CN115786490A - Marker for diabetes complicated tuberculosis and application thereof - Google Patents

Abstract

The invention belongs to the technical field of medical diagnosis, and particularly discloses a marker for diabetes complicated tuberculosis and application thereof. The invention discovers for the first time that the transcription level and the protein level of activin A receptor IIA (ACVR 2A), angiostatin (Angiostatin) and chemokine ligand 14 (CXCL 14) are inversely proportional to the severity of diabetes-complicated tuberculosis, and the activin A receptor IIA, the Angiostatin (Angiostatin) and the chemokine ligand 14 (CXCL 14) can be used as a marker of the diabetes-complicated tuberculosis to indirectly reflect whether people have the diabetes-complicated tuberculosis and the development of the disease, so that the diagnosis and treatment can be carried out in time. A new detection kit for the diabetes complicated tuberculosis can be prepared according to the three markers and applied to public medical treatment. The three markers can effectively improve the diagnosis efficiency of the diabetes combined tuberculosis, and have high sensitivity and strong specificity.

Description

Marker for diabetes complicated tuberculosis and application thereof

Technical Field

The invention relates to the technical field of medical diagnosis, in particular to a marker for diabetes complicated tuberculosis and application thereof.

Background

Tuberculosis is a main infectious disease which is harmful to human health, and according to the worldwide report of tuberculosis in 2021 by world health organization, about 1000 million tuberculosis patients and 20 hundred million people are infected with latent tuberculosis in 2020 world. Approximately 16% of tuberculosis patients are complicated by diabetes, and the dual burden of diabetes combined with tuberculosis has become a major public health challenge worldwide.

The risk of tuberculosis of the diabetic patients is 3 times that of the ordinary people, and the failure of tuberculosis treatment is increased by 2 times. Compared with the patients with simple tuberculosis, the patients with diabetes and tuberculosis have worse treatment outcome. Thus, the world health organization has identified diabetes as a neglected, important, recurring risk factor for tuberculosis. In general, diabetes can affect the clinical manifestations and therapeutic response of tuberculosis, which can lead to worsening glycemic control in diabetic patients. The complex interplay between the two poses a serious challenge for worldwide ending tuberculosis.

Patients with diabetes and tuberculosis are easy to delay the disease condition and aggravate the treatment burden. Therefore, early discovery and early treatment are the key points for preventing and treating diabetes complicated with tuberculosis. However, diabetes mellitus complicated with tuberculosis lacks an efficient and accurate diagnostic method at present. The existing diagnosis mode of diabetes combined tuberculosis mainly carries out diagnosis of blood sugar and mycobacterium tuberculosis separately, and the diagnosis method has long time consumption and high cost and cannot meet the requirements of quick diagnosis and economy of patients.

Therefore, the development of the biomarker and the detection method for early diagnosis of the diabetes-complicated tuberculosis with high sensitivity and high specificity, the development of the sensitive and efficient diagnostic reagent for the diabetes-complicated tuberculosis and the great significance for preventing and treating the diabetes-complicated tuberculosis are realized.

Disclosure of Invention

In view of the shortcomings of the prior art, the invention aims to provide a marker for diabetes complicated tuberculosis and application thereof, and provides a novel specific marker and a detection means with high sensitivity and high specificity for diagnosis and curative effect evaluation of diabetes complicated tuberculosis.

To achieve the above objects and other related objects, the present invention provides, in a first aspect, use of activin a receptor iia (ACVR 2A), chemokine ligand 14 (CXCL 14), and/or Angiostatin (Angiostatin) as biomarkers in preparing or screening test kits for diagnosing diabetes complicated tuberculosis and/or for evaluating prognosis and treatment effect of diabetes complicated tuberculosis.

Further, the diabetes includes type ii diabetes, and the tuberculosis includes pulmonary tuberculosis.

Furthermore, the detection kit is used for quantitatively detecting the expression level of activin A receptor IIA, chemokine ligand 14 and/or angiostatin in a sample.

Further, the expression levels of activin A receptor IIA, chemokine ligand 14, and angiostatin were inversely correlated with the severity of diabetes complicated with tuberculosis.

Further, when the expression levels of activin a receptor iia, chemokine ligand 14, and angiostatin are decreased to the expression levels of normal people, it is suggested that the therapeutic effect of diabetes complicated with tuberculosis is better or basically cured.

Further, if the expression level A1 of the activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin is significantly higher than a reference control value A0, the probability that the detected object has diabetes combined tuberculosis is higher than that of a normal population, and the reference control value A0 is the expression level of the diabetes combined tuberculosis marker activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin in a healthy population without diabetes combined tuberculosis or a patient only having diabetes or tuberculosis.

