Biomarker for diagnosing short-term and long-term tuberculosis infection

Abstract

The invention belongs to the technical field of biology, and particularly relates to a biomarker for differential diagnosis of short-term and long-term tuberculosis infection. Differential protein in blood of a mycobacterium tuberculosis recent infected person and a mycobacterium tuberculosis long-term infected person is determined through proteomics, and a diagnostic biomarker is provided for identifying mycobacterium tuberculosis recent infection and mycobacterium tuberculosis long-term infection. The biomarker provided by the invention has better diagnosis efficiency no matter in individual diagnosis or combined diagnosis, and the AUC value of the biomarker is greater than or close to 0.8. The biomarker can be used for rapidly, efficiently, sensitively and specifically identifying recent infected persons of mycobacterium tuberculosis, and a diagnostic basis is provided for early intervention of tuberculosis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a biomarker for the differential diagnosis of recent and long-term tuberculosis infection. Background Technology

[0002] Tuberculosis (TB) is a chronic respiratory infectious disease caused by Mycobacterium tuberculosis (MTB). The most common type is pulmonary tuberculosis, with typical symptoms including cough, sputum production, fever, night sweats, and hemoptysis. The World Health Organization (WHO) estimates that in 2021, there were 10.6 million new cases of TB worldwide, resulting in approximately 1.4 million deaths. Studies show that one-third of the population exposed to Mycobacterium tuberculosis will become infected, while the remaining exposed individuals will clear the pathogen through an immune response. Less than 10% of those infected develop TB (Bloom BR, Small PM. The evolving relation between humans and Mycobacterium tuberculosis. N Engl J Med, 1998, 338(10):677-8.). Tuberculosis (TB) is not only an infectious disease but also an immune disease. Humans exhibit genetic differences in their resistance to Mycobacterium tuberculosis (MTB) infection, and the varying immune responses to MTB ultimately determine its occurrence, development, and outcome. Studies have found that individuals recently infected with Mycobacterium tuberculosis have a higher risk of progressing to TB than those infected later. The high-risk period for TB progression is within the first 1-2 years after initial infection, but the risk of TB is low in those infected later. Therefore, early diagnosis and timely intervention in recent TB infections are crucial for reducing TB incidence. However, current clinical diagnostic methods can identify whether or not Mycobacterium tuberculosis is present, but cannot differentiate between recent and distant infections. Therefore, it is clinically necessary to identify diagnostic markers for distinguishing between recent and distant Mycobacterium tuberculosis infections.

[0003] Proteomics technology can be used to study the structure, function, efficacy and interaction of proteins in depth (Voyer P, Champoux N, Desrosiers J, et al. RADAR: a measure of the sixth vital sign? [J]. Clinical Nursing Research, 2016, 25(1): 9-29). The proteome is located downstream of the genome and transcriptome. Analyzing it can identify differentially expressed proteins caused by post-translational modifications, changes in the activity and function of biological systems during DNA and RNA translation, thereby discovering the link between proteins and diseases (Ouyang Hui, Fu Haiyan, Du Yingrong. Advances in the application of omics technology in essential hypertension [J]. Journal of Practical Cardiovascular and Cerebrovascular Diseases, 2022, 30(9): 118-123.). It also helps researchers better understand the function and structure of proteins and provides valuable information for clinical practice (JHHan, EEZimmerman, N. Cutler, et al., Delirium in older emergency department patients: recognition, risk factors, and psychomotor). Subtypes, Acad[J]. Emerg. Med. 2009, 16(3): 193-200.). Because it reflects different stages of cell differentiation and state, its characteristics are multifaceted and dynamic in time and space, providing opportunities for further elucidation of complex biological processes. In the past few years, proteomics has been used to analyze differentially expressed proteins in cancer, diabetes, Alzheimer's disease, polycythemia vera, and even in animal and plant research. Proteomics has advantages that conventional marker detection methods do not have, providing researchers with a new perspective to explore the network regulation between proteins, which is of great significance for the early diagnosis of postoperative cognitive dysfunction and the search for new biomarkers. Currently, proteomics has a wide range of applications, including: four clinical applications of proteomics technology: (1) identification of proteins in a specific biological sample; (2) detection of protein expression profiles describing specific states of organisms or cells; (3) determination of protein interactions in specific functional protein networks; and (4) precise localization of protein post-translational modifications (Kosteria I, Anagnostopoulos AK, Kanaka-Gantenbein C, et al. The use of proteomics in assisted reproduction[J].in vivo,2017,31(3):267-283.).

