Markers for diagnosing active tuberculosis and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a group of markers for diagnosing active tuberculosis and application. The biomarker is easy and convenient to sample, only peripheral blood needs to be taken for detection, and the result reporting speed is high. The biomarker disclosed by the invention is high in accuracy: when a subject's working characteristic curve (ROC) analysis shows that when GCH1, GK, MTHFD2 and SLC7A6 are combined, it can be known through database screening that the ROC for diagnosing active tuberculosis patients in TB patients, HC control and LTBI patients is 0.906, the specificity is 93.9, and the sensitivity is 85.7; clinical tests prove that the ROC of active tuberculosis patients in TB patients, HC control and LTBI patients is 0.942, the specificity is 90.9, and the sensitivity is 85.7.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a group of markers for diagnosing active tuberculosis and applications thereof. Background Art

[0002] Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (M.tb) that seriously endangers human health and is a major threat to the global public health system. The 2024 Global Tuberculosis Report shows that in 2023, there will be 10.8 million new cases of tuberculosis, 8.2 million confirmed cases, and 1.25 million deaths worldwide. The situation of tuberculosis prevention and control remains severe. The diagnosis of tuberculosis currently relies mainly on bacteriology and molecular biology detection technologies (Industry Guidelines for Diagnosis of Pulmonary Tuberculosis, WS-288-2017). In countries with a high burden of tuberculosis, smear microscopy is widely used, but it is labor-intensive, has low sensitivity, and cannot rule out non-tuberculous mycobacterial infection (Zhang Wei et al., Research Progress in Laboratory Diagnostic Methods and Detection Technologies for Mycobacterium tuberculosis Infection, International Journal of Respiratory Diseases, 2019, 39: 1586-1591); sputum culture is the gold standard for diagnosing tuberculosis. Even if a relatively rapid liquid culture method (such as modified Roche medium) is used to culture Mycobacterium tuberculosis, it takes at least 2 weeks to obtain a positive result, and it takes about 45 days to report a negative result, thus affecting the efficiency of early diagnosis and treatment (Pai M, Schito M. Tuberculosis diagnostics in 2015: landscape, priorities, needs, and prospects [J]. J Infect Dis, 2015, 211 (Suppl 2): ​​S2l-S28.). The new generation of Xpert Ultra molecular detection methods recommended by the World Health Organization are often limited by the type of specimens and the method of sampling. In addition, the results of the tuberculin test and the interferon-γ release experiment cannot distinguish between active tuberculosis and latent infection. The imaging technology-assisted diagnosis of tuberculosis is similar to the imaging manifestations of other lung diseases and has low specificity. At present, the diagnosis method for patients with active tuberculosis mainly relies on pathogen detection and imaging examinations, but no method has high sensitivity and specificity for active tuberculosis. In order to control the spread of tuberculosis, the World Health Organization (WHO) also recommends early active screening of suspected tuberculosis patients, but the existing diagnostic technology is far from meeting clinical needs.

[0003] Biomarkers can reflect disease status, risk of progression, biological effects after treatment, therapeutic effects and prognosis assessment, and play an important role in disease diagnosis, treatment and prevention, providing a key basis for the development of new anti-tuberculosis drugs and vaccines. In response to this situation, WHO has developed a target product profile (TPP), a set of standards for defining the key characteristics that an ideal diagnostic tool should have (Sarah C Charnaud, V. Moorthy et al. "WHO target product profiles to shape global research and development." Bulletin of the World Health Organization (2023).). TPP emphasizes the use of non-sputum samples for testing, and requires that the detection method is simple and rapid, suitable for use in resource-limited areas. Therefore, finding new tuberculosis biomarkers that are stably expressed in non-sputum samples is of great significance for improving the existing diagnostic system and enhancing diagnostic efficiency.

[0004] Therefore, it is necessary to explore rapid and effective detection methods to diagnose active tuberculosis. Summary of the invention

[0005] The present invention found that the expression level of the gene has significant changes in the peripheral blood of active tuberculosis patients and is significantly correlated with the progress of anti-tuberculosis treatment with high sensitivity and specificity. Based on this, the present invention was completed.

