "expression." Other examples include the visualization of binding ligands, such as hybridization probes or antibodies, to genes or other oligonucleotides, proteins, or protein fragments. Therefore, the intensity of spots on microarrays, hybridization blots (e.g., Northern blotting), or immunoblotting (e.g., Western blotting), or bead arrays, or the intensity as analyzed by PCR, is included within the scope of the biomolecular term "expression" as described.

[0051] The term "oligonucleotide" refers to polynucleotides, generally probes or primers, including but not limited to: single-stranded deoxynucleotides, single-stranded or double-stranded nucleotides, RNA:DNA hybrids, and double-stranded DNA. Oligonucleotides, such as single-stranded DNA probes, are often synthesized using chemical methods, such as by using commercially available automated oligonucleotide synthesizers, or by a variety of other methods, including in vitro expression systems, recombinant technologies, and expression in cells and organisms.

[0052] When used in the singular or plural, the term "polynucleotide" generally refers to any polynucleotide or polydeoxynucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. This includes, but is not limited to, single-stranded and double-stranded DNA, DNA including single-stranded and double-stranded regions, single-stranded and double-stranded RNA, RNA including single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or more generally double-stranded or include single-stranded and double-stranded regions. It also includes triple-stranded regions comprising RNA or DNA or both RNA and DNA. In particular, it includes mRNA, cDNA, and genomic DNA. The term includes DNA and RNA comprising one or more modified bases such as tritium bases or rare bases such as inosine. The polynucleotides of the present invention can comprise coding or non-coding sequences, or sense or antisense sequences.

[0053] As used herein, “polypeptide” refers to oligopeptides, peptides, or protein sequences, or fragments thereof, and naturally occurring, recombinant, synthetic, or semi-synthetic molecules. The term “polypeptide” as used herein refers to the amino acid sequence of a naturally occurring protein molecule, and “polypeptide” and similar terms are not intended to limit the amino acid sequence to the complete natural amino acid sequence of the full-length molecule. It should be understood that each use of “polypeptide” or similar term herein includes the full-length sequence and any fragments, derivatives, or variants thereof.

[0054] The term "methylation" used in this article refers to a natural modification of DNA. In eukaryotes, it primarily refers to the process by which a methyl group is attached to the 5-carbon position of the cytosine at the 5' end of a CpG dinucleotide, transforming it into 5-methylcytosine (5-mC), under the action of methylated CpG-binding domains (MBD) and DNA methyltransferases (DNMT). DNA methylation is a well-studied form of epigenetics with multifaceted biological significance. It is closely related to normal embryonic development, gene expression regulation, X chromosome inactivation in female individuals, inhibition of parasitic DNA sequences, imprinted genes, and genome structural stability. Significant progress has been made in research on DNA methylation mechanisms, DNA methyltransferases, methylation transcriptional repression mechanisms, the relationship between methylation and tumors and diseases, and detection methods. DNA methylation research is becoming an important frontier area in disease and tumor research.

[0055] As used herein, the term "sample" refers to a biological sample obtained or derived from a source of purpose as described herein. In some embodiments, the source of purpose includes an organism, such as an animal or a human. In some embodiments, the biological sample includes biological tissue or fluid. In some embodiments, the biological sample may be or includes bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; body fluids containing cells; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; skin swabs; oral swabs; nasal swabs; washings or lavages, such as catheter lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions and / or excretions; and / or cells therein, etc. In some embodiments, the biological sample is or includes cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from the source of purpose by any suitable means. For example, in some embodiments, primary biological samples are obtained by methods selected from: biopsy (e.g., fine-needle aspiration or tissue biopsy), surgical tissue, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term "sample" refers to a preparation obtained through processing (e.g., by removing one or more components of the primary sample and / or by adding one or more reagents to the primary sample). For example, semi-permeable membrane filtration is used. Such "processed samples" may contain, for example, nucleic acids or proteins extracted from the sample or obtained by techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components of the primary sample. In specific embodiments of the invention, the sample is blood or airway epithelial tissue.

[0056] Reagent test kit

[0057] This invention provides a kit for diagnosing asthma in subjects, the kit being used to determine the expression levels and / or methylation levels of the aforementioned biomarkers. The kit may include materials and reagents suitable for selectively detecting the presence of biomarkers or biomarker groups for diagnosing asthma in samples derived from subjects.

[0058] In a further embodiment, the kit may contain instructions for appropriate operating parameters in the form of labels or product inserts. For example, the instructions may include information or guidance on how to collect samples, how to determine the levels of one or more biomarkers in the samples, or how to correlate the levels of one or more biomarkers in the samples with a subject having asthma.