Further, if the expression level A1 level of activin a receptor iia, chemokine ligand 14, and/or angiostatin drops to the control reference value A0, it is suggested that the therapeutic effect of the subject for diabetes complicated with tuberculosis is better or substantially cured.

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

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

Further, the detection kit is used for detecting the genes, mRNA, cDNA, proteins or the combination thereof of the activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin.

Further, the detection kit comprises:

(a) Specific antibodies, specific binding molecules for activin A receptor IIA, chemokine ligand 14 and/or angiostatin;

and/or (b) a primer or primer pair, probe or chip of mRNA or cDNA of activin A receptor IIA, chemokine ligand 14, and/or angiostatin, said chip comprising a nucleic acid chip and a protein chip.

Further, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically bound with activin A receptor IIA, chemokine ligand 14 and/or angiostatin polynucleotides, and the polynucleotides are mRNA or cDNA.

Further, the protein chip comprises a substrate and specific antibodies spotted on the substrate, wherein the specific antibodies comprise specific antibodies to activin A receptor IIA, chemokine ligand 14 and/or angiostatin.

Further, the detection reagent in the detection kit is coupled with or carries a detectable label.

Further, the detectable label is selected from at least one of an isotope, a fluorophore, a chemiluminescent group, an enzyme.

Further, the diagnosis includes early diagnosis and auxiliary diagnosis.

Further, the detection kit comprises: a primer pair for amplifying activin a receptor iia, a primer pair for amplifying chemokine ligand 14, a specific antibody for detecting angiostatin, or a combination thereof.

Further, the primer pair for amplifying the activin A receptor IIA is SEQ ID No.1 and SEQ ID No.2, and the primer pair for amplifying the chemokine ligand 14 is SEQ ID No.3 and SEQ ID No.4.

Further, the antibody is a monoclonal antibody or a polyclonal antibody.

Further, the method for detecting the expression levels of the activin A receptor IIA, the chemokine ligand 14 and the angiostatin in the sample by the detection kit is any one of real-time fluorescent quantitative PCR, immunoblotting test, immunochemiluminescence method, immunoturbidimetry, immunoblotting method and enzyme-linked immunosorbent assay.

In a second aspect, the invention provides a detection kit for diagnosing diabetes complicated with tuberculosis and/or for evaluating prognosis and treatment effect of diabetes complicated with tuberculosis by detecting the expression level of activin A receptor IIA, chemokine ligand 14 and/or angiostatin.

Further, the diabetes includes type ii diabetes, and the tuberculosis includes pulmonary tuberculosis.

Furthermore, the detection kit is used for quantitatively detecting the expression level of activin A receptor IIA, chemokine ligand 14 and/or angiostatin in a sample.

Further, the expression levels of activin A receptor IIA, chemokine ligand 14, and angiostatin were inversely correlated with the severity of diabetes complicated with tuberculosis.

Further, when the expression levels of activin a receptor iia, chemokine ligand 14, and angiostatin are decreased to the expression levels of normal people, it is suggested that the therapeutic effect of diabetes complicated with tuberculosis is better or basically cured.

Further, if the expression level A1 of the activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin is significantly higher than a reference control value A0, the probability of the detection object generating the diabetes-associated tuberculosis is larger than that of a normal population, and the reference control value A0 is the expression level of the diabetes-associated tuberculosis marker activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin in a healthy population without diabetes-associated tuberculosis or a patient only suffering from diabetes or tuberculosis.

Further, if the expression level A1 level of activin a receptor iia, chemokine ligand 14, and/or angiostatin drops to the control reference value A0, it is suggested that the therapeutic effect of the subject for diabetes complicated with tuberculosis is better or substantially cured.

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

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

Further, the detection kit is used for detecting genes, mRNA, cDNA, proteins or combinations thereof of activin A receptor IIA, chemokine ligand 14 and/or angiostatin.

Further, the detection kit comprises:

(a) Specific antibodies, specific binding molecules for activin a receptor iia, chemokine ligand 14, and/or angiostatin;

and/or (b) a primer or primer pair, probe or chip of activin A receptor IIA, chemokine ligand 14, and/or mRNA or cDNA of angiostatin, said chip comprising a nucleic acid chip and a protein chip.

Further, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically combined with activin A receptor IIA, chemokine ligand 14 and/or angiostatin polynucleotides, and the polynucleotides are mRNA or cDNA.

