Biomarker for differential diagnosis of active tuberculosis patients

Abstract

The invention belongs to the technical field of biology, and particularly relates to a biomarker for differential diagnosis of pulmonary tuberculosis in different stages. The marker provided by the invention is an SCF expression level, and active tuberculosis patients can be screened from healthy people and tuberculosis patients; patients in two different stages, namely an incubation stage and an active stage, can be screened from pulmonary tuberculosis patients. And when the SCF protein expression level is less than or equal to 9.712 pg / ml, judging that the subject is an active tuberculosis patient. In the screening group, LTBI vs TB has an area under an ROC curve of 0.9006, a sensitivity of 88.46% and a specificity of 83.33%. In a verification group, LTBI vs TB, the area under an ROC curve is 0.9432, the sensitivity is 90.91%, and the specificity is 100.00%. The marker detection method is convenient to operate, simple in process, low in cost and good in sensitivity and specificity, and can be put into use in the market.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a biomarker for differential diagnosis of active pulmonary tuberculosis patients. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (MTB, referred to as "tuberculosis bacillus"). MTB can infect any organ of the human body. Clinically, the disease caused by MTB infection in the lungs is called pulmonary tuberculosis (PTB), which is also the most common. MTB infection in other parts of the body is called extrapulmonary tuberculosis.

[0003] Latent Tuberculosis Infection (LTBI) and Active Tuberculosis (ATB) are two different states of tuberculosis. Latent tuberculosis refers to a state in which an individual is infected with Mycobacterium tuberculosis but has not yet developed active tuberculosis. In this state, the infected person usually has no symptoms and does not spread the disease (Sripathi Chandragupthan. "Deep Insight to Mycobacterium Tuberculosis (Mtb): Causes, Symptoms, Diagnostics, Treatment, Risk Assessment & Recent Advances." Journal of Medical Science And Clinical Research (2023).). Active tuberculosis refers to a state in which Mycobacterium tuberculosis reproduces in the body and causes clinical symptoms. This stage is the most serious stage of pulmonary tuberculosis, and patients will have obvious symptoms such as persistent cough, sputum, weight loss, night sweats and fever, chest pain and dyspnea (Huang Sheng, Luo Danlin. Diagnostic value of anti-lipoarabinomannan antibody IgG, tuberculosis infection T cell spot test and adenosine deaminase for active pulmonary tuberculosis [J]. Journal of Clinical Internal Medicine, 2020.). Lesions at this stage include infiltrative lesions, tuberculomas, cavitation, etc. (W. Aitken. "The Science and Practice of Medicine." Glasgow Medical Journal (1864).. William Aitken, MU, JDdinbu.). Active pulmonary tuberculosis requires timely treatment to prevent worsening and spread of the disease (Nabila Rahmadhani. "KEWASPADAAN PERAWATDALAM PENULARAN PENYAKIT TUBERKULOSIS (TB) PARU SAAT MELAKSANAKAN TUGASDI RUMAHSAKIT." (2020).). The diagnosis of active pulmonary tuberculosis requires the comprehensive use of clinical symptoms, imaging examinations, laboratory tests, and pathological examinations to ensure accuracy and reduce the risk of misdiagnosis and missed diagnosis. In areas with a high tuberculosis burden, it is also necessary to select appropriate test combinations based on specific circumstances to improve diagnostic efficiency and accuracy.

[0004] The protein composition in serum plays an important role in disease diagnosis and research. By analyzing the differential expression of serum proteins, protein markers associated with specific diseases can be identified, thereby providing important information for early diagnosis, treatment and prognosis assessment of the disease (Yang Yanling, Cao Xuebin. Application of plasma / serum proteomics in cardiovascular disease [J]. Journal of PLA Postgraduate Medical School, 2011.). Olink proteomics technology, namely Olink proximity extension assay (PEA), is a technology for detecting and quantifying proteins. This technology uses oligonucleotide-labeled antibodies to recognize target proteins. When the antibody binds to the target protein, the oligonucleotide is extended by DNA polymerase to generate specific DNA amplicons, whose concentration can be measured by quantitative PCR, thereby inferring the initial concentration of the protein (Alejandro Correa Rojo, D. Heylen et al. "Towards Building a Quantitative Proteomics Toolbox in Precision Medicine: A Mini-Review." Frontiers in Physiology (2021).). Olink technology has high sensitivity and specificity, and can detect related proteins in multiple fields including cardiovascular metabolism, cell regulation, immune system, oncology, inflammation, metabolism and neurology, and has great potential in biomarker discovery and disease mechanism research (Wen Zhaoyu. Relationship between neuroinflammatory markers and postoperative delirium in elderly patients with hip replacement [D]. Henan University, 2023.).

