Markers for diabetes mellitus combined with tuberculosis and uses thereof

Abstract

The application belongs to the technical field of medical diagnosis, and specifically discloses a diabetes mellitus combined with tuberculosis marker and application thereof. It is found for the first time that the transcription level and protein level of activin A receptor 2A (ACVR2A), angiostatin and chemokine ligand 14 (CXCL14) are inversely proportional to the severity of diabetes mellitus combined with tuberculosis, and can be used as a diabetes mellitus combined with tuberculosis marker to indirectly reflect whether the crowd is suffering from diabetes mellitus combined with tuberculosis and the development of the disease, so as to timely diagnose and treat. A new diabetes mellitus combined with tuberculosis detection kit can be prepared according to the three markers, and is applied to public medical treatment. The three markers can effectively improve the diagnosis efficiency of diabetes mellitus combined with tuberculosis, and have high sensitivity and strong specificity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical diagnosis, in particular to a marker for diabetes combined with tuberculosis and use thereof. BACKGROUND

[0002] Tuberculosis is a major infectious disease that endangers human health. According to the Global Tuberculosis Report 2021 of the World Health Organization, there were about 10 million tuberculosis patients worldwide in 2020, and 2 billion people were latent tuberculosis infected. About 16% of tuberculosis patients are complicated with diabetes, and the double burden of diabetes combined with tuberculosis has become a major global public health challenge.

[0003] Diabetes patients have a 3-fold higher risk of developing tuberculosis, and tuberculosis treatment failure is increased by 2-fold. Compared with simple tuberculosis patients, the treatment outcome of diabetes combined with tuberculosis patients is poorer. Therefore, the World Health Organization has identified diabetes as an important, neglected and re-emerging risk factor for tuberculosis. In general, diabetes can affect the clinical manifestations and treatment response of tuberculosis, and tuberculosis can worsen the blood glucose control of diabetes patients. The complex interaction between the two poses a serious challenge to the global goal of ending tuberculosis.

[0004] Diabetes patients combined with tuberculosis are prone to delay the disease and increase the treatment burden. Therefore, early detection and early treatment are the key to preventing and treating diabetes combined with tuberculosis. However, there is currently a lack of efficient and accurate diagnostic methods for diabetes combined with tuberculosis. The existing diagnosis method for diabetes combined with tuberculosis is mainly to separately diagnose blood glucose and Mycobacterium tuberculosis. This diagnosis method is time-consuming and expensive, and cannot meet the needs of rapid diagnosis and economy of patients.

[0005] Therefore, it is of great significance to develop a biomarker and detection method for early diagnosis of diabetes combined with tuberculosis with high sensitivity and high specificity, and to develop a sensitive and efficient diagnostic reagent for diabetes combined with tuberculosis. SUMMARY

[0006] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a marker for diabetes combined with tuberculosis and use thereof, to provide a new, high-sensitivity and high-specificity specific marker and detection means for the diagnosis and efficacy evaluation of diabetes combined with tuberculosis.

[0007] To achieve the above-mentioned purposes and other related purposes, the first aspect of the present application provides the use of Activin A Receptor 2A (ACVR2A), Chemokine Ligand 14 (CXCL14) and / or Angiostatin as a biomarker in the preparation or screening of a detection kit for diagnosing diabetes combined with tuberculosis and / or for evaluating the prognosis and treatment effect of diabetes combined with tuberculosis.

[0008] Further, the diabetes includes type II diabetes, and the tuberculosis includes pulmonary tuberculosis.

[0009] Further, the detection kit is used for quantitatively detecting the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin in the sample.

[0010] Further, the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and Angiostatin are negatively correlated with the severity of diabetes complicated with tuberculosis.

[0011] Further, when the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and Angiostatin decrease to the expression levels of normal population, it indicates that the treatment effect of diabetes complicated with tuberculosis is better or the diabetes complicated with tuberculosis is basically cured.

[0012] Further, if the expression levels A1 of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin are significantly higher than the control reference value A0, the probability of the detection object suffering from diabetes complicated with tuberculosis is greater than that of normal population, and the control reference value A0 is the expression level of diabetes complicated with tuberculosis marker Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin in non-diabetes complicated with tuberculosis healthy population or diabetes or tuberculosis patients.

[0013] Further, if the expression levels A1 of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin decrease to the control reference value A0, it indicates that the treatment effect of diabetes complicated with tuberculosis of the detection object is better or the diabetes complicated with tuberculosis is basically cured.

[0014] Further, the sample is selected from at least one of blood sample, serum sample, tissue sample and body fluid sample.