Further, the protein chip comprises a substrate and specific antibodies spotted on the substrate, wherein the specific antibodies comprise specific antibodies to activin A receptor IIA, chemokine ligand 14 and/or angiostatin.

Further, the detection reagent in the detection kit is coupled with or carries a detectable label.

Further, the detectable label is selected from at least one of an isotope, a fluorophore, a chemiluminescent group, an enzyme.

Further, the diagnosis includes early diagnosis and auxiliary diagnosis.

Further, the detection kit comprises: a primer pair for amplifying activin a receptor iia, a primer pair for amplifying chemokine ligand 14, a specific antibody for detecting angiostatin, or a combination thereof.

Further, the primer pair for amplifying the activin A receptor IIA is SEQ ID No.1 and SEQ ID No.2, and the primer pair for amplifying the chemokine ligand 14 is SEQ ID No.3 and SEQ ID No.4.

Further, the antibody is a monoclonal antibody or a polyclonal antibody.

Further, the method for determining the expression levels of activin A receptor IIA, chemokine ligand 14 and angiostatin in the sample by the detection kit is any one of real-time fluorescent quantitative PCR, immunoblot assay, immunochemiluminescence method, immunoturbidimetry, immunoblot assay and enzyme-linked immunosorbent assay.

As described above, the marker for diabetes combined tuberculosis and the use thereof of the present invention have the following beneficial effects:

the invention discovers three serum marker genes of diabetes-complicated tuberculosis based on serological research, and the three serum marker genes can be used for diagnosing the diabetes-complicated tuberculosis and/or the treatment effect of the diabetes-complicated tuberculosis according to the transcription and/or protein level; the three genes discovered by the invention are respectively: the transcriptional level and the protein level of activin A receptor IIA (ACVR 2A), chemokine ligand 14 (CXCL 14) and Angiostatin (Angiostatin) are inversely proportional to the severity degree of diabetes complicated tuberculosis, and can indirectly reflect whether the crowd has the diabetes complicated tuberculosis and the disease development so as to diagnose and treat the diabetes complicated tuberculosis in time. Meanwhile, the transcription level and protein translation of the three markers are remarkably reduced in the patients with diabetes and tuberculosis through transcriptomics and proteomics, and the patients are recovered to be normal along with the prolonging of the treatment time. Based on the above, the invention provides the method for preparing the new diabetes-associated tuberculosis detection kit by using the three marker genes as biomarkers for diagnosis, prognosis and curative effect evaluation of the diabetes-associated tuberculosis and preparing the new diabetes-associated tuberculosis detection kit according to the two genes, which can be widely used in public medical treatment, can effectively improve the diagnosis efficiency of the diabetes-associated tuberculosis, and has high sensitivity and strong specificity.

Drawings

FIG. 1 shows the protein levels of three markers ACVR2A, CXCL14 and Angiostatin in example 1 of the invention during treatment of patients with Chinese diabetes complicated by tuberculosis.

FIG. 2 is a graph showing the ROC curves of ACVR2A, CXCL14 and Angiostatin in the Chinese patient data in example 1 of the present invention.

FIG. 3 shows a graph of the transcription levels and ROC during treatment of patients with diabetes mellitus combined with tuberculosis as marker ACVR2A in Indonesia in example 2 of the present invention.

FIG. 4 is a graph showing the transcription levels and ROC curves of ACVR2A in patients with south non-diabetic complicated tuberculosis and different control groups in example 3 of the present invention.

FIG. 5 is a graph showing the transcription levels and ROC curves of ACVR2A in the Romania diabetic combination tuberculosis patients and different control groups in example 4 of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The inventor of the present application extensively and deeply studied, and found for the first time three diabetes mellitus combined tuberculosis markers existing in blood: activin a receptor iia (ACVR 2A), CXC chemokine 14 (CXCL 14), and Angiostatin (Angiostatin). The diabetes-associated tuberculosis marker is specifically and highly expressed in the blood of a patient with diabetes-associated tuberculosis, and therefore, can be used as a specific marker for diagnosing (especially early diagnosis and/or auxiliary diagnosis) the diabetes-associated tuberculosis. Meanwhile, the experimental result of the embodiment of the application shows that compared with healthy people, patients with tuberculosis only or diabetic patients, the expression level of the diabetes-complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin in the patients with the diabetes-complicated tuberculosis is remarkably reduced, and the diabetes-complicated tuberculosis markers can be used as biomarkers for diagnosis, prognosis and curative effect evaluation of the diabetes-complicated tuberculosis. The present invention has been completed based on this finding.

Term(s) for

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which specific information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject.