[0004] Olink proteomics leverages its core patented protein detection technique—Proximity Extension Assay (PEA)—to achieve precise detection of over 3000 proteins in samples ranging from 1 to 6 μL. PEA is a dual-recognition immunoassay where two matched antibodies simultaneously bind to a target, each labeled with a unique DNA oligonucleotide. Proteins in solution bring the two antibodies close together, causing their DNA oligonucleotides to hybridize, serving as a template for a DNA polymerase-dependent extension step. This creates a double-stranded DNA "barcode" that is unique to the specific antigen and proportional in number to the initial concentration of the target protein. PCR amplification is performed immediately after hybridization and extension, and the amplicon is finally quantified using microfluidic qPCR. PEA successfully combines antibody-based immunoassays with the powerful capabilities of quantitative real-time PCR (qPCR) and next-generation sequencing (NGS) technology, resulting in a multi-channel and highly specific approach (Liu Huan. Study on plasma immunoproteomics of Behcet's disease patients based on PEA technology [D]. Henan: Zhengzhou University, 2022.). PEA overcomes the limitations of traditional proteomics detection technologies, such as limited sensitivity, low specificity, limited dynamic range, large sample consumption, and low throughput. To understand complex, real-time human biology, it can simultaneously quantify thousands of protein biomarkers, effectively screening markers for disease diagnosis. It features high specificity and multiple channels, providing innovative proteomics detection solutions for precision medicine, translational medicine, and drug development (Hou Mengyuan. Proteomics Analysis of Inflammatory Factors in Plasma of Elderly Patients with Delirium After General Anesthesia [D]. Henan: Henan University, 2023).

[0005] The human immune system plays a crucial role in resisting Mycobacterium tuberculosis infection, the pathogenesis and prognosis of tuberculosis. From an immunological perspective, tuberculosis is an immune disorder closely related to immune responses. Cytokines, as effector and regulatory factors secreted by various immune cells, play a role in transmitting information between cells, and the disruption of their network is related to the pathogenesis of tuberculosis (Dong Haiping, Wang Caizhu, Liang Xiaocheng, et al. The significance of plasma cytokines in the recurrence of pulmonary tuberculosis [J]. Modern Hospital, 2019, 19(7):1015-1018.). Cytokines are low-molecular-weight soluble polypeptides produced by various cells (mainly immune cells) that can participate in immune responses. They are classified into interleukins, tumor necrosis factors, interferons, etc., and can mediate physiological processes such as innate immunity and specific immune responses. They also participate in inducing apoptosis and stimulating hematopoiesis. In conclusion, cytokines may have certain auxiliary diagnostic value for pulmonary tuberculosis and lung cancer. However, due to the wide range of sources of cytokines and the many influencing factors, there are currently no biomarkers that can be used to distinguish between recent and long-term Mycobacterium tuberculosis infection. Summary of the Invention

[0006] This invention uses proteomics to identify differentially expressed proteins in the blood of individuals recently infected with Mycobacterium tuberculosis (MBTB) and those with long-term MBTB infection, providing diagnostic biomarkers for differentiating between these two conditions. These biomarkers can be used to more quickly, efficiently, sensitively, and specifically identify MBTB-infected individuals, providing a diagnostic basis for early intervention in tuberculosis. Based on this, this invention was completed.

[0007] In a first aspect, the present invention provides a biomarker for differentiating between recent and long-term tuberculosis infection, wherein the biomarker is selected from one or more of CCL3, TNFα and / or CCL20; when the concentration of the biomarker protein is below a critical value, it indicates recent pulmonary tuberculosis infection.

[0008] Furthermore, when the concentration of TNFα protein in the subject's biological sample was below 93.37 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0009] Furthermore, when the concentration of CCL20 protein in the subject's biological sample was below 149.55 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0010] Furthermore, when the concentration of CCL3 protein in the subject's biological sample was below 170.65 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0011] Furthermore, the biological sample is derived from the patient's whole blood, serum, or plasma.

[0012] Preferably, the biological sample is derived from the patient's plasma.

[0013] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0014] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, and extrapulmonary infection.