[0006] In a first aspect, the present invention provides a group of markers for diagnosing active tuberculosis, wherein the markers are selected from any one of GCH1, GK, MTHFD2 or SLC7A6, or a combination of GCH1, GK, MTHFD2 and SLC7A6.

[0007] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, if the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0008] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is a patient with active tuberculosis.

[0009] Furthermore, when the marker for diagnosing active tuberculosis is GK, if the expression level of GK in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0010] Furthermore, when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is a patient with active tuberculosis.

[0011] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, if the expression level of MTHFD2 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0012] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis.

[0013] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, if the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, the subject is a patient with active tuberculosis.

[0014] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

[0015] Furthermore, when the markers for diagnosing active tuberculosis are a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

[0016] Furthermore, the biological sample of the subject is selected from blood.

[0017] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0018] In a second aspect, the present invention provides use of the marker described in the first aspect of the present invention in the preparation of a reagent for diagnosing active tuberculosis patients.

[0019] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, if the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0020] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is a patient with active tuberculosis.

[0021] Furthermore, when the marker for diagnosing active tuberculosis is GK, if the expression level of GK in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0022] Furthermore, when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is a patient with active tuberculosis.

[0023] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, if the expression level of MTHFD2 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0024] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis.

[0025] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, if the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, the subject is a patient with active tuberculosis.

[0026] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

[0027] Furthermore, when the markers for diagnosing active tuberculosis are a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

[0028] Furthermore, the biological sample of the subject is selected from blood.

[0029] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0030] In a third aspect, the present invention provides a kit for diagnosing active tuberculosis patients, wherein the kit contains a reagent for detecting the expression level of the marker described in the first aspect.

[0031] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, if the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0032] Furthermore, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is a patient with active tuberculosis.

[0033] Furthermore, when the marker for diagnosing active tuberculosis is GK, if the expression level of GK in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0034] Furthermore, when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is a patient with active tuberculosis.

[0035] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, if the expression level of MTHFD2 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, then the subject is an active tuberculosis patient.

[0036] Furthermore, when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis.

[0037] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, if the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, the subject is a patient with active tuberculosis.

[0038] Furthermore, when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

[0039] Furthermore, when the markers for diagnosing active tuberculosis are a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

[0040] Furthermore, the biological sample of the subject is selected from blood.

[0041] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0042] Furthermore, the kit may be one or more of a nucleic acid detection kit, an immunofluorescence kit, a gene chip detection kit, a molecular hybridization kit and / or an in situ hybridization staining kit.

[0043] Furthermore, the diagnostic method of the kit includes one or more of PCR method / qPCR method, linear probe method, high-resolution melting curve method and / or gene chip method.

[0044] Beneficial Effects

[0045] The biomarker applied in the present invention is easy to sample, only peripheral blood needs to be taken for detection, and the results are reported quickly. The biomarker applied in the present invention has high accuracy: the receiver operating characteristic curve (ROC) analysis shows that when GCH1, GK, MTHFD2 and SLC7A6 are combined, it can be learned through database screening that the ROC for diagnosing active tuberculosis patients in TB patients, HC controls and LTBI patients is 0.906, the specificity is 93.9, and the sensitivity is 85.7; it is verified in clinical trials that the ROC for diagnosing active tuberculosis patients in TB patients, HC controls and LTBI patients is 0.942, the specificity is 90.9, and the sensitivity is 85.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 To screen for differentially expressed metabolism-related genes in patients with active tuberculosis compared with healthy samples.

[0047] Note: A. Volcano plot shows the changes in gene expression in active tuberculosis patients compared with healthy samples; B. Venn diagram shows the common genes of differentially expressed genes and metabolism-related genes; C. Heat map shows the expression of metabolism-related differentially expressed genes.

[0048] Figure 2To screen metabolism-related genes with the ability to diagnose active tuberculosis using machine learning models.

[0049] Note: A. LASSO coefficient path diagram shows the change curve of each independent variable coefficient; B. Cross-validation curve of LASSO regression model; C. Error rate and confidence interval of random forest model; D. Bar graph shows the importance score of each gene; E. Veen graph shows the common genes screened by two machine learning models; F. Expression of four key genes in GSE83456 dataset; G. ROC curve shows the diagnostic ability of four genes in GSE83456 dataset.