The term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or a gene or gene portion, and also includes the presence of a detection substance associated with expression, such as cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and the chromogenic portion of the binding ligand are all included within the scope of "expression".

The term "reference value" or "control reference value" refers to a value that is statistically correlated with a particular result when compared to the results of the analysis. In an optimized embodiment, the reference value is determined from a statistical analysis comparing the expression of the diabetes-associated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin and performing a study with known clinical outcomes. Of course, this ratio can be determined on a case-by-case basis.

Diabetes complicated tuberculosis marker ACVR2A, CXCL14 and/or Angiostatin

As used herein, the term "diabetes-associated tuberculosis marker of the present invention" refers to one or more biomarkers selected from the group consisting of: ACVR2A, CXCL14 and Angiostatin. The term includes the full spectrum of diabetes mellitus associated with tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin and fragments thereof, as well as the completed amino acid sequences, secreted proteins, mutants thereof, and functionally active fragments thereof.

ACVR2A (NCBI Gene ID: 92), activin A Receptor Type 2A, the gene encodes a Receptor that mediates the function of Activin, 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 receptors are primarily involved in ligand binding and include an extracellular ligand-binding domain, a transmembrane domain, and a cytoplasmic serine threonine kinase domain. The relevant pathways include BMP signaling and telomere end packaging. The gene ontology annotation associated with this gene includes transferase activity, transfer phosphorus-containing groups, and protein tyrosine kinase activity. An important homologous gene of this gene is ACVR2B. Adipogenesis is mediated by GDF 6.

CXCL14 (NCBI gene ID: 9547), C-X-C Motif Chemokine Ligand 14, CXC Chemokine 14, which is a family of cytokine genes that encode secreted proteins that are involved in immune regulation and inflammatory processes. The protein encoded by the gene is structurally related to a cytokine CXC (Cys-X-Cys) subfamily. Members of this subfamily are characterized by two cysteines separated by one amino acid. The cytokine has chemotactic activity for monocytes but not lymphocytes, dendritic cells, neutrophils or macrophages. It is postulated that this cytokine is involved in monocyte macrophage homeostasis, rather than inflammation. Can promote thermogenic activity of brown adipose tissue, browning of white adipose tissue and enrichment of M2 type macrophage, and can change glucose balance in organism. Is inversely related to human obesity and accompanying type 2 diabetes.

Angiostatin and Angiostatin have anti-inflammatory and anti-fibrosis effects, and have therapeutic potential for diabetic nephropathy. Reduce vascular leakage, which is associated with diabetic macular edema. At the same time, anti-angiogenic drugs can improve tuberculosis treatment.

In the present invention, the terms "diabetes-associated tuberculosis marker gene" and "polynucleotide of diabetes-associated tuberculosis marker" and "protein of diabetes-associated tuberculosis marker", "protein of the present invention", or "polypeptide of the present invention" are interchangeable.

It is understood that nucleotide substitutions in codons are acceptable when encoding the same amino acid; conservative amino acid substitutions resulting from nucleotide substitutions are also acceptable. The proteins of the invention also include conservative variants thereof, such as conservative polypeptides.

When the amino acid or nucleotide fragment of the diabetes complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin is obtained, a specific probe can be designed according to the sequence. The full-length nucleic acid sequence or a fragment thereof can be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the nucleotide sequences, especially the open reading frame sequences, of ACVR2A, CXCL14 and/or Angiostatin, which are markers of diabetes combined tuberculosis disclosed by the invention, and relevant sequences can be obtained by amplification using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template.

In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. At present, the DNA sequence encoding the protein of the present invention (or its fragments, derivatives) can be obtained completely by chemical synthesis.

The polynucleotide sequences of the present invention can be used to express or produce recombinant markers ACVR2A, CXCL14 and/or Angiostatin for diabetes complicated tuberculosis by conventional recombinant DNA techniques.

Specific antibodies

In the present invention, the terms "antibody of the invention" and "antibody specific for an anti-diabetic combined tuberculosis marker" are used interchangeably.

The invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for the diabetes-associated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin polypeptides, as well as antibodies capable of binding to the gene products of the human diabetes-associated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin in modified or unmodified form. The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments.

The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. For example, purified human diabetes mellitus combined with tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin gene products, or fragments with antigenicity, can be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing human diabetes mellitus combined with tuberculosis markers ACVR2A, CXCL14, and/or Angiostatin proteins or antigenic fragments can be used to immunize animals to produce antibodies.