[0005] Stem Cell Factor (SCF) is an important multifunctional cytokine that mainly exerts its effects by binding to the c-kit receptor. SCF plays a key role in the hematopoietic system, especially in the proliferation, differentiation and survival of early hematopoietic stem cells and primitive hematopoietic progenitor cells (Bin Shen, Wenhong Jiang et al. "Residues 39-56 of Stem Cell Factor Protein Sequence Are Capable of Stimulating the Expansion of Cord Blood CD34+Cells." PLoS ONE (2015).). It is produced by a variety of cells, including bone marrow mesenchymal cells, T lymphocytes, hepatocytes and fibroblasts (Anna Hordyjewska, etal. "Characteristics of hematopoietic stem cells of umbilical cord blood." Cytotechnology (2014)..Anna Hordyjewska, ). SCF has extensive and important biological functions in the hematopoiesis and immune systems. Its in-depth research provides important clues for understanding related physiological and pathological processes and provides potential targets for the treatment of diseases. Summary of the invention

[0006] The present invention found that the expression levels of SCF (Stem cell factor) protein in healthy samples, serum of patients with latent pulmonary tuberculosis and active pulmonary tuberculosis were significantly different, and SCF protein can be used to differentiate and diagnose patients with active pulmonary tuberculosis. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a marker for differential diagnosis of active pulmonary tuberculosis patients, wherein the marker is SCF protein.

[0008] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤10.28 pg / ml, the subject is differentially diagnosed as a pulmonary tuberculosis patient.

[0009] Further, when the SCF protein expression level in the subject's biological sample is >9.712 pg / ml and the SCF protein expression level in the subject's biological sample is <10.28 pg / ml, the subject is differentially diagnosed as a patient with latent pulmonary tuberculosis.

[0010] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤9.712 pg / ml, the subject is judged to be a patient with active pulmonary tuberculosis.

[0011] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0012] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0013] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy and septic tuberculosis.

[0014] Furthermore, the biological sample of the subject is selected from blood and / or urine.

[0015] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0016] In a second aspect, the present invention provides use of the marker described in the first aspect in preparing a reagent for differential diagnosis of active pulmonary tuberculosis.

[0017] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤10.28 pg / ml, the subject is differentially diagnosed as a pulmonary tuberculosis patient.

[0018] Further, when the SCF protein expression level in the subject's biological sample is >9.712 pg / ml and the SCF protein expression level in the subject's biological sample is <10.28 pg / ml, the subject is differentially diagnosed as a patient with latent pulmonary tuberculosis.

[0019] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤9.712 pg / ml, the subject is judged to be a patient with active pulmonary tuberculosis.

[0020] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0021] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0022] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy and septic tuberculosis.

[0023] Furthermore, the biological sample of the subject is selected from blood and / or urine.

[0024] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0025] In a third aspect, the present invention provides a kit for differential diagnosis of active pulmonary tuberculosis patients, wherein the kit contains a reagent for detecting the expression amount of the marker described in the first aspect.

[0026] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤10.28 pg / ml, the subject is differentially diagnosed as a pulmonary tuberculosis patient.

[0027] Further, when the SCF protein expression level in the subject's biological sample is >9.712 pg / ml and the SCF protein expression level in the subject's biological sample is <10.28 pg / ml, the subject is differentially diagnosed as a patient with latent pulmonary tuberculosis.

[0028] Furthermore, when the SCF protein expression level in the subject's biological sample is ≤9.712 pg / ml, the subject is judged to be a patient with active pulmonary tuberculosis.

[0029] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0030] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0031] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy and septic tuberculosis.

[0032] Furthermore, the biological sample of the subject is selected from blood and / or urine.

[0033] Furthermore, the subject's blood sample is at least one of peripheral blood, plasma and / or serum.

[0034] Furthermore, the kit may be an ELISA detection kit and / or a colloidal gold detection kit.

[0035] Furthermore, the diagnostic method of the kit includes a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

[0036] Beneficial Effects

[0037] The marker provided by the present invention can screen out active pulmonary tuberculosis patients from healthy persons and pulmonary tuberculosis patients; among pulmonary tuberculosis patients, patients in two different stages, latent stage and active stage, can be screened out. The detection method of the marker is easy to operate, simple in process, low in cost, good in sensitivity and specificity, and can be put into use in the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Comparison of SCF expression levels and ROC curve analysis among the healthy group, latent infection group, and active pulmonary tuberculosis group in the screening test.