[0050] Figure 3 To construct a nomogram for predicting active tuberculosis.

[0051] Note: A. Construction of a nomogram based on four metabolism-related genes; B. The combined diagnostic ability of four genes in the GSE83456 dataset.

[0052] Figure 4 The diagnostic ability of four genes for active tuberculosis for the new dataset.

[0053] Note: A. Expression of four key genes in PBMC of HC controls, LTBI and TB patients in GSE107991 dataset; B. Diagnostic ability of four gene combinations in GSE107991 dataset for TB and HC controls; C. Diagnostic ability of four gene combinations in GSE107991 dataset for LTBI and TB patients; D. Diagnostic ability of four gene combinations in GSE107991 dataset for HC controls, LTBI and TB.

[0054] Figure 5 Four genes were validated for expression and analyzed for diagnostic ability on PBMCs collected clinically.

[0055] Note: A. Expression levels of GCH1, GK, MTHFD2 and SLC7A6 genes in PBMCs of HC controls, LTBI and TB patients; B. Diagnostic ability of the four-gene combination. DETAILED DESCRIPTION

[0056] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0057] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0058] Example 1: Standards for elimination

[0059] A. Inclusion Criteria

[0060] 1) Patients with active tuberculosis: According to the industry standard "WS-288-2017 Diagnosis of Pulmonary Tuberculosis", patients with clinical symptoms of tuberculosis and chest imaging showing active tuberculosis lesions are included, and one of the following conditions is met: positive sputum smear for Mycobacterium tuberculosis, positive culture for Mycobacterium tuberculosis, positive molecular biological detection for Mycobacterium tuberculosis, or positive histopathology.

[0061] 2) Healthy samples: no clinical symptoms of tuberculosis, no abnormalities in imaging examinations, negative tuberculosis-infected T cell tests, and no previous history of tuberculosis or contact with tuberculosis.

[0062] 3) Latently infected persons: no clinical symptoms of tuberculosis, no abnormalities in imaging examinations, and positive tuberculosis infection T cell test.

[0063] B. Exclusion criteria

[0064] Age <18 years, anti-TB treatment for more than 2 weeks.

[0065] C. Screening Queue

[0066] Dataset GSE83456: 61 healthy samples (HC) that meet the criteria; 92 active tuberculosis patients (TB) that meet the criteria; peripheral blood mononuclear cells (PBMCs) transcriptome sequencing data were collected to form the dataset GSE83456.

[0067] Dataset GSE107991: 12 healthy samples (HC) that meet the criteria; 21 latent tuberculosis patients that meet the criteria; 21 active tuberculosis patients (LITB) that meet the criteria; peripheral blood mononuclear cells (PBMCs) transcriptome sequencing data were collected to form the dataset GSE107991.

[0068] D. Verification Queue

[0069] There were 14 healthy samples that met the criteria; 11 active tuberculosis patients that met the criteria; and 11 latent tuberculosis patients that met the criteria.

[0070] Example 2 Screening test - healthy and active tuberculosis samples

[0071] (1) Test method

[0072] A. Transcriptome data acquisition

[0073] The transcriptome data used in this application were downloaded from the GEO dataset, with accession numbers GSE83456.

[0074] B. Differential gene expression analysis

[0075] The peripheral blood mononuclear cells (PBMCs) transcriptome sequencing dataset GSE83456 was used as the training set, and the R package “limma” was used to analyze differentially expressed genes (DEGs) according to the screening criteria of P < 0.05 and |fold change|> 1.5.

[0076] The intersection of 1932 known metabolism-related genes and 785 DEGs was taken to obtain 41 metabolism-related DEGs (MRDEGs), which were used to elucidate the changes of metabolism-related genes after Mtb infection.

[0077] C. Screening of metabolism-related genes using LASSO regression and random forest models

[0078] LASSO regression and random forest algorithms were used to construct a machine learning model for 41 MRDEGs to further identify key metabolic genes associated with active tuberculosis.

[0079] (2) Test results

[0080] Analysis of differentially expressed genes showed that 546 up-regulated genes and 239 down-regulated genes were identified in patients with active tuberculosis compared with healthy controls ( Figure 1 (A).