[0015] Further, the sample is derived from a mammal; preferably, the mammal includes human or non-human mammal; more preferably, the non-human mammal is preferably a primate other than human, such as monkey, ape.

[0016] Further, the detection kit is used for detecting the gene, mRNA, cDNA, protein or combination thereof of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin.

[0017] Further, the detection kit comprises:

[0018] (a) specific antibody, specific binding molecule of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin;

[0019] and / or (b) primers or primer pairs, probes or chips of mRNA or cDNA of Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin, wherein the chips comprise nucleic acid chips and protein chips.

[0020] Further, the nucleic acid chips comprise substrates and specific oligonucleotide probes spotted on the substrates, wherein the specific oligonucleotide probes comprise probes specifically binding to polynucleotides of Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin, wherein the polynucleotides are mRNA or cDNA.

[0021] Further, the protein chips comprise substrates and specific antibodies spotted on the substrates, wherein the specific antibodies comprise antibodies specifically binding to Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin.

[0022] Further, the detection reagents in the detection kits are coupled with or carry detectable labels.

[0023] Further, the detectable labels are selected from at least one of isotopes, fluorophores, chemiluminescent groups, enzymes.

[0024] Further, the diagnosis comprises early diagnosis and auxiliary diagnosis.

[0025] Further, the detection kits comprise primer pairs for amplifying Activin A receptor IIA, primer pairs for amplifying Chemokine ligand 14, specific antibodies for detecting Angiostatin, or combinations thereof.

[0026] Further, the primer pairs for amplifying Activin A receptor IIA are SEQ ID NO. 1 and SEQ ID NO. 2, and the primer pairs for amplifying Chemokine ligand 14 are SEQ ID NO. 3 and SEQ ID NO. 4.

[0027] Further, the antibodies are monoclonal antibodies or polyclonal antibodies.

[0028] Further, the methods for determining expression levels of Activin A receptor IIA, Chemokine ligand 14 and Angiostatin in samples in the detection kits are selected from any one of real-time fluorescent quantitative PCR, immunoblotting test, immunochemiluminescence method, immunoturbidimetry, immunoblotting method, enzyme-linked immunosorbent assay.

[0029] The second aspect of the present application provides a detection kit for diagnosing diabetes mellitus complicated with tuberculosis, and / or for evaluating prognosis and therapeutic effect of diabetes mellitus complicated with tuberculosis, by detecting expression levels of Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin.

[0030] Further, the diabetes includes type II diabetes, and the tuberculosis includes pulmonary tuberculosis.

[0031] Further, the detection kit is used for quantitatively detecting the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin in the sample.

[0032] Further, the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and Angiostatin are negatively correlated with the severity of diabetes complicated with tuberculosis.

[0033] Further, when the expression levels of Activin A Receptor IIA, Chemokine Ligand 14 and Angiostatin decrease to the expression levels of normal population, it indicates that the treatment effect of diabetes complicated with tuberculosis is better or the diabetes complicated with tuberculosis is basically cured.

[0034] Further, if the expression level A1 of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin is significantly higher than the control reference value A0, the probability of diabetes complicated with tuberculosis of the detection object is greater than that of normal population, and the control reference value A0 is the expression level of diabetes complicated with tuberculosis marker Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin in non-diabetes complicated with tuberculosis healthy population or diabetes or tuberculosis patients.

[0035] Further, if the expression level A1 of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin decreases to the control reference value A0, it indicates that the treatment effect of diabetes complicated with tuberculosis of the detection object is better or the diabetes complicated with tuberculosis is basically cured.

[0036] Further, the sample is selected from at least one of blood sample, serum sample, tissue sample and body fluid sample.

[0037] Further, the sample is derived from a mammal; preferably, the mammal includes human or non-human mammal; more preferably, the non-human mammal is preferably primate other than human, such as monkey, ape.

[0038] Further, the detection kit is used for detecting the gene, mRNA, cDNA, protein or combination thereof of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin.

[0039] Further, the detection kit comprises:

[0040] (a) specific antibody, specific binding molecule of Activin A Receptor IIA, Chemokine Ligand 14 and / or Angiostatin;

[0041] and / or (b) primers or primer pairs, probes or chips of mRNA or cDNA of Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin, wherein the chips comprise nucleic acid chips and protein chips.

[0042] Further, the nucleic acid chips comprise substrates and specific oligonucleotide probes spotted on the substrates, wherein the specific oligonucleotide probes comprise probes specifically binding to polynucleotides of Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin, wherein the polynucleotides are mRNA or cDNA.