[0015] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tracheal or bronchial tuberculosis, and tuberculous pleurisy.

[0016] Secondly, the present invention provides the application of a biomarker in the preparation of a reagent for differentiating between recent and long-term tuberculosis infection, wherein the biomarker is one or more of CCL3, TNFα and / or CCL20, and the reagent contains a reagent capable of detecting the levels of CCL3, TNFα and CCL20 in a patient's biological sample; when the concentration of the biomarker protein is below a critical value, it indicates recent pulmonary tuberculosis infection.

[0017] Furthermore, when the concentration of TNFα protein in the subject's biological sample was below 93.37 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0018] Furthermore, when the concentration of CCL20 protein in the subject's biological sample was below 149.55 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0019] Furthermore, when the concentration of CCL3 protein in the subject's biological sample was below 170.65 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0020] Furthermore, the biological sample is derived from the patient's whole blood, serum, or plasma.

[0021] Preferably, the biological sample is derived from the patient's plasma.

[0022] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0023] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, and extrapulmonary infection.

[0024] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tracheal or bronchial tuberculosis, and tuberculous pleurisy.

[0025] Thirdly, the present invention provides a kit for differentiating between recent and long-term tuberculosis infection, the kit comprising reagents for detecting the levels of CCL3, TNFα and CCL20 in a patient's biological sample; when the concentration of the biomarker protein is below a critical value, it indicates recent pulmonary tuberculosis infection.

[0026] Furthermore, when the concentration of TNFα protein in the subject's biological sample was below 93.37 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0027] Furthermore, when the concentration of CCL20 protein in the subject's biological sample was below 149.55 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0028] Furthermore, when the concentration of CCL3 protein in the subject's biological sample was below 170.65 pg / ml, it indicated that the subject had recently been infected with pulmonary tuberculosis.

[0029] Furthermore, the biological sample is derived from the patient's whole blood, serum, or plasma.

[0030] Preferably, the biological sample is derived from the patient's plasma.

[0031] Furthermore, the tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis.

[0032] Furthermore, the Mycobacterium tuberculosis infection includes primary infection, secondary infection, and extrapulmonary infection.

[0033] Furthermore, the pulmonary tuberculosis includes primary pulmonary tuberculosis, hematogenous disseminated pulmonary tuberculosis, secondary pulmonary tuberculosis, tracheal or bronchial tuberculosis, and tuberculous pleurisy.

[0034] Furthermore, the kit may be one or more of the following: ELISA detection kit, colloidal gold detection kit, immunohistochemistry kit, immunofluorescence kit, and / or in situ hybridization staining kit.

[0035] Furthermore, the diagnostic methods of the kit include one or more of the following: direct method, indirect method, double-antibody sandwich method, and / or competitive method.

[0036] Beneficial effects

[0037] 1. This invention is the first to use TNF-α, CCL3, and CCL20 as differential diagnostic markers for different stages of pulmonary tuberculosis infection, distinguishing between recent and long-term pulmonary tuberculosis infection. Both alone and in combination, they exhibit good diagnostic efficacy, with AUC values ​​greater than or close to 0.8. Among them, TNF-α, when used alone as a diagnostic marker, shows a specificity as high as 100%.

[0038] 2. This invention applies proteomics technology to detect differentially expressed protein biomarkers related to pulmonary tuberculosis. It has high sensitivity and specificity, is suitable for the detection of pulmonary tuberculosis, and provides high accuracy in the detection results, which helps to improve detection efficiency.

[0039] 3. This invention applies differentially expressed protein biomarkers related to pulmonary tuberculosis to the detection of pulmonary tuberculosis. The detection has the advantages of simple sample processing, easy acquisition, small sample volume, and good repeatability. Attached Figure Description

[0040] Figure 1 The expression levels of CCL3 protein in the three groups of people.

[0041] Figure 2 The expression levels of CCL20 protein in the three groups of people.

[0042] Figure 3 The expression levels of TNF-α protein in the three groups are shown.

[0043] Figure 4 The expression level of CCL3 protein in patients with recent and long-term pulmonary tuberculosis infection.

[0044] Figure 5 The expression level of CCL20 protein in patients with recent and long-term pulmonary tuberculosis infection.

[0045] Figure 6 The expression level of TNF-α protein in patients with recent and long-term pulmonary tuberculosis infection.