[0039] Figure 2 Comparison of SCF expression levels and ROC curve analysis between the latent infection group and the active pulmonary tuberculosis group in the screening test.

[0040] Figure 3 Comparison of SCF expression levels and ROC curve analysis among the healthy group, latent infection group, and active pulmonary tuberculosis group in the validation experiment.

[0041] Figure 4 Comparison of SCF expression levels between the latent infection group and the active pulmonary tuberculosis group and ROC curve analysis. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] Explanation of terms

[0045] The "subjects" in the present invention are healthy persons and tuberculosis patients who meet the inclusion criteria.

[0046] Example 1: Standards for elimination

[0047] Research subjects: Eligible research subjects who visited Beijing Chest Hospital affiliated to Capital Medical University, including 48 patients with active pulmonary tuberculosis, 20 patients with latent tuberculosis infection and 20 healthy subjects.

[0048] Healthy controls: aged 18-65 years; no clinical symptoms of tuberculosis; negative interferon-γ release test; no lung lesions found in chest X-ray or CT; no other serious medical history.

[0049] Patients with latent tuberculosis infection: no clinical symptoms of tuberculosis; positive interferon-γ release test, no history of tuberculosis treatment.

[0050] Active tuberculosis patients: aged 18 years or above; with clinical symptoms of tuberculosis; lung lesions found on chest X-ray or CT.

[0051] Example 2 Screening Group Sample Collection and Olink Proteomics Technology Detection

[0052] A. Sample Collection

[0053] Venous blood was collected from all subjects who met the criteria, and plasma was separated and cryopreserved.

[0054] There were 12 healthy cases, 12 patients with latent infection, and 26 patients with active pulmonary tuberculosis.

[0055] B. Incubation

[0056] (1) Prepare incubation mix (400ul): Target 96IncubationSolution、 Target 96Incubation Stabilizer, Target 96A-probes and Target 96B-probes;

[0057] (2) Mix the incubation mixture and add it to the incubation plate. Set up control wells and sample quality control wells.

[0058] C. Extension

[0059] (1) Prepare the extension mix: High Purity Water, Target 96PEASolution, Target 96PEA Enzyme and Target 96PCR Polymerase;

[0060] (2) centrifuging the incubation plate and adding the mixed amplification mixture to the well plate;

[0061] (3) Perform PCR amplification.

[0062] D. Detection

[0063] (1) Injection of 96.96 Dynamic Array TM Integrated fluid circuit (IFC);

[0064] (2) Prepare the detection mix: Target 96Detection Solution、HighPurity Water、 Target 96Detection Enzyme and Target 96PCR Polymerase;

[0065] (3) adding the mixed test mixture to the sample plate;

[0066] (4) Take out the incubation plate from the PRC instrument and transfer the solution in the wells to the sample plate;

[0067] (5) Transferring the solutions in the primer plate and sample plate to the Dynamic Array IFC wells;

[0068] (6) Place the chip into Q100 and run the program.

[0069] E. Calculation and Analysis

[0070] (1) Process Q100 (qPCR) data using Olink NPX Signature software;

[0071] (2) IPC Normalized was used to normalize the NPX data obtained by the bioinformatics analysis process.

[0072] F. Test Results

[0073] A total of 47 differentially expressed proteins were screened out in the comparison among the healthy group, latent infection group and active tuberculosis patient group, including IL8, MCP-3, CDCP1, CD244, IL7, LAP, TGF-beta-1, IL6, CXCL11, AXIN1, TRAIL, CXCL9, CST5, OSM, CXCL1, CCL4, CD6, SCF, IL18, TGF-alpha, MCP-4, CCL19, PD-L1, Beta-NGF, CXCL5, TRANCE, HGF, IL-24, CCL23, CCL3, Flt3L, CXCL6, CXCL10, 4E-BP1, SIRT2, EN-RAGE, FGF-19, LIF, CASP-8, TNFRSF9, NT-3, TWEAK, ST1A1, STAMBP, CSF-1, DNER, IFN-gamma and TNF.

[0074] Among them, through further statistical analysis, a significantly differentially expressed protein SCF was screened out among the healthy group, latent infection group and active tuberculosis group for the diagnosis of active tuberculosis.