[0043] Further, the protein chips comprise substrates and specific antibodies spotted on the substrates, wherein the specific antibodies comprise antibodies specifically binding to Activin A receptor IIA, Chemokine ligand 14 and / or Angiostatin.

[0044] Further, the detection reagents in the detection kits are coupled with or carry detectable labels.

[0045] Further, the detectable labels are selected from at least one of isotopes, fluorophores, chemiluminescent groups, enzymes.

[0046] Further, the diagnosis comprises early diagnosis and auxiliary diagnosis.

[0047] Further, the detection kits comprise primer pairs for amplifying Activin A receptor IIA, primer pairs for amplifying Chemokine ligand 14, specific antibodies for detecting Angiostatin, or combinations thereof.

[0048] Further, the primer pairs for amplifying Activin A receptor IIA are SEQ ID NO. 1 and SEQ ID NO. 2, and the primer pairs for amplifying Chemokine ligand 14 are SEQ ID NO. 3 and SEQ ID NO. 4.

[0049] Further, the antibodies are monoclonal antibodies or polyclonal antibodies.

[0050] Further, the methods for determining the expression levels of Activin A receptor IIA, Chemokine ligand 14 and Angiostatin in the samples in the detection kits are selected from any one of real-time fluorescent quantitative PCR, immunoblotting test, immunochemiluminescence method, immunoturbidimetry, immunoblotting method, enzyme-linked immunosorbent assay.

[0051] As described above, the markers of diabetes mellitus complicated with tuberculosis and uses thereof of the present application have the following beneficial effects:

[0052] The present application is based on the serological research that three serum marker genes of diabetes mellitus combined with tuberculosis are found, and according to the transcription and / or protein level, the three serum marker genes can be used for diagnosing diabetes mellitus combined with tuberculosis and / or treatment effect of diabetes mellitus combined with tuberculosis; the three genes found by the present application are respectively: Activin A receptor 2A (ACVR2A), chemokine ligand 14 (CXCL14) and Angiostatin, and the transcription level and protein level of the three genes are inversely proportional to the severity of diabetes mellitus combined with tuberculosis, which can indirectly reflect whether the crowd is suffering from diabetes mellitus combined with tuberculosis and the development of the disease, so as to diagnose and treat in time. Meanwhile, through transcriptomics and proteomics, the transcription level and protein translation of the three markers are significantly reduced in patients with diabetes mellitus combined with tuberculosis, and recover to normal with the extension of treatment time. Based on this, the present application proposes that the three marker genes are used as biomarkers for diagnosing, prognosis and efficacy evaluation of diabetes mellitus combined with tuberculosis, and a new diabetes mellitus combined with tuberculosis detection kit is prepared according to the two genes, which can be widely used in public medical treatment, can effectively improve the diagnosis efficiency of diabetes mellitus combined with tuberculosis, and has high sensitivity and strong specificity. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 The figure shows the protein level of the three markers ACVR2A, CXCL14 and Angiostatin in Chinese patients with diabetes mellitus combined with tuberculosis during treatment in Example 1 of the present application.

[0054] Figure 2 The figure shows the ROC curve of ACVR2A, CXCL14 and Angiostatin in the data of Chinese patients in Example 1 of the present application.

[0055] Figure 3 The figure shows the transcription level and ROC curve of the marker ACVR2A of Indonesian patients with diabetes mellitus combined with tuberculosis during treatment in Example 2 of the present application.

[0056] Figure 4 The figure shows the transcription level and ROC curve of ACVR2A in South African patients with diabetes mellitus combined with tuberculosis and different control groups in Example 3 of the present application.

[0057] Figure 5 The figure shows the transcription level and ROC curve of ACVR2A in Romanian patients with diabetes mellitus combined with tuberculosis and different control groups in Example 4 of the present application. DETAILED DESCRIPTION

[0058] Following, the present application will be described in greater detail by way of specific embodiments, and thus, those skilled in the art can easily understand other advantages and functions of the present application from the contents disclosed in the specification. The present application can also be implemented or applied in other different embodiments, and the details in the specification can be modified or changed in various ways based on different views and applications without departing from the spirit of the present application.