[0081] The intersection of 1932 known metabolism-related genes and 785 DEGs was taken to obtain 41 metabolism-related DEGs (MRDEGs), of which 7 genes were downregulated and 34 genes were upregulated ( Figure 1 Medium B and Figure 1 Middle C).

[0082] The LASSO regression and random forest algorithms were used to construct machine learning models for 41 MRDEGs. Figure 2 Middle A and Figure 2 As shown in Figure B, LASSO regression analysis screened out 12 candidate genes with diagnostic value. At the same time, the random forest model identified 6 genes with importance scores above 4.0 that were associated with active tuberculosis patients ( Figure 2 Middle C and Figure 2 After integrating the results of the two machine learning algorithms, four key metabolism-related genes with potential for active tuberculosis diagnosis were obtained, including GCH1, GK, MTHFD2, and SLC7A6 ( Figure 2 Middle E).

[0083] The expression levels of these genes in the training set were analyzed. Compared with HC controls, GCH1, GK, and MTHFD2 were upregulated, while SLC7A6 was downregulated in TB patients ( Figure 2 Middle F).

[0084] Receiver operating characteristic (ROC) curves were drawn to evaluate the prognostic value of each gene. The areas under the ROC curve (AUC) of GCH1, GK, MTHFD2, and SLC7A6 were 0.883, 0.897, 0.919, and 0.946, respectively. Figure 2 This indicates that the four genes have good diagnostic efficacy for patients with active tuberculosis.

[0085] A diagnostic nomogram was constructed using the training set to evaluate the efficacy of the four genes in diagnosing active tuberculosis patients. Figure 3 As shown in Figure A, the expression level of each gene is converted into a score, and the scores of the four genes are aggregated to predict the possibility of active tuberculosis. The AUC value of the ROC curve is 0.979 ( Figure 3 Middle B), indicating that the combined diagnosis of the four genes has a good diagnostic performance, with a specificity of 100.0 and a sensitivity of 87.0, and the diagnostic effect is better than that of a single gene.

[0086] Example 3 Screening test - healthy, latent tuberculosis and active tuberculosis samples

[0087] (1) Test method

[0088] The transcriptome data used in this application were downloaded from the GEO dataset, with accession numbers GSE83456.

[0089] The test method was the same as in Example 2. The expression levels of four genes in different groups of samples were analyzed.

[0090] (2) Test results

[0091] Compared with healthy samples and LTBI samples, the expression levels of GCH1, GK, and MTHFD2 were significantly increased in the TB group, while the expression level of SLC7A6 was significantly decreased in the TB group ( Figure 4 This is consistent with the results of Example 2.

[0092] The diagnostic ability of 4 genes was evaluated. Figure 4 In BD, the ROC of the four gene combination for diagnosing active TB patients in TB patients and HC controls was 0.940, the specificity was 85.7, and the sensitivity was 100.0; the ROC of diagnosing active TB patients in TB patients and LTBI patients was 0.887, the specificity was 85.7, and the sensitivity was 90.5; the ROC of diagnosing active TB patients in TB patients, HC controls, and LTBI patients was 0.906, the specificity was 93.9, and the sensitivity was 85.7; all showed high diagnostic ability.

[0093] Example 3 Validation Test - Healthy, Latent Tuberculosis and Active Tuberculosis Samples

[0094] (1) Test method

[0095] There were 14 healthy samples that met the criteria; 11 active tuberculosis patients that met the criteria; and 11 latent tuberculosis patients that met the criteria.

[0096] The peripheral blood of the subjects was collected and centrifuged; the ring-shaped milky white mononuclear cell layer was aspirated, the cell pellet was resuspended, and centrifuged again;

[0097] The cell pellet was reselected and then centrifuged;

[0098] Take the supernatant, add isopropanol and centrifuge to precipitate RNA;

[0099] Reselect the precipitate, centrifuge and repeat the process, dissolve the precipitate and determine the RNA concentration;

[0100] Reverse transcription and real-time quantitative PCR (RT-qPCR) analysis were performed.

[0101] Two-sided unpaired t-test was used to compare the differences between the two groups. The differences were considered statistically significant when *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

[0102] (2) Test results

[0103] like Figure 5 As shown in Figure A, compared with healthy samples, the expression levels of GCH1, GK and MTHFD2 in PBMCs of patients with active tuberculosis were significantly increased, while the expression level of SLC7A6 was significantly decreased; compared with latent tuberculosis patients, the expression levels of GCH1, GK and MTHFD2 in PBMCs of patients with active tuberculosis were increased, while the expression level of SLC7A6 was decreased.