[0059] In another embodiment, the kit may contain one or more containers with a marker sample for use as a reference standard, a suitable control, or for calibration to detect the marker in the test sample.

[0060] The term "subject" refers to any animal (e.g., mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, etc. Furthermore, a subject is a human subject. The terms "subject," "individual," and "patient" are used interchangeably herein.

[0061] Unless otherwise stated, the present invention will be practiced using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which fall within the scope of the art. Such techniques are well described in the literature, for example: *Molecular Cloning: A Laboratory Manual*, 2nd edition, Sambrook et al., 1989; *Oligonucleotide Synthesis*, edited by MJ Gait, 1984; *Animal Cell Culture*, edited by R.R. Freshney, 1987; *Methods in Enzymology*, Academic Press, Inc.; *Handbook of Experimental Immunology*, 4th edition, edited by D.M. Weir & C. Blackwell, Blackwell Science Inc., 1987; *Gene Transfer Vectors for Mammalian Cells*, edited by J.M. Miller & M.P. Calos, 1987; *Current Protocols in Molecular Biology*, edited by F.M. Ausubel et al., 1987; and *PCR: The Polymerase Chain Reaction*, edited by Mullis et al., 1994.

[0062] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. 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.

[0063] Example 1: Differential Expression Gene Analysis Based on Aberrant Methylation Modification

[0064] I. Experimental Methods

[0065] 1. NCBI GEO (Gene Expression Omnibus) Data Retrieval and Analysis

[0066] The GEO (Gene Expression Omnibus) database is developed and maintained by NCBI (National Center for Biotechnology Information). As the largest database of gene expression data, GEO primarily contains microarray data, but also includes some non-microarray data, such as SAGE (Gene Expression Serial Analysis) data, SARST (Synthetic Tagged Sequence Analysis) data, MS (Mass Spectrometry) data, proteomics data, and next-generation high-throughput sequencing data (MPSS, massively parallel sequencing technology).

[0067] (1) Search keywords

[0068] (Asthma)AND"Homo sapiens"[porgn:__txid9606]

[0069] (2) Sample selection strategies in the study

[0070] Data sets that meet the following criteria and are restricted to the study types "Expression profiling by array" and "Methylation profiling by array" will be included in the study: ① The selected dataset must be whole-genome mRNA transcriptome data and DNA methylation data; ② These data are from asthma and control airway epithelial samples; ③ This study considers both standardized or raw datasets; After screening, one set of mRNA dataset and one set of methylation dataset were obtained, which are listed in Table 1 and Table 2, respectively.

[0071] Table 1. mRNA datasets retrieved from the GEO database

[0072]

[0073] Table 2. Methylation datasets retrieved from the GEO database.

[0074]

[0075] 2. Results of integrated analysis based on high-throughput transcriptome and methylation data

[0076] (1) Differential analysis of mRNA

[0077] The GSE64913 dataset was downloaded from the GEO database. Probes were mapped to genes, and the average value of multiple probes for a single gene was taken as the gene expression level. Differential expression analysis of the mRNA dataset was performed using the limma package in R-4.0.5. The screening criteria were set as P.Value < 0.05 & |logFC| > 0.5. Analysis yielded 245 differentially expressed genes, including 113 upregulated and 132 downregulated genes.

[0078] (2) Differential methylation analysis

[0079] Differential methylation analysis was performed on the methylation data using the CHAMP package. The screening criteria were P.Value < 0.05 & |deltaBeta| > 0.1, resulting in 8447 differential methylation sites and a total of 3494 differentially methylated genes, including 1351 hypermethylated genes and 2143 hypomethylated genes.

[0080] (3) Differentially expressed genes modified by aberrant methylation

[0081] By taking the intersection of differentially expressed mRNA genes and differentially methylated genes, differentially expressed genes regulated by abnormal methylation were obtained, resulting in 10 genes downregulated by hypermethylation and 14 genes upregulated by hypomethylation.

[0082] 3. Diagnostic efficacy analysis

[0083] Based on the expression levels of differentially expressed genes with abnormal methylation modifications involved in this invention, ROC curves were plotted using the pROC package in the R language.

[0084] II. Experimental Results

[0085] (1) The expression of the genes (i.e., markers) of the present invention is shown in Table 3. (Table 3 and Appendix) Figure 1 Appendix Figure 2 Appendix Figure 3 and appendix Figure 4 It can be seen that, compared with the normal control, PDK4 and TM4SF1 were significantly upregulated in asthma patients, while WIF1 and WNT5A were significantly downregulated.