[0059] The present inventors have made extensive and in-depth research and for the first time discovered three diabetes combined with tuberculosis markers existing in blood: Activin A Receptor 2A (ACVR2A), CXC Chemokine 14 (CXCL14) and Angiostatin. The diabetes combined with tuberculosis markers are specifically highly expressed in the blood of patients with diabetes combined with tuberculosis, and thus can be used as specific markers for the diagnosis (especially early diagnosis and / or auxiliary diagnosis) of diabetes combined with tuberculosis. Meanwhile, the experimental results of the embodiments of the present application show that compared with healthy people, only tuberculosis patients or diabetes patients, the expression amount of diabetes combined with tuberculosis markers ACVR2A, CXCL14 and / or Angiostatin in patients with diabetes combined with tuberculosis is significantly reduced, which can be used as a biomarker for the diagnosis, prognosis and efficacy evaluation of diabetes combined with tuberculosis. On this basis, the present application is completed.

[0060] The term

[0061] The term "sample" or "specimen" as used herein refers to material specifically associated with a subject from which particular information about the subject can be determined, calculated or inferred. The sample can be constituted in whole or in part by biological material from the subject.

[0062] The term "expression" includes the production of mRNA from a gene or portion of a gene, and includes the production of a protein encoded by the RNA or gene or portion of a gene, and also includes the appearance of detection substances associated with expression, such as the binding of a cDNA, a binding ligand (such as an antibody) to a gene or other oligonucleotide, a protein or protein fragment, and the visualization of a binding ligand.

[0063] The term "reference value" or "control reference value" refers to a value that is statistically related to a particular result when compared to the results of an analysis. In an optimized embodiment, the reference value is determined based on a statistical analysis comparing the expression of diabetes combined with tuberculosis markers ACVR2A, CXCL14 and / or Angiostatin, and known clinical outcome studies. Of course, this value can be determined according to specific circumstances.

[0064] Diabetes combined with tuberculosis markers ACVR2A, CXCL14 and / or Angiostatin

[0065] As used herein, the term "diabetes mellitus co-tuberculosis markers of the invention" refers to one or more biomarkers selected from the group consisting of: ACVR2A, CXCL14 and Angiostatin. The term includes the full field of diabetes mellitus co-tuberculosis markers ACVR2A, CXCL14 and / or Angiostatin and fragments thereof, as well as complete amino acid sequences, secreted proteins thereof, mutants, and functionally active fragments thereof.

[0066] ACVR2A (NCBI Gene ID: 92), Activin A Receptor Type 2A, Activin A Receptor Type 2A, the gene encodes a receptor that mediates the function of activin, which is a member of the transforming growth factor-beta superfamily, involved in a variety of biological processes. The encoded protein is a transmembrane serine threonine kinase receptor that mediates signal transduction by forming heterodimeric complexes with type I and type II receptors and ligands in a cell-specific manner. The encoded type II receptor is mainly involved in ligand binding, including an extracellular ligand binding domain, a transmembrane domain and a cytoplasmic serine threonine kinase domain. Related pathways include BMP signaling and telomere end packaging. Gene ontology annotations associated with this gene include transferase activity, transfer phosphorus-containing groups, and protein tyrosine kinase activity. An important homolog of this gene is ACVR2B. Mediates adipogenesis by GDF6.

[0067] CXCL14 (NCBI Gene ID: 9547), C-X-C Motif Chemokine Ligand 14, CXC Chemokine 14, the antibacterial gene belongs to the cytokine gene family, encoding a secreted protein involved in immune regulation and inflammatory processes. The protein encoded by this gene is structurally related to the CXC (Cys-X-Cys) subfamily of cytokines. Members of this subfamily are characterized by two cysteines separated by an amino acid. This cytokine has chemotactic activity for monocytes, but not for lymphocytes, dendritic cells, neutrophils, or macrophages. It is postulated to be involved in the homeostasis of mononuclear phagocytes rather than inflammation. It can promote the thermogenic activity of brown adipose tissue, the browning of white adipose tissue, and the enrichment of M2 macrophages and alter the balance of glucose in the body. It is negatively associated with human obesity and accompanying type 2 diabetes.

[0068] Angiostatin, Angiostatin, has anti-inflammatory and anti-fibrotic effects, and has therapeutic potential for diabetic nephropathy. Reduces vascular leakage, which is associated with diabetic macular edema. At the same time, anti-angiogenic drugs can improve tuberculosis treatment.

[0069] In the present application, the terms "diabetes mellitus-associated tuberculosis marker gene" and "polynucleotide of the diabetes mellitus-associated tuberculosis marker" and "diabetes mellitus-associated tuberculosis marker protein" and "protein of the present application" or "polypeptide of the present application" are used interchangeably.

[0070] It is to be understood that substitution of nucleotides in a codon is acceptable when it codes for the same amino acid; substitution of nucleotides resulting in conservative amino acid substitution is also acceptable. The proteins of the present application also include conservative variants thereof, such as conservative polypeptides.