[0046] Figure 7 To screen the ROC analysis results in the experiment.

[0047] Figure 8 To verify the differences in TNF-α, CCL3, and CCL20 protein expression between patients with recent and long-term pulmonary tuberculosis infection in the experiment.

[0048] Figure 9 To verify the ROC analysis results in the experiment. Detailed Implementation

[0049] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, 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.

[0050] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0051] Example 1: Screening of differentially expressed proteins between recent and long-term pulmonary tuberculosis infections based on proteomics technology. 1.1 Inclusion and grouping of study subjects.

[0052] The study included individuals aged 30-60 years who had recently been infected with pulmonary tuberculosis, had long-term pulmonary tuberculosis, or were healthy controls and who visited the Beijing Chest Hospital affiliated with Capital Medical University. Specific groupings are as follows:

[0053] (1) Group of individuals with recent tuberculosis infection (15 individuals): Individuals who were negative for IGRA (interferon-gamma release assay) at baseline and turned positive after 12 months, or who were negative for IGRA 12 months prior and turned positive after 24 months, were defined as having a recent infection. For these individuals, the first positive sample after the negative sample was used for analysis.

[0054] (2) Long-term tuberculosis infection group (15 people): Doctors and nurses who have worked in tuberculosis hospitals for at least 2 years, who were IGRA positive at baseline and remained positive for 24 months, indicating infection for at least 2 years.

[0055] (3) Healthy control group (15 people): healthy people with no history of active tuberculosis, no clinical symptoms, and normal IGRA test results.

[0056] 1.2 Blood collection, preservation, and IGRA test

[0057] Anticoagulated venous blood was collected from all study subjects and aliquoted into three types of culture tubes: "N," "T," and "P." "N" tubes contained Tris, "T" tubes contained Mycobacterium tuberculosis-specific antigen, and "P" tubes contained phytohemagglutinin (PHA). After inverting and mixing, the tubes were incubated, centrifuged, and the supernatant was collected. The supernatant from each of the three tubes was aliquoted into new centrifuge tubes for ELISA and proteomics assays, and then frozen until use. All IGRA-related reagents were purchased from Redbury Biopharmaceutical Co., Ltd., and the procedures were strictly followed according to the instructions.

[0058] 1.3 Screening for differentially expressed proteins using proteomics techniques

[0059] (1) Data Preprocessing: Protein levels were quantified using the Olink Target 96 Inflammation Panel (OlinkProteomics AB, Uppsala, Sweden) according to the manufacturer's instructions. The Olink panel is based on Proximity Extended Analysis (PEA) technology. PEA allows for the simultaneous analysis of up to 92 analytes. In short, oligonucleotide-tagged antibody probe pairs are allowed to bind to target proteins in the sample. When two antibodies bind tightly, DNA oligonucleotides hybridize and extend via DNA polymerization to form a polymerase chain reaction (PCR) reporter sequence. The sequence is detected, amplified, and quantified using a microfluidic real-time PCR instrument. Protein abundance is reported as Normalized Protein Expression (NPX), which is an arbitrary unit in the Log2 scale. A high NPX value indicates a high protein concentration. However, NPX values ​​cannot be compared between different proteins.

[0060] (2) Quality Control Analysis: Data quality control (QC) was performed in two steps: First, the test control, incubation control, and the standard deviation of each run for both were determined. Only runs with a standard deviation of less than 0.2 for each control passed quality control. Second, each sample was checked for quality using the test control and incubation control 2. All samples in the run were compared to the calculated run median for each control. If a sample deviated from the median of the well plates of the two control groups by more than 0.3 NPX, QC failed, and a QC warning was issued in the data output file. All samples were successfully measured.

[0061] 1.4 Statistical Analysis

[0062] Experimental data are expressed as standard deviation ± mean. Skewness coefficient, kurtosis coefficient, and Shapiro-Wilk test were used to determine normality. For normally distributed data, one-way ANOVA was used for comparisons, and the Tukey test was used for comparisons between two or more groups. For non-normally distributed data, the Nemenyi test and Kruskal-Wallis test were used for comparisons between two groups. P < 0.05 was considered statistically significant. This allowed for the screening of potential differentially expressed protein biomarkers.