Antibodies against human diabetes-associated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein can be used in immunohistochemical techniques to detect human diabetes-associated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein in a sample, particularly a tissue sample or a blood sample. Since the diabetes combined tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein exist in mononuclear cells in a blood sample, the expression level can be a detection object.

Detection method

Based on the low expression of the diabetes-complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin in blood, the invention also provides a corresponding method for diagnosing the diabetes-complicated tuberculosis. The invention relates to a diagnostic test method for quantitatively detecting the protein level or mRNA level of human diabetes combined tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin. These assays are well known in the art. The protein level or mRNA level of the human diabetes complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin detected in the test can be used for diagnosing (including early diagnosis and auxiliary diagnosis) whether the diabetes complicated tuberculosis exists.

A preferred method is PCR based quantitative detection of mRNA or cDNA.

A preferred method is to perform sequencing quantitative detection on mRNA or cDNA.

One preferred method is to quantitatively detect the protein of a marker for diabetes combined with tuberculosis.

Preferably, one method for detecting the presence of the diabetic tuberculosis complex protein in a sample is by using specific antibodies, which comprises: and (3) contacting the sample with an antibody specific to the diabetes-complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein, observing whether an antibody complex is formed, wherein the formed antibody complex represents that the diabetes-complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein exists in the sample.

The marker protein of diabetes combined tuberculosis or polynucleotide thereof can be used for diagnosing the diabetes combined tuberculosis. Part or all of the polynucleotides of the present invention can be used as probes fixed on a microarray or a DNA chip for analyzing differential expression analysis of genes in a single nuclear cell and gene diagnosis. The antibody for resisting diabetes combined tuberculosis can be fixed on a protein chip and used for detecting diabetes combined tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin protein in a sample, and based on the research of the invention, the expression level (mRNA level or protein level) of ACVR2A, CXCL14 and/or Angiostatin gene is remarkably reduced in a diabetes combined tuberculosis patient. Therefore, the ACVR2A, CXCL14 and/or Angiostatin can be used as markers for detecting or diagnosing (especially auxiliary diagnosis and/or early diagnosis) the diabetes combined with tuberculosis. When detecting, if the ratio of the expression level of the marker gene (i.e. ACVR2A, CXCL14 and/or Angiostatin) to the corresponding expression level in the normal population is significantly changed, the risk of diabetes complicated with tuberculosis can be considered to be increased.

According to the experimental results of the present invention, a preferred method for diagnosing patients with diabetic complications is based on the geometric mean of transcription level. Preferably, the method comprises the following criteria: if the geometric mean of the transcription levels of the ACVR2A, CXCL14 and/or Angiostatin genes in the blood of the test subject is reduced, the test subject can be preliminarily predicted to be diabetic patients with tuberculosis. In addition, in view of the obvious ascending trend of the transcription level of the marker genes (ACVR 2A, CXCL14 and/or Angiostatin) in the diabetes-complicated tuberculosis patients during the treatment process and at the completion of the treatment, the expression level of the marker genes can be detected to reflect or evaluate the treatment effect of the diabetes-complicated tuberculosis. Wherein, when the expression level of the marker gene is increased to the level of normal people, the effect of treating the diabetes combined tuberculosis is better or basically cured.

Detection kit

Based on the correlation between the diabetes-complicated tuberculosis markers and the diabetes-complicated tuberculosis, the diabetes-complicated tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin can be used as diagnostic markers of the diabetes-complicated tuberculosis.

The invention also provides a kit for diagnosing the diabetes complicated with tuberculosis, which contains a detection reagent used for detecting the diabetes complicated with tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin gene, mRNA, cDNA, protein or the combination thereof.

Preferably, the kit contains an antibody or immunoconjugate of the anti-diabetic combined tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin of the invention, or an active fragment thereof; or a primer pair, a probe or a chip which specifically amplifies the mRNA or cDNA of the diabetes mellitus combined tuberculosis markers AACVR2A, CXCL14 and/or Angiostatin.

In another preferred embodiment, the kit further comprises a label or a specification, wherein the label or the specification indicates that the kit is used for diagnosing diabetes complicated with tuberculosis and/or evaluating the treatment effect of diabetes complicated with tuberculosis.

The main advantages of the invention include:

(a) The ACVR2A, CXCL14 and/or Angiostatin are biomarkers of diabetes complicated tuberculosis, which are discovered by the inventor for the first time, can be applied to diagnosis of diabetes complicated tuberculosis, and provide reference basis for more accurate and earlier diagnosis.

(b) The diabetes combined tuberculosis markers ACVR2A, CXCL14 and/or Angiostatin exist in mononuclear cells in blood, so that the diabetes combined tuberculosis markers can be conveniently sampled and detected.