[0071] In the case where the amino acid or nucleotide fragment of the diabetes mellitus-associated tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin is obtained, a specific probe can be designed based on the sequence. The full-length sequence of the nucleic acid or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification, primers can be designed based on the nucleotide sequence of the diabetes mellitus-associated tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin disclosed in the present application, particularly the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art is used as a template to amplify the relevant sequence.

[0072] In addition, the relevant sequence, particularly a fragment having a short length, can be synthesized by an artificial synthesis method. At present, it is possible to obtain a DNA sequence encoding the protein of the present application (or a fragment or derivative thereof) by chemical synthesis alone.

[0073] The polynucleotide sequence of the present application can be used to express or produce a recombinant diabetes mellitus-associated tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin by a conventional DNA recombination technique.

[0074] Specific antibodies

[0075] In the present application, the terms "antibody of the present application" and "specific antibody against the diabetes mellitus-associated tuberculosis marker" are used interchangeably.

[0076] The present application also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the polypeptide of the diabetes mellitus-associated tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin, and antibodies capable of binding to the gene product of human diabetes mellitus-associated tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin in a modified or unmodified form. The present application includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments having immunological activity.

[0077] Antibodies of the present application can be prepared by various techniques known to those skilled in the art. For example, purified human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin gene products or fragments having antigenicity can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin protein or fragments having antigenicity can be used to immunize an animal to produce antibodies.

[0078] Antibodies against human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin protein can be used in immunohistochemical techniques to detect human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin protein in a sample, especially a tissue sample or a blood sample. Since diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin protein is present in mononuclear cells in blood samples, the amount of expression can be detected.

[0079] Detection methods

[0080] Based on the low expression of diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin in blood, the present application also provides corresponding methods for diagnosing diabetes mellitus. The present application relates to diagnostic test methods for quantitatively detecting the protein level or mRNA level of human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin. These tests are well known in the art. The human diabetes mellitus marker ACVR2A, CXCL14 and / or Angiostatin protein level or mRNA level detected in the test can be used to diagnose (including early diagnosis and auxiliary diagnosis) whether there is diabetes mellitus.

[0081] A preferred method is to quantitatively detect mRNA or cDNA by PCR.

[0082] A preferred method is to quantitatively detect mRNA or cDNA by sequencing.

[0083] A preferred method is to quantitatively detect the protein of the diabetes mellitus marker.

[0084] Preferably, a method for detecting the presence of the diabetes-tuberculosis marker protein in a sample is to use specific antibodies to detect, which comprises: contacting the sample with the diabetes-tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin protein specific antibodies, and observing whether antibody complexes are formed, the formation of antibody complexes represents the presence of the diabetes-tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin protein in the sample.

[0085] The diabetes-tuberculosis marker protein or its polynucleotide can be used for the diagnosis of diabetes-tuberculosis. Part or all of the polynucleotide of the present application can be immobilized on a microarray or DNA chip as a probe for differential expression analysis and gene diagnosis of genes in single nucleated cells. Anti-diabetes-tuberculosis antibodies can be immobilized on a protein chip for detecting the diabetes-tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin protein in a sample. Based on the research of the present application, the expression level (mRNA level or protein level) of ACVR2A, CXCL14 and / or Angiostatin gene is significantly reduced in diabetes-tuberculosis patients. Therefore, ACVR2A, CXCL14 and / or Angiostatin can be used as a marker for detecting or diagnosing (especially auxiliary diagnosis and / or early diagnosis) diabetes-tuberculosis. In detection, if the expression level of the marker gene (i.e. ACVR2A, CXCL14 and / or Angiostatin) is significantly changed compared with the corresponding expression level in normal population, it can be considered as an increased risk of diabetes-tuberculosis.

[0086] According to the experimental results of the present application, a preferred method for diagnosing diabetes-tuberculosis patients is based on the geometric mean of the transcription level. Preferably, the method comprises the following judgment criteria: if the geometric mean of the transcription level of ACVR2A, CXCL14 and / or Angiostatin gene in the blood of the detection object is reduced, the detection object can be preliminarily judged as a diabetes-tuberculosis patient. In addition, in view of the significant upward trend of the transcription level of the marker gene (ACVR2A, CXCL14 and / or Angiostatin) in diabetes-tuberculosis patients during treatment and at the end of treatment, the expression level of the marker gene can be detected to reflect or evaluate the treatment effect of diabetes-tuberculosis. When the expression level of the marker gene rises to the level of the normal population, it indicates that the treatment effect of diabetes-tuberculosis is good or the patient is basically cured.