[0086] Table 3. Expression of markers

[0087] Gene logFC AveExpr P.Value up / down PDK4 0.540 5.376 0.000 up TM4SF1 0.641 4.106 0.000 up WIF1 -0.822 3.763 0.012 down WNT5A -0.672 4.465 0.000 down

[0088] (2) The diagnostic efficacy of the genes (i.e., biomarkers) of the present invention is shown in Table 4. In assessing the diagnostic efficacy, the first factor considered is the AUC value; the higher the AUC value, the better the diagnostic efficacy. Secondly, sensitivity and specificity can also be considered. (Table 4 and Appendix) Figure 5 Appendix Figure 6 Appendix Figure 7 Appendix Figure 8 Appendix Figure 9 and appendix Figure 10 It can be seen that the biomarkers of the present invention have good diagnostic efficacy for diagnosing asthma, and the diagnostic efficacy of a combination of multiple biomarkers is better than that of a single biomarker.

[0089] Table 4. Diagnostic efficacy of the biomarkers of this invention in the GSE64913 dataset.

[0090]

[0091]

[0092] Example 2: Real-time PCR Validation

[0093] I. Experimental Materials

[0094] 1. Sample List

[0095] The laboratory provided 30 human blood samples, including 17 control samples (sample names 1-17) and 13 samples from asthma patients (sample names 18-30).

[0096] 2. Main reagents for the experiment

[0097] Table 5 List of Reagents Used

[0098]

[0099] 3. Main experimental instruments

[0100] Table 6 List of Instruments Used

[0101] Instrument Name Instrument Model factory centrifuge Centrifuge 5424R Eppendorf NanoVue Plus 28956057 BIOCHROM LTD Real-time PCR instrument ABI7300 Applied Biosystems

[0102] II. Experimental Methods

[0103] 1. Primer design

[0104] Primers for Real-Time PCR detection of the target gene are listed in Table 7. The primers in Table 7 were synthesized by BOMIDE.

[0105] Table 7 Primer sequences

[0106]

[0107] 2. Experimental Procedure

[0108] (1) Extract total RNA from the sample

[0109] ① Add 0.75 mL of lysis buffer RLS to every 0.25 mL of liquid sample, and pipette the liquid sample several times to help lyse the cells in the sample. Repeat every 5–10 × 10⁻⁶ mL. 6 Add at least 0.75 mL of lysis buffer RLS to each cell. The final volume ratio of lysis buffer RLS to liquid sample is always 3:1.

[0110] ② Add 0.75 mL of lysis buffer RLS to the EP tube, then add 0.25 mL of blood sample, shake vigorously for 30 seconds to mix, and incubate at 15-30℃ for 10 minutes to allow complete decomposition of ribosomes.

[0111] ③ Add 0.2 mL of chloroform to every 0.75 mL of lysis buffer RLS, shake vigorously for 15 s and let stand at room temperature for 5 min.

[0112] ④ Centrifuge at 12000 rpm for 10 min at 4℃. The sample will separate into three layers: a lower organic phase, a middle layer, and an upper colorless aqueous phase. RNA is present in the upper aqueous phase. The volume of the aqueous phase layer is approximately 70% of the volume of the added RLS. Transfer the aqueous phase to a new tube for the next step.

[0113] ⑤ Add 1 volume of 70% ethanol and mix by inverting (precipitation may occur at this time); transfer the resulting solution and any possible precipitate into the adsorption column RA (the adsorption column is placed inside the collection tube), centrifuge at 12000 rpm for 45 s, discard the waste liquid, and put the adsorption column back into the collection tube.

[0114] ⑥ Add 0.5 mL of protein removal solution RE, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0115] ⑦ Add 0.5 mL of rinsing buffer RW, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0116] ⑧ Add 0.5 mL of rinsing buffer RW, centrifuge at 12000 rpm for 45 s, and discard the waste liquid.

[0117] ⑨ Place the adsorption column RA back into the collection tube and centrifuge at 13000 rpm for 2 min to remove as much of the washing solution as possible, so as to avoid residual ethanol in the washing solution inhibiting the downstream reaction.

[0118] ⑩ Remove the adsorption column RA and place it in an RNase-free centrifuge tube. Add 30-50 μL of RNase-free water (preheating in a 65-70°C water bath beforehand will improve the effect) to the middle of the adsorption membrane according to the expected RNA yield. Incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 1 minute. If more RNA is needed, the resulting solution can be added back to the adsorption column and centrifuged for 1 minute, or another 30 μL of RNase-free water can be added and centrifuged for 1 minute. Combine the two eluents.