[0063] 1.5 Results

[0064] 1.5.1 Screening of differentially expressed proteins

[0065] By comparing protein expression levels among three groups—recently infected with pulmonary tuberculosis, long-term infected with pulmonary tuberculosis, and healthy controls—significantly differentially expressed biomarkers were identified. Significant differences were found in the distribution of proteomics data among the three groups. Further analysis selected the most significantly differentially expressed biomarkers (TNF-α, CCL3, and CCL20), and one-way ANOVA was performed using GraphPad software to determine the statistical significance among the three groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0066] Figure 1 The study showed significant differences in the expression levels of TNF-α, CCL3, and CCL20 proteins among the three groups. Specifically, compared to the other two groups, the expression of CCL3, TNF-α, and CCL20 was low in the recent tuberculosis infection group.

[0067] Figure 2 The study showed significant differences in these three differentially expressed proteins between patients with recent and distant pulmonary tuberculosis (TB). Specifically, TNF-α, CCL3, and CCL20 were highly expressed in the distant TB infection group and lowly expressed in the recent TB infection group.

[0068] 1.5.2 Diagnostic efficacy analysis of differentially expressed biomarkers

[0069] For the differentially expressed proteins, SPSS software was used to perform receiver operating characteristic (ROC) analysis based on a logistic model.

[0070] The results showed that, within a 95% confidence interval, the AUC for the diagnosis of the three protein biomarkers, alone or in combination, ranged from 0.800 to 0.929, as detailed in Table 1. This indicates that the three differentially expressed protein biomarkers obtained through screening have high accuracy in differentiating between recent and long-term pulmonary tuberculosis infection, and are of great significance for the diagnosis of pulmonary tuberculosis.

[0071] Table 1. ROC data of differentially expressed proteins

[0072]

[0073]

[0074] Example 2 Validation of CCL3, CCL20, and TNF-α expression levels in plasma of patients under different infection states of pulmonary tuberculosis 2.1 Study subject inclusion and grouping

[0075] The study included individuals aged 30-80 years who had recently been infected with pulmonary tuberculosis or had been infected with pulmonary tuberculosis in the long term and were treated at Beijing Chest Hospital, affiliated with Capital Medical University. Specific groupings are as follows:

[0076] (4) Recent tuberculosis infection group (33 individuals): Individuals who were negative at baseline and turned positive after 12 months, or who were negative IGRA 12 months ago and turned positive after 24 months, were defined as recently infected. For these individuals, the first positive sample after the negative sample was used for analysis.

[0077] (5) Long-term tuberculosis infection group (45 people): Doctors, nurses and other staff who have worked in tuberculosis hospitals for at least 2 years, who were IGRA positive at baseline and remained positive for 24 months, indicating infection for at least 2 years.

[0078] 2.2 Blood collection, preservation, and IGRA test

[0079] Heparin lithium-anticoagulated venous blood was collected from all study subjects and aliquoted into three types of culture tubes: "N," "T," and "P." The "N" tubes contained Tris, the "T" tubes contained Mycobacterium tuberculosis-specific antigen, and the "P" tubes contained phytohemagglutinin (PHA). After inverting and mixing, the tubes were incubated, centrifuged, and the supernatant was collected. The supernatant from each of the three tubes was aliquoted into new centrifuge tubes for ELISA and proteomics assays, and then frozen until use. All IGRA-related reagents were purchased from Redbury Biopharmaceutical Co., Ltd., and the procedures were strictly followed according to the instructions.

[0080] 2.3 Plasma Sample Analysis

[0081] The collected plasma samples were analyzed using Luminex's commercially available Bio-Rad genotyping combination (TNF-α, CCL3, CCL20) to identify differentially expressed biomarkers associated with recent and long-term tuberculosis infection. The specific procedures were as follows, according to the manufacturer's instructions:

[0082] 2.3.1 Sample preparation and standard preparation

[0083] Plasma sample: Centrifuge, collect the supernatant, dilute with Sample Diluent, and finally load the diluted sample for testing.

[0084] 2.3.2 Preparation of Standard Products

[0085] Add Standard Diluent to the standard vials, vortex, ice bath, and then dilute standard samples S1-S8.

[0086] 2.3.3 Sample incubation

[0087] Take the microbeads and shake them, then dilute the microbeads with Assay Buffer;

[0088] The diluted microbeads were shaken again, added to the well plate, and washed.