[0087] Detection kit

[0088] Based on the correlation between the diabetes mellitus combined with tuberculosis marker and diabetes mellitus combined with tuberculosis, the diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin can be used as a diagnostic marker of diabetes mellitus combined with tuberculosis.

[0089] The present application also provides a kit for diagnosing diabetes mellitus combined with tuberculosis, which comprises a detection reagent for detecting the diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin gene, mRNA, cDNA, protein, or a combination thereof.

[0090] Preferably, the kit comprises an antibody or immunoconjugate of the diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin of the present application, or an active fragment thereof; or a primer or primer pair, a probe or a chip for specifically amplifying the mRNA or cDNA of the diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin.

[0091] In another preferred embodiment, the kit further comprises a label or instructions indicating that the kit is used for diagnosing diabetes mellitus combined with tuberculosis and / or evaluating the therapeutic effect of diabetes mellitus combined with tuberculosis.

[0092] The main advantages of the present application include:

[0093] (a) ACVR2A, CXCL14 and / or Angiostatin are the first diabetes mellitus combined with tuberculosis biomarkers discovered by the present inventors, which can be applied to the diagnosis of diabetes mellitus combined with tuberculosis, providing a reference for more accurate and earlier diagnosis.

[0094] (b) The diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin exists in mononuclear cells in the blood, so it can be conveniently sampled and detected.

[0095] (c) The diabetes mellitus combined with tuberculosis marker ACVR2A, CXCL14 and / or Angiostatin can be used as a biomarker for effectively evaluating the therapeutic effect of diabetes mellitus combined with tuberculosis, and also as a biomarker for effectively evaluating the prognosis of diabetes mellitus combined with tuberculosis.

[0096] The present application will be further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts. The materials, reagents, instruments, etc. used in the examples, if not otherwise specified, can be obtained from commercial channels.

[0097] General methods

[0098] Real-time fluorescent quantitative PCR

[0099] Real-time fluorescent quantitative PCR is an experimental method that applies fluorescent quantitative technique to polymerase chain reaction. A fluorescent dye called SYBR Green I is used in this experiment. In the PCR reaction system, SYBR Green I emits fluorescent signal specifically after participating in DNA double strands, while the SYBR dye molecules not incorporated into the strands do not emit any fluorescent signal. Because this method makes the increase of fluorescent signal synchronous with the increase of PCR product, that is, the fluorescent signal intensity emitted by the fluorescent dye is proportional to the DNA yield. Therefore, the detection of the fluorescent signal intensity in the PCR process can determine the initial concentration of the target sequence, so as to achieve the purpose of quantification.

[0100] Enzyme-linked immunosorbent assay

[0101] Enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA or ELASA for short) refers to the qualitative and quantitative detection method of immunoreaction by combining soluble antigens or antibodies to polystyrene solid phase carrier and using antigen-antibody specific binding. The basic principle of this method is: ① make antigens or antibodies bind to the surface of certain solid phase carrier and maintain their immunocompetence. ② make antigens or antibodies connected with certain enzymes into enzyme-labeled antigens or antibodies, which retain their immunocompetence and enzyme activity.

[0102] Example 1

[0103] Changes of ACVR2A, CXCL14 and Angiostatin protein levels in patients with diabetes mellitus complicated with tuberculosis during treatment

[0104] In this embodiment, the changes of ACVR2A, CXCL14 and Angiostatin protein levels in whole blood samples of the study subjects, i.e. diabetic patients with tuberculosis, during the treatment process were studied.

[0105] The violin plot shows the changes of ACVR2A, CXCL14 and Angiostatin protein levels in diabetic patients with tuberculosis during the treatment process, and the Wilcoxon rank sum test was used to calculate the differences between the two time points.

[0106] In this embodiment, the expression of genes in 4 Chinese diabetic patients with tuberculosis before, during and after treatment was determined.

[0107] As shown in Figure 1 As shown in A, 1B and 1C, the results show that for the protein levels of ACVR2A, CXCL14 and Angiostatin in diabetic patients with tuberculosis: as the treatment progresses, the protein levels of ACVR2A, CXCL14 and Angiostatin in diabetic patients with tuberculosis continue to rise, and there is a significant difference.

[0108] According to the geometric mean of the relative expression amount of ACVR2A, CXCL14 and Angiostatin protein during the treatment and after the treatment of diabetic patients with tuberculosis, the receiver operating characteristic curve analysis was performed using R language.