[0119] (2) Reverse transcription to synthesize mRNA and cDNA

[0120] mRNA reverse transcription was performed using the FastKing cDNA First-Strand Synthesis Kit (catalog number: KR116). First, genomic DNA was removed. In a test tube, 2.0 μL of 5×gDNA Buffer, 1 μg of Total RNA, and RNase-Free ddH2O were added to bring the total volume to 10 μL. The mixture was heated in a water bath at 42°C for 3 min. Then, 2.0 μL of 10×King RT Buffer, 1.0 μL of FastKing RT Enzyme Mix, 2.0 μL of FQ-RT Primer Mix, and 5.0 μL of RNase-Free ddH2O were added to the same test tube, bringing the total volume to 20 μL. The mixture was then heated in a water bath at 42°C for 15 min and then at 95°C for 3 min. For long-term storage of the synthesized cDNA, please store at -20°C or lower.

[0121] (3) Real-Time PCR

[0122] ① mRNA fluorescence quantitative detection

[0123] A. Instruments and Analytical Methods

[0124] An ABI 7300 real-time PCR instrument was used, employing 2- △△CT The method is used to perform relative quantitative analysis of the data.

[0125] B. The operation process is as follows:

[0126] (I) Reaction System

[0127] Amplification was performed using SuperReal PreMix Plus (SYBR Green) (catalog number: FP205), and the experimental procedures were performed according to the product instructions. The RealTime reaction system is listed in Table 8.

[0128] Table 8 RealTime Reaction System

[0129]

[0130]

[0131] (II) Amplification Procedure

[0132] 95℃ for 15 min, (95℃ for 10 sec, 55℃ for 30 sec, 72℃ for 32 sec) × 40 cycles, 95℃ for 15 sec, 60℃ for 60 sec, 95℃ for 15 sec.

[0133] (III) Primer Screening

[0134] After mixing the cDNA from each sample, the mixture was used as a template for 10-fold serial dilution. 2 μL of each diluted sample was then used as a template for amplification using the target gene primer and the internal reference gene primer, respectively. Melting curve analysis was performed at 60-95℃, and primers were screened based on high amplification efficiency and a single peak in the melting curve.

[0135] (iv) Real-Time PCR Detection of Samples

[0136] Each sample's cDNA was diluted 3-10 times, and 2 μL was used as a template for amplification using the target gene primer and the internal control gene primer (see Table 7). Melting curve analysis was performed at 60-95℃. The design for Real-Time PCR detection of the samples is shown in Table 9.

[0137] Table 9. Sample Real-Time PCR Detection Design

[0138] template Sample cDNA Sample cDNA Number of repeated detection channels 3 3 Primers Target gene primers Internal reference gene primers

[0139] 3. Experimental Results

[0140] (1) The results of RNA concentration detection and 1.5% agarose RNA electrophoresis detection are listed in Tables 10 and 11.

[0141] Table 10 Results of RNA Concentration and Purity

[0142]

[0143]

[0144] Note: Dissolving RNA in water will result in a lower A260 / 280 ratio;

[0145] Note: A: Concentration not up to standard; B: A260 / A280 unqualified; C: Electrophoresis pattern unqualified; H: Qualified;

[0146] Sample evaluation criteria:

[0147] 1. Concentration > 30 ng / uL;

[0148] 2.1.8 <A260 / A280<2.0;

[0149] 3. The electrophoresis image shows three relatively clear bands.

[0150] Electrophoresis typically detects 28S, 18S, and 5.8S sedimentation coefficient rRNA, so there will be three bands. However, the third band may not be visible due to the small amount. In actual experiments, the presence of the first or second band is considered acceptable.

[0151] Table 11 Electrophoresis Sample Loading Details

[0152]

[0153]

[0154] (2) Real-Time PCR Detection Results and Analysis of Each Sample

[0155] ①From the appendix Figure 11 Appendix Figure 12 Appendix Figure 13 Appendix Figure 14 Appendix Figure 15 Appendix Figure 16 Appendix Figure 17 Appendix Figure 18 Appendix Figure 19 and attached Figure 20 It can be seen that the biomarkers PDK4, WIF1 and WNT5A of the present invention achieved specific amplification.

[0156] ② Relative quantitative analysis results of each sample

[0157] Based on the original RealTime PCR test results, according to 2 -△△ct The relative quantitative calculation formula, i.e.