(c) The markers ACVR2A, CXCL14 and/or Angiostatin for the diabetes complicated tuberculosis can be used as biomarkers for effectively evaluating the treatment effect of the diabetes complicated tuberculosis and can also be used as biomarkers for effectively evaluating the prognosis effect of the diabetes complicated tuberculosis.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures for which specific conditions are not noted in the following examples are generally performed according to conventional conditions, for example, as described in Sambrook et al, molecular cloning: A Laboratory Manual (New York: cold spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight. Materials, reagents, instruments and the like used in examples are commercially available unless otherwise specified.

General procedure

Real-time fluorescent quantitative PCR

Real-time fluorescence quantitative PCR is an experimental method for applying a fluorescence quantitative technology to polymerase chain reaction. A fluorescent dye called SYBR Green I was used in this experiment. In a PCR reaction system, SYBRGreen I specifically participates in a DNA double strand and then emits a fluorescent signal, and SYBR dye molecules which are not doped into the strand do not emit any fluorescent signal. Because this method allows the increase in fluorescence signal to be synchronized with the increase in PCR product, i.e., the intensity of the fluorescence signal emitted by the fluorochrome is proportional to the DNA yield. Therefore, the initial concentration of the target sequence can be obtained by detecting the intensity of the fluorescence signal in the PCR process, thereby achieving the purpose of quantification.

Enzyme linked immunosorbent assay

Enzyme linked immunosorbent assay (abbreviated as ELISA or ELASA) refers to a qualitative and quantitative detection method in which soluble antigen or antibody is bound to a solid-phase carrier such as polystyrene, and immunoreaction is carried out by utilizing the specific binding of antigen and antibody. The basic principle of this method is: (1) allowing the antigen or antibody to bind to the surface of a solid support and retain its immunological activity. (2) The antigen or antibody is linked with certain enzyme to form enzyme labeled antigen or antibody, which retains both its immunological activity and enzyme activity.

Example 1

Changes in the levels of ACVR2A, CXCL14 and Angiostatin protein during treatment of patients with diabetes complicated by tuberculosis

In this example, the subjects were the changes in the levels of ACVR2A, CXCL14 and Angiostatin during the treatment of whole blood samples from a group of patients with diabetes and tuberculosis.

The violin diagram shows the changes of ACVR2A, CXCL14 and Angiostatin proteins during the treatment of diabetes combined tuberculosis, and the difference between the two time points is calculated by the rank sum test (Wilcoxon rank test).

In this example, the expression of genes in 4 cases of Chinese diabetes patients with tuberculosis before treatment, during treatment and after recovery was determined.

As shown in fig. 1A, 1B and 1C, the results indicate protein levels of ACVR2A, CXCL14 and Angiostatin for patients with diabetes complicated with tuberculosis: as treatment progressed, ACVR2A, CXCL14 and Angiostatin protein levels continued to rise with significant variability in diabetic patients with tuberculosis.

And (3) performing analysis on a working characteristic curve of the subject by using an R language according to a geometric mean value of relative expression amounts of ACVR2A, CXCL14 and Angiostatin proteins during and after the treatment period of the diabetes combined tuberculosis.

The Receiver Operating Characteristic (ROC) reflects the balance between sensitivity and specificity, the area under the ROC curve is an important test accuracy index, and the larger the area under the ROC curve is, the higher the diagnostic value of the test is.

Sensitivity (true positive rate): if the patient is actually ill, the patient is correctly judged as the percentage of ill according to the test standard, and the sensitivity is better if the sensitivity is larger, and the ideal sensitivity is 100%.

Specificity (true negative rate): the percent of the disease-free rate is judged correctly according to the test standard, the larger the specificity is, the better the ideal specificity is 100%.

The ROC curves are shown in fig. 2A, 2B and 2C. The areas under the ROC curve are 0.938, 0.812 and 0.812 respectively, which shows that the diagnosis of the diabetes complicated with tuberculosis patients according to the expression levels of the ACVR2A, the CXCL14 and the Angiostatin proteins has higher accuracy.

Example 2

Changes in ACVR2A transcript levels during treatment of diabetic patients with tuberculosis

In this example, the subjects were the ACVR2A protein level changes during the treatment of whole blood samples from diabetic patients with tuberculosis.

The violin diagram shows the change of the ACVR2A gene in the treatment process of diabetes combined tuberculosis, and the difference between two time points is calculated by the rank sum test (Wilcoxon rank test).