[0109] The receiver operating characteristic (ROC) reflects the balance between sensitivity and specificity, and the area under the ROC curve is an important test accuracy index. The larger the area under the ROC curve, the greater the diagnostic value of the test.

[0110] Sensitivity (true positive rate): The percentage of actual patients who are correctly judged as having the disease according to the test standard. The greater the sensitivity, the better, and the ideal sensitivity is 100%.

[0111] Specificity (true negative rate): The percentage of actual non-diseased individuals who are correctly judged as non-diseased according to the test standard. The greater the specificity, the better, and the ideal specificity is 100%.

[0112] The ROC curve is shown in Figure 2 As shown in A, 2B and 2C, the area under the ROC curve is 0.938, 0.812 and 0.812, respectively, which indicates that the diagnosis of diabetic patients with tuberculosis based on the expression of ACVR2A, CXCL14 and Angiostatin protein has high accuracy.

[0113] Example 2

[0114] Changes in ACVR2A transcript levels in patients with diabetes mellitus and tuberculosis during treatment

[0115] In this example, the change in ACVR2A protein levels in whole blood samples from patients with diabetes mellitus and tuberculosis during treatment was investigated.

[0116] The violin plot shows the change in ACVR2A gene expression in patients with diabetes mellitus and tuberculosis during treatment, and the Wilcoxon rank sum test was used to calculate the difference between the two time points.

[0117] In this example, the expression of the ACVR2A gene was measured in 29 Indonesian patients with diabetes mellitus and tuberculosis before, during, and after treatment.

[0118] As shown in Figure 3 A, the results show that for the ACVR2A gene transcript levels in patients with diabetes mellitus and tuberculosis: ACVR2A protein levels continue to rise in patients with diabetes mellitus and tuberculosis as treatment progresses.

[0119] Based on the geometric mean of the relative expression of the ACVR2A gene during and after treatment of diabetes mellitus and tuberculosis, a receiver operating characteristic curve analysis was performed using R language.

[0120] The ROC curve is shown in Figure 3 B. The area under the ROC curve was 0.631, indicating that the diagnosis of patients with diabetes mellitus and tuberculosis based on the expression of the ACVR2A gene has high accuracy.

[0121] Example 3

[0122] ACVR2A is a serum marker for diabetes mellitus and tuberculosis

[0123] 1.1 Study subjects

[0124] In this example, the study subjects were a group of patients with diabetes mellitus and tuberculosis, a group of patients with only diabetes or tuberculosis, and a healthy control group.

[0125] The group of patients with diabetes mellitus and tuberculosis included 15 patients who were clinically diagnosed with diabetes mellitus and tuberculosis in South Africa, were HIV-negative, were not pregnant or taking corticosteroids, and had no other serious illnesses. The age range was 27-57 years, with an average age of 46 years. There were 7 males and 8 females.

[0126] The diabetes mellitus group included 33 patients with a clinical glycated hemoglobin (HbA1c) of ≥6.5% or a fasting blood glucose of ≥7 mmol / L, and had no other serious illnesses. The age range was 29-64 years, with an average age of 49 years. There were 15 males and 18 females.

[0127] Tuberculosis patients group, 11 Mycobacterium tuberculosis detection positive and imaging detection patients, no other serious illness. Age is divided into 31-56 years old, the average age is 48 years old. 2 male 9 female.

[0128] Healthy control group, 24 without serious illness. Age is divided into 30-70 years old, the average age is 42 years old. 12 male 12 female.

[0129] 1.2 Detection of ACVR2A expression in venous blood

[0130] For each study object, venous blood is collected at the time of diagnosis, introduced into PAXgene blood RNA tube (PreAnalytiX), and used for RNA-seq analysis, using polyA tail library preparation method and single read sequencing. The sequencing FASTQ file is compared with the human genome, and transcription quantification, differential gene expression, biological characteristics and machine learning are performed according to the fixed method.

[0131] 1.3 Results

[0132] The results of RNA-seq analysis show that the expression of ACVR2A gene is significantly reduced in the group of diabetes combined with tuberculosis, and the relative expression of ACVR2A gene in the group of diabetes combined with tuberculosis is significantly lower than that in the group of only diabetes or tuberculosis (P<0.05). Figure 4 A).

[0133] The results show that there is a significant difference in the expression of ACVR2A gene compared with the healthy control group and the group of only diabetes or tuberculosis. ACVR2A gene has high accuracy for diagnosing diabetes combined with tuberculosis.