[0104] like Figure 5As shown in B, the four gene combinations have a ROC of 0.974, a specificity of 93.9, and a sensitivity of 85.7 for diagnosing active TB patients in TB patients and HC controls; a ROC of 0.909, a specificity of 100.0, and a sensitivity of 92.9 for diagnosing active TB patients in TB patients and LTBI patients; and a ROC of 0.942, a specificity of 90.9, and a sensitivity of 85.7 for diagnosing active TB patients in TB patients, HC controls, and LTBI patients. All of them showed high diagnostic ability.

Claims

1. A group of markers for diagnosing active tuberculosis, wherein the markers are selected from any one of GCH1, GK, MTHFD2 or SLC7A6, or a combination of GCH1, GK, MTHFD2 and SLC7A6.

2. A group of markers for diagnosing active tuberculosis as claimed in claim 1, when the marker for diagnosing active tuberculosis is GCH1, the expression level of GCH1 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is GK, the expression level of GK in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, the expression level of MTHFD2 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, the expression level of MTHFD2 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; When the marker for active tuberculosis is SLC7A6, the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

3. A group of markers for diagnosing active tuberculosis as claimed in claim 1, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

4. The group of markers for diagnosing active tuberculosis according to claim 1, wherein the biological sample of the subject is selected from blood, and the blood of the subject is at least one of peripheral blood, plasma and / or serum.

5. Use of the marker as claimed in claim 1 in preparing a reagent for diagnosing active tuberculosis patients.

6. Use of the marker as claimed in claim 5 in the preparation of a reagent for diagnosing active tuberculosis patients, when the marker for diagnosing active tuberculosis is GCH1, the expression level of GCH1 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is GK, the expression level of GK in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, the expression level of MTHFD2 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; When the marker for diagnosing active tuberculosis is SLC7A6, the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

7. Use of the marker according to claim 5 in the preparation of a reagent for diagnosing active tuberculosis patients, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is an active tuberculosis patient; when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is an active tuberculosis patient. The subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

8. A kit for diagnosing active tuberculosis patients, the kit comprising a reagent for detecting the expression level of the marker described in claim 1; the diagnostic method of the kit comprises one or more of PCR method / qPCR method, linear probe method, high-resolution melting curve method and / or gene chip method.

9. A kit for diagnosing active tuberculosis patients as claimed in claim 8, when the marker for diagnosing active tuberculosis is GCH1, the expression level of GCH1 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is GK, the expression level of GK in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, the expression level of MTHFD2 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, the expression level of MTHFD2 in the biological sample of the subject is significantly higher than that in healthy samples and patients with latent tuberculosis, then the subject is a patient with active tuberculosis; When the marker for active tuberculosis is SLC7A6, the expression level of SLC7A6 in the subject's biological sample is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is a combination of GCH1, GK, MTHFD2 and SLC7A6, the expression level of GCH1 in the subject's biological sample is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of GK is significantly higher than that in healthy samples and latent tuberculosis patients, the expression level of MTHFD2 is significantly higher than that in healthy samples and latent tuberculosis patients, and the expression level of SLC7A6 is significantly lower than that in healthy samples and latent tuberculosis patients, then the subject is a patient with active tuberculosis.

10. A kit for diagnosing active tuberculosis patients as claimed in claim 8, when the marker for diagnosing active tuberculosis is GCH1, when the difference between the Ct value of GCH1 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is GK, when the difference between the Ct value of GK expression detected by PCR and the Ct value of the internal reference GAPDH is less than 5.5 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is MTHFD2, when the difference between the Ct value of MTHFD2 expression detected by PCR and the Ct value of the internal reference GAPDH is less than 9 cycles, the subject is a patient with active tuberculosis; when the marker for diagnosing active tuberculosis is SLC7A6, when the difference between the Ct value of SLC7A6 expression detected by PCR and the Ct value of the internal reference GAPDH is greater than 8 cycles, the subject is a patient with active tuberculosis.

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