[0158] ;

[0159] The relative quantitative results of the target gene for each sample were calculated, that is, the difference in the mRNA transcription level of the target gene between each other sample and the control sample.

[0160] From the appendix Figure 21 Appendix Figure 22 and attached Figure 23 As can be seen, RealTime PCR confirmed that PDK4, WIF1, and WNT5A were differentially expressed in asthma patients, which is consistent with the trend shown in previous experiments.

[0161] ③ Diagnostic efficacy analysis

[0162] Table 12. Area below the ROC curve for PDK4 in diagnosing asthma.

[0163]

[0164] a. Assuming nonparametric properties; b. Null hypothesis: True region = 0.5.

[0165] Table 13 shows the area below the ROC curve for WIF1 in diagnosing asthma.

[0166]

[0167] a. Assuming nonparametric properties; b. Null hypothesis: True region = 0.5.

[0168] Table 14. Area below the ROC curve for WNT5A in diagnosing asthma.

[0169]

[0170] a. Assuming nonparametric properties; b. Null hypothesis: True region = 0.5.

[0171] The diagnostic efficacy of PDK4, WIF1, and WNT5A is listed in Tables 12, 13, and 14. (See Table 12 and Appendix...) Figure 24 As can be seen, the diagnostic efficacy of PDK4 is as follows: AUC = 0.923, specificity = 0.824, and sensitivity = 0.923. (See Table 13 and Appendix...) Figure 25 As can be seen, the diagnostic efficacy of WIF1 is as follows: AUC 0.756, specificity 0.706, and sensitivity 0.769. (See Table 14 and Appendix...) Figure 26 As can be seen, WNT5A has an AUC of 0.769, a specificity of 0.765, and a sensitivity of 0.769 in terms of diagnostic efficacy. This indicates that PDK4, WIF1, and WNT5A have good diagnostic efficacy for asthma.

[0172] Therefore, compared with the prior art, the beneficial effects of the present invention are as follows: The reagent for detecting biomarkers in the present invention is a reagent for detecting the expression level and / or methylation level of biomarkers in a sample. It can be used in a kit for diagnosing asthma. By using this kit to diagnose asthma, early and accurate diagnosis of asthma can be achieved, with good diagnostic efficacy. This allows for the screening of drugs for the prevention or treatment of asthma. It solves the problems of inaccurate judgment and inconvenient operation of traditional techniques that rely on clinical manifestations, pulmonary function tests, or peak flow rate measurements to diagnose asthma. It fills the gap in kits for diagnosing asthma and provides a fast and convenient method for diagnosing asthma.

[0173] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting the expression level of a biomarker mRNA in a sample in the preparation of a kit for diagnosing asthma, characterized in that: The biomarkers are a combination of PDK4, WIF1, and WNT5A, and the sample is blood.

2. The application of the reagent for detecting the expression level of biomarker mRNA in a sample according to claim 1 in the preparation of a kit for diagnosing asthma, characterized in that: The kit is a qPCR kit.

3. The application of the reagent for detecting the expression level of biomarker mRNA in a sample according to claim 1 in the preparation of a kit for diagnosing asthma, characterized in that: The reagents are those used to detect the expression level of biomarkers via polymerase chain reaction or DNA microarray.

4. The application of the reagent for detecting the expression level of biomarker mRNA in a sample according to claim 3 in the preparation of a kit for diagnosing asthma, characterized in that: The polymerase chain reaction is any one of real-time quantitative reverse transcription polymerase chain reaction, reverse transcription polymerase chain reaction, or competitive polymerase chain reaction.

5. The application of the reagent for detecting the expression level of biomarker mRNA in a sample according to claim 1 in the preparation of a kit for diagnosing asthma, characterized in that: The reagents include probes and / or primers that are specific to the marker.

6. The application of a biomarker in constructing a diagnostic system for asthma, characterized in that: The biomarkers are a combination of PDK4, WIF1, and WNT5A, and the diagnostic system includes: The detection unit includes a biomarker detection module, which detects the mRNA expression level of biomarkers in a blood sample. The analysis unit takes the expression level of the markers detected by the detection unit as the input variable and inputs it into the asthma diagnostic model for analysis. The asthma diagnostic model is any one or more of XGBoost, Random Forest, glmnet, cforest, machine learning classification and regression tree, treebag, K-adjacency, neural network, radial support vector machine, linear support vector machine, Naive Bayes, or multilayer perception. The assessment unit outputs the risk value of asthma for the individual corresponding to the sample.

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