In this example, the expression of genes in 29 Indonesia patients with diabetes complicated with tuberculosis before treatment, during treatment and after recovery was determined.

As shown in fig. 3A, the results indicate the transcription level of ACVR2A gene for patients with diabetes complicated with tuberculosis: as treatment progresses, ACVR2A protein levels continue to increase in patients with diabetic complications.

And (3) performing analysis on a working characteristic curve of the testee by using an R language according to a geometric mean value of relative expression of the ACVR2A gene during and after the treatment period of the diabetes combined tuberculosis.

The ROC curve is shown in fig. 3B. The areas under the ROC curves are respectively 0.631, which shows that the diagnosis of the diabetes mellitus complicated by tuberculosis patients according to the expression quantity of the ACVR2A gene has higher accuracy.

Example 3

ACVR2A is a serum marker for diabetes complicated tuberculosis

1.1 study object

In this example, the subjects were a group of patients with diabetes combined with tuberculosis, a group with diabetes alone or tuberculosis, and a healthy control group.

The group of patients with diabetes complicated with tuberculosis comprises 15 patients with diabetes complicated with tuberculosis in south Africa clinically diagnosed, which have negative HIV, no pregnancy or are taking corticosteroids, and no other serious diseases. The age is 27-57 years, and the average age is 46 years, 7 men and 8 women.

In the group of diabetes patients, 33 patients with clinical glycosylated hemoglobin (HbA 1 c) more than or equal to 6.5 percent or fasting blood glucose more than or equal to 7mmol/L have no other serious diseases. The age is 29-64 years, and the average age is 49 years. 15 male and 18 female.

The tuberculosis patient group has 11 positive tuberculosis mycobacteria detection patients and imaging detection patients without other serious diseases. The age is 31-56 years, and the average age is 48 years. 2 male and 9 female.

Healthy controls, 24 had no severe disease. The age is 30-70 years, and the average age is 42 years. 12 male and 12 female.

1.2 ACCRV2A expression level detection in venous blood

For each subject, venous blood was collected at diagnosis, introduced into PAXgene blood RNA tubes (PreAnalytiX), and used for RNA-seq analysis, using polyA tail library preparation methods and single read sequencing. Sequencing FASTQ files were aligned to the human genome and transcription quantification, differential gene expression, biological characterization and machine learning were performed according to a fixed method.

1.3 results

The RNA-seq analysis result shows that the expression level of the ACVR2A gene is obviously reduced in the diabetes and tuberculosis group, and the relative expression level of the ACVR2A gene in the diabetes and tuberculosis group is obviously higher than that in a healthy control group, a group only suffering from diabetes or tuberculosis (figure 4A).

The results show that: compared with a healthy control group and a group only suffering from diabetes or tuberculosis, the ACVR2A gene has a significant difference in expression level. The ACVR2A gene has higher accuracy when being used for diagnosing the diabetes complicated with tuberculosis.

And performing the analysis of the working characteristic curve of the testee by using an R language according to the geometric mean value of the relative expression quantity of the ACVR2A gene in the blood of the diabetes combined tuberculosis group, the diabetes only or tuberculosis group and the healthy control group.

The ROC curve is shown in fig. 4B. The area under the ROC curve is 0.682, which shows that the diagnosis of the diabetes mellitus with tuberculosis patients according to the expression level of the ACVR2A gene has higher accuracy.

Example 4

ACVR2A is a serum marker for diabetes combined tuberculosis

1.1 study subjects

In this example, the subjects were diabetic patients with tuberculosis, diabetic patients alone or tuberculosis patients, and healthy controls.

The group of patients with diabetes complicated with tuberculosis comprises 15 patients with diabetes complicated with tuberculosis, which are clinically diagnosed as Romani, and HIV is negative, and the patients are not pregnant or are taking corticosteroids, and have no other serious diseases. The age is 22-64 years, and the average age is 47 years. 13 male and 2 female.

In the group of patients with diabetes, 19 patients with clinical glycosylated hemoglobin (HbA 1 c) of not less than 6.5% or fasting blood glucose of not less than 7mmol/L have no other serious diseases. The age is 38-65 years, and the average age is 55 years. 14 male and 5 female.

In the tuberculosis patient group, 10 positive tuberculosis mycobacteria detection patients and imaging detection patients have no other serious diseases. The age is 30-64 years, and the average age is 43 years. 6 male and 4 female.

Healthy control group, 12 had no serious disease. The age is 38-61 years, and the average age is 46 years. 10 male and 2 female.