[0075] like Figure 1 As shown in A, the concentration of SCF protein in the serum of active tuberculosis patients was significantly lower than that in the healthy group and the latent infection group (P less than 0.0001) when compared between the active tuberculosis group, the latent infection group and the healthy group, indicating that the concentration of SCF protein can be used to diagnose active pulmonary tuberculosis: when the expression level of SCF protein in the biological sample of the subject is ≤9.820pg / ml, the subject is judged to be a patient with active pulmonary tuberculosis. When the expression level of SCF protein in the biological sample of the subject is ≤10.28pg / ml, the subject is differentially diagnosed as a patient with pulmonary tuberculosis. When the expression level of SCF protein in the biological sample of the subject is >9.820pg / ml and the expression level of SCF protein in the biological sample of the subject is <10.28pg / ml, the subject is differentially diagnosed as a patient with latent pulmonary tuberculosis.

[0076] like Figure 1As shown in Figure B, ROC curves were drawn based on the concentration of SCF in the serum of healthy group (HC), latent infection group (LTBI) and active tuberculosis group (TB). In the screening group, the area under the ROC curve for LTBI vs TB was 0.9006, the sensitivity was 88.46%, and the specificity was 83.33%. In the screening group, the area under the ROC curve for HC vs TB was 0.8333, the sensitivity was 73.08%, and the specificity was 83.33%.

[0077] A significantly differentially expressed protein, SCF, was screened out in samples from patients in the latent tuberculosis infection group and the active pulmonary tuberculosis group for differential diagnosis of active pulmonary tuberculosis and latent pulmonary tuberculosis.

[0078] like Figure 2 As shown in A, when the SCF protein was compared between the latent infection group and the active pulmonary tuberculosis group, the concentration in the serum of patients with active tuberculosis was significantly lower than that in the latent infection group (P less than 0.0001), indicating that the concentration of SCF protein can be used to diagnose different stages of pulmonary tuberculosis patients: latent infection stage and active pulmonary tuberculosis stage; when the SCF protein expression level in the subject's biological sample is ≤9.820pg / ml, the subject is judged to be an active pulmonary tuberculosis patient; otherwise, he is a patient in the latent infection stage.

[0079] like Figure 2 As shown in B, the ROC curve was drawn based on the concentration of SCF in the serum of patients with latent infection and active pulmonary tuberculosis. In the screening group, the area under the ROC curve for LTBI vs TB was 0.9006, the sensitivity was 88.46%, and the specificity was 83.33%.

[0080] Example 3: Sample collection of validation group and Olink proteomics technology detection

[0081] A. Sample Collection

[0082] Venous blood was collected from all subjects who met the criteria, and plasma was separated and cryopreserved.

[0083] There were 8 healthy cases, 8 patients with latent infection, and 22 patients with active pulmonary tuberculosis.

[0084] B. Incubation

[0085] (3) Prepare incubation mix (400ul): Target 96IncubationSolution、 Target 96Incubation Stabilizer, Target 96A-probes and Target 96B-probes;

[0086] (4) Mix the incubation mixture and add it to the incubation plate, setting up control wells and sample quality control wells.

[0087] C. Extension

[0088] (4) Prepare the extension mix: High Purity Water, Target 96PEASolution, Target 96PEA Enzyme and Target 96PCR Polymerase;

[0089] (5) Centrifuging the incubation plate and adding the mixed amplification mixture to the well plate;

[0090] (6) Perform PCR amplification.

[0091] D. Detection

[0092] (7) Infusion of 96.96 Dynamic Array TM Integrated fluid circuit (IFC);

[0093] (8) Prepare detection mix: Target 96Detection Solution、HighPurityWater、 Target 96Detection Enzyme and Target 96PCR Polymerase;

[0094] (9) adding the mixed detection mixture to the sample plate;

[0095] (10) Take out the incubation plate from the PRC instrument and transfer the solution in the wells to the sample plate;

[0096] (11) Transferring the solutions in the primer plate and sample plate to the DynamicArray IFC wells;

[0097] (12) Place the chip into Q100 and run the program.

[0098] E. Calculation and Analysis

[0099] (3) Q100 (qPCR) data were processed using Olink NPX Signature software;

[0100] (4) IPC Normalized was used to normalize the NPX data obtained by the bioinformatics analysis process.

[0101] F. Test Results

[0102] A total of 47 differentially expressed proteins were screened out in the comparison among the healthy group, latent infection group and active tuberculosis patient group, including: IL8, MCP-3, CDCP1, CD244, IL7, LAP, TGF-beta-1, IL6, CXCL11, AXIN1, TRAIL, CXCL9, CST5, OSM, CXCL1, CCL4, CD6, SCF, IL18, TGF-alpha, MCP-4, CCL19, PD-L1, Beta-NG F, CXCL5, TRANCE, HGF, IL-24, CCL23, CCL3, Flt3L, CXCL6, CXCL10, 4E-BP1, SIRT2, EN-RAGE, FGF-19, LIF, CASP-8, TNFRSF9, NT-3, TWEAK, ST1A1, STAMBP, CSF-1, DNER, IFN-gamma and TNF; 1 significantly differentially expressed protein SCF was screened out for the diagnosis of active pulmonary tuberculosis.