[0134] According to the geometric mean of the relative expression of ACVR2A gene in the blood of the group of diabetes combined with tuberculosis, the group of only diabetes or tuberculosis, and the healthy control group, the receiver operating characteristic curve analysis is performed using R language.

[0135] The ROC curve is shown in Figure 4 B. The area under the ROC curve is 0.682, which indicates that the diagnosis of diabetes combined with tuberculosis patients based on the expression of ACVR2A gene has high accuracy.

[0136] Example 4

[0137] ACVR2A is a serum marker for diabetes combined with tuberculosis

[0138] 1.1 Study object

[0139] In this embodiment, the study objects are the group of diabetes combined with tuberculosis patients, the group of only diabetes or tuberculosis, and the healthy control group.

[0140] The group of patients with diabetes mellitus and tuberculosis included 15 patients with clinically diagnosed diabetes mellitus and tuberculosis in Romania, HIV-negative, without pregnancy or taking corticosteroids, without other serious diseases. The age was between 22 and 64 years, with an average age of 47 years. There were 13 men and 2 women.

[0141] The group of patients with diabetes mellitus included 19 patients with clinically diagnosed diabetes mellitus, with glycosylated hemoglobin (HbA1c) ≥ 6.5% or fasting glucose ≥ 7 mmol / L, without other serious diseases. The age was between 38 and 65 years, with an average age of 55 years. There were 14 men and 5 women.

[0142] The group of patients with tuberculosis included 10 patients with positive detection of Mycobacterium tuberculosis and imaging detection, without other serious diseases. The age was between 30 and 64 years, with an average age of 43 years. There were 6 men and 4 women.

[0143] The group of healthy controls included 12 patients without serious diseases. The age was between 38 and 61 years, with an average age of 46 years. There were 10 men and 2 women.

[0144] 1.2 Detection of the expression of ACVR2A in venous blood

[0145] For each study subject, venous blood was collected at the time of diagnosis, introduced into PAXgene Blood RNA Tubes (PreAnalytiX), and used for RNA-seq analysis using the polyA tail library preparation method and single-read sequencing. The sequencing FASTQ files were aligned to the human genome, and transcription quantification, differential gene expression, biological properties, and machine learning were performed according to a fixed method.

[0146] 1.3 Results

[0147] The results of the RNA-seq analysis showed that the expression of the ACVR2A gene was significantly reduced in the group of patients with diabetes mellitus and tuberculosis, and the relative expression of the ACVR2A gene in the group of patients with diabetes mellitus and tuberculosis was significantly lower than that in the group of healthy controls and the group of patients with only diabetes mellitus or tuberculosis (p < 0.05). Figure 5 A).

[0148] The results showed that there was a significant difference in the expression of the ACVR2A gene compared to the group of healthy controls and the group of patients with only diabetes mellitus or tuberculosis. The ACVR2A gene has high accuracy for the diagnosis of diabetes mellitus and tuberculosis.

[0149] According to the geometric mean of the relative expression of the ACVR2A gene in the blood of the group of patients with diabetes mellitus and tuberculosis, the group of patients with only diabetes mellitus or tuberculosis, and the group of healthy controls, the receiver operating characteristic curve analysis was performed using R language.

[0150] The ROC curve is shown in Figure 1. Figure 5The area under the ROC curve is 0.733, which indicates that the expression level of ACVR2A is highly accurate for diagnosing diabetes patients with TB.

[0151] Example 5

[0152] A kit for detecting diabetes patients with TB

[0153] A kit for detecting diabetes patients with TB is prepared, which comprises:

[0154] (a) a container and primers specific to the markers of diabetes patients with TB in the container, which comprises:

[0155] The primer pair for amplifying ACVR2A is SEQ ID NO. 1 and SEQ ID NO. 2;

[0156] The primer pair for amplifying CXCL14 is SEQ ID NO. 3 and SEQ ID NO. 4;

[0157] Specific antibodies for detecting Angiostatin.

[0158] Table 1

[0159]

[0160] (b) and a label or instructions indicating that the kit is used for detecting diabetes patients with TB.

[0161] The above examples are only illustrative of the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. Use of a reagent for detecting the expression level of Activin A Receptor IIA in the preparation of a test kit for diagnosing diabetes complicated with tuberculosis.

2. Use according to claim 1, characterized in that: The test kit comprises: (a) an antibody specific to Activin A Receptor IIA; and / or (b) a primer or probe for mRNA or cDNA of Activin A Receptor IIA.

3. Use according to claim 1, characterized in that: The test kit comprises a chip for mRNA or cDNA of Activin A Receptor IIA, which comprises a nucleic acid chip and a protein chip.

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