1.2ACVR2A expression level detection in venous blood

For each subject, venous blood was collected at diagnosis, introduced into PAXgene blood RNA tubes (PreAnalytiX), and used for RNA-seq analysis, using polyA tail library preparation methods and single read sequencing. Sequencing FASTQ files were aligned to the human genome and transcription quantification, differential gene expression, biological characterization and machine learning were performed according to a fixed method.

1.3 results

The RNA-seq analysis result shows that the expression level of the ACVR2A gene is obviously increased in the diabetes and tuberculosis group, and the relative expression level of the ACVR2A gene in the diabetes and tuberculosis group is obviously higher than that in a healthy control group, a group only suffering from diabetes or tuberculosis (figure 5A).

The results show that: compared with a healthy control group and a group only suffering from diabetes or tuberculosis, the ACVR2A gene has a significant difference in expression level. The ACVR2A gene has higher accuracy when being used for diagnosing the diabetes complicated with tuberculosis.

And performing the analysis of the working characteristic curve of the testee by using R language according to the geometric mean value of the relative expression quantity of the ACVR2A gene in the blood of the diabetes combined tuberculosis group, the diabetes only or tuberculosis group and the healthy control group.

The ROC curve is shown in FIG. 5B. The area under the ROC curve is 0.733, which shows that the diagnosis of the diabetes mellitus combined with tuberculosis patients according to the expression quantity of the ACVR2A factor has higher accuracy.

Example 5

Kit for detecting diabetes complicated tuberculosis

Preparing a kit for detecting diabetes combined tuberculosis, wherein the kit comprises:

(a) A container and a primer positioned in the container and specifically aiming at a diabetes combined tuberculosis marker, wherein the primer comprises the following components:

the primer pair for amplifying the ACVR2A is SEQ ID NO.1 and SEQ ID NO.2;

the primer pair for amplifying CXCL14 is SEQ ID NO.3 and SEQ ID NO.4;

antibodies specific for the detection of Angiostatin.

TABLE 1

(b) And a label or instruction indicating that the kit is used for detecting diabetes combined tuberculosis.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Those skilled in the art can modify or change the above-described embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Claims (10)

1. The application of activin A receptor IIA (ACVR 2A), chemokine ligand 14 (CXCL 14) and/or Angiostatin (Angiostatin) as biomarkers in preparing or screening a detection kit for diagnosing diabetes complicated with tuberculosis and/or evaluating prognosis and treatment effect of diabetes complicated with tuberculosis.

2. Use according to claim 1, characterized in that: the detection kit is used for quantitatively detecting the expression level of activin A receptor IIA, chemokine ligand 14 and/or angiostatin in a sample.

3. Use according to claim 2, characterized in that: the expression levels of activin A receptor IIA, chemokine ligand 14 and angiostatin are inversely related to the severity of diabetes complicated with tuberculosis.

4. Use according to claim 2, characterized in that: the sample is selected from at least one of blood sample, serum sample, tissue sample and body fluid sample.

5. Use according to claim 1, characterized in that: the detection kit comprises:

(a) Specific antibodies, specific binding molecules for activin A receptor IIA, chemokine ligand 14 and/or angiostatin;

(b) And/or (b) a primer or primer pair, probe or chip of mRNA or cDNA of activin A receptor IIA, chemokine ligand 14, and/or angiostatin, said chip comprising a nucleic acid chip and a protein chip.

6. A detection kit, which is characterized in that: the detection kit is used for diagnosing the diabetes complicated with tuberculosis and/or evaluating the prognosis and treatment effect of the diabetes complicated with tuberculosis by detecting the expression level of the activin A receptor IIA, the chemokine ligand 14 and/or the angiostatin.

7. The detection kit according to claim 6, characterized in that: the detection kit is used for quantitatively detecting the expression level of activin A receptor IIA, chemokine ligand 14 and/or angiostatin in a sample.

8. The test kit according to claim 7, characterized in that: the expression levels of activin A receptor IIA, chemokine ligand 14 and angiostatin are inversely related to the severity of diabetes complicated tuberculosis.

9. The test kit according to claim 7, characterized in that: the sample is selected from at least one of blood sample, serum sample, tissue sample and body fluid sample.

10. The detection kit according to claim 6, characterized in that: the detection kit comprises:

(a) Specific antibodies, specific binding molecules for activin a receptor iia, chemokine ligand 14, and/or angiostatin;

(b) And/or (b) a primer or primer pair, probe or chip of mRNA or cDNA of activin A receptor IIA, chemokine ligand 14, and/or angiostatin, said chip comprising a nucleic acid chip and a protein chip.

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