[0103] The results of the healthy group, latent infection group and active tuberculosis group were statistically analyzed, and the screened significantly differentially expressed protein SCF was verified for its effectiveness in diagnosing active tuberculosis.

[0104] like Figure 3 As shown in A, the concentration of SCF protein in the serum of active tuberculosis patients was significantly lower than that in the healthy group and the latent infection group (P less than 0.0001) in comparison between the active tuberculosis group, the latent infection group and the healthy group, indicating that the concentration of SCF protein can be used to diagnose active pulmonary tuberculosis: when the expression level of SCF protein in the biological sample of the subject is ≤9.712pg / ml, the subject is judged to be a patient with active pulmonary tuberculosis. When the expression level of SCF protein in the biological sample of the subject is ≤10.28pg / ml, the subject is differentially diagnosed as a patient with pulmonary tuberculosis. When the expression level of SCF protein in the biological sample of the subject is >9.712pg / ml and the expression level of SCF protein in the biological sample of the subject is <10.28pg / ml, the subject is differentially diagnosed as a patient with latent pulmonary tuberculosis.

[0105] like Figure 3As shown in Figure B, the ROC curve was drawn based on the concentration of SCF in the serum of the healthy group (HC), latent infection group (LTBI) and active tuberculosis group (TB). In the validation group, the area under the ROC curve for LTBI vs TB was 0.9432, the sensitivity was 90.91%, and the specificity was 100.00%. In the validation group, the area under the ROC curve for HC vs TB was 0.9432, the sensitivity was 90.91%, and the specificity was 100.00%.

[0106] In the samples of patients with latent tuberculosis infection and active pulmonary tuberculosis, the screened out significantly differentially expressed protein SCF was verified to be effective in differential diagnosis of active pulmonary tuberculosis and latent pulmonary tuberculosis.

[0107] like Figure 4 As shown in A, when the SCF protein was compared between the latent infection group and the active pulmonary tuberculosis group, the concentration in the serum of patients with active tuberculosis was significantly lower than that in the latent infection group (P less than 0.0001), indicating that the concentration of SCF protein can be used to diagnose different stages of pulmonary tuberculosis patients: latent infection stage and active pulmonary tuberculosis stage; when the SCF protein expression level in the subject's biological sample is ≤9.712pg / ml, the subject is judged to be an active pulmonary tuberculosis patient; otherwise, he is a patient in the latent infection stage.

[0108] like Figure 4 As shown in Figure B, the ROC curve was drawn based on the concentration of SCF in the serum of patients with latent infection and active pulmonary tuberculosis. In the validation group, the area under the ROC curve for LTBI vs TB was 0.9432, the sensitivity was 90.91%, and the specificity was 100.00%.

Claims

1. A marker for differential diagnosis of active pulmonary tuberculosis patients, wherein the marker is SCF (Stem cell factor) protein.

2. Provide a use of the marker described in claim 1 in the preparation of a reagent for differential diagnosis of active tuberculosis.

3. A kit for differential diagnosis of active pulmonary tuberculosis patients, the kit comprising a reagent for detecting the expression amount of the marker as claimed in claim 1.

4. The marker according to any one of claims 1 to 3, wherein when the SCF protein expression level in the biological sample of a pulmonary tuberculosis patient is ≤9.712 pg / ml, the pulmonary tuberculosis patient is judged to be an active pulmonary tuberculosis patient.

5. The pulmonary tuberculosis according to any one of claims 1 to 3 is an infection caused by the pathogen Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

6. Pulmonary tuberculosis as described in any one of claims 1 to 3, including primary pulmonary tuberculosis, secondary pulmonary tuberculosis, blood type disseminated pulmonary tuberculosis, tracheobronchial tuberculosis, tuberculous pleurisy and septic tuberculosis.

7. The marker according to any one of claims 1 to 3, wherein the biological sample of the subject is selected from blood and / or urine; the blood sample of the subject is at least one of peripheral blood, plasma and / or serum.

8. The kit as claimed in claim 3, which can be an ELISA detection kit and / or a colloidal gold detection kit.

9. The kit according to claim 3, wherein the diagnostic method of the kit comprises a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

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