[0089] Add the prepared standards, samples, references, and Blank to the well plate, seal with film, shake, protect from light, and incubate at room temperature.

[0090] 2.3.4 Incubation and Antibody Detection

[0091] Discard the sample and wash it.

[0092] Dilute DetectionAntibody with Antibody Diluent as per the instructions.

[0093] Add diluted DetectionAntibody to each well, seal with a sealing film, shake, protect from light, and incubate at room temperature.

[0094] 2.3.5 Color Development

[0095] Discard the antibody test result and wash.

[0096] Dilute Streptavidin-PE using Assay Buffer as per the instructions.

[0097] Add diluted Streptavidin-PE to each well, seal with film, shake, protect from light, incubate at room temperature;

[0098] washing;

[0099] Add Assay Buffer to each well to resuspend, cover with sealing film, incubate at room temperature, away from light, and shake.

[0100] The value is read from the calibrated Luminex 200 machine.

[0101] 2.3.6 Data Analysis

[0102] The fluorescence of the samples and standards tested in this experiment was automatically calculated and optimized by software after being detected by the Luminex 200 detector.

[0103] 2.4 Data Analysis

[0104] Statistical analysis was performed using GraphPad Prism and MedCalc-version software. For normally distributed data, t-tests were used for comparison; for non-normally distributed data, the Mann-Whitney test was used to compare the two groups. Statistical significance was determined by P < 0.05, indicating a statistically significant difference. This process was used to screen for potential differentially expressed protein biomarkers and to validate biomarkers for diagnosing recent and long-term pulmonary tuberculosis infections.

[0105] 2.5 Results

[0106] like Figure 4 As shown, there were significant differences in plasma expression levels of CCL3, CCL20, and TNF-α between the group of patients recently infected with pulmonary tuberculosis and the group of patients infected in the long term with pulmonary tuberculosis.

[0107] Further ROC-based comparisons were used to validate the diagnostic efficacy of CCL3, CCL20, and TNF-α in patients with recent and long-term pulmonary tuberculosis infection. Figure 5As shown, the AUC value of CCL3 was 0.859 (95% confidence interval, 0.760-0.928; sensitivity, 86.36%; specificity, 75.00%); the AUC value of CCL20 was 0.783 (95% confidence interval, 0.674-0.870; sensitivity, 68.18%; specificity, 93.75%); and the AUC value of TNF-α was 0.851 (95% confidence interval, 0.751-0.922; sensitivity, 56.82%; specificity, 100.00%).

[0108] The combined diagnostic efficacy of CCL3, CCL20, and TNF-α was evaluated. A combined diagnostic model was constructed using multivariate logistic regression analysis, and ROC curves for the combined diagnosis were plotted. The AUC values ​​of each combination are shown in Table 2. Among the combined biomarkers, CCL3+CCL20 showed the highest diagnostic efficacy, with an AUC value of 0.863. The results validated the diagnostic efficacy of the biomarker combination in a expanded clinical sample, confirming its feasibility for application in actual clinical diagnosis. A TNF-α protein concentration below 93.37 pg / ml indicates recent infection with pulmonary tuberculosis; a CCL20 protein concentration below 149.55 pg / ml indicates recent infection with pulmonary tuberculosis; and a CCL3 protein concentration below 170.65 pg / ml indicates recent infection with pulmonary tuberculosis.

[0109] Table 2. ROC data of differentially expressed proteins

[0110]

Claims

1. A biomarker for differential diagnosis of recent and long-term tuberculosis infection, wherein the biomarker is selected from one or more of CCL3, TNFα and / or CCL20; when the biomarker protein concentration is lower than a critical value, it indicates recent pulmonary tuberculosis infection.

2. The biomarker according to claim 1, when the TNFα protein concentration in the subject's biological sample is lower than 93.37 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL20 protein concentration in the subject's biological sample is lower than 149.55 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL3 protein concentration in the subject's biological sample is lower than 170.65 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis.

3. The biomarker according to claim 2, wherein the biological sample is from whole blood, serum or plasma of a patient.

4. The biomarker according to claim 1, wherein the pulmonary tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

5. Use of a biomarker in the preparation of a reagent for differential diagnosis of recent and long-term tuberculosis infection, wherein the biomarker is one or more of CCL3, TNFα and / or CCL20, and the reagent contains a reagent that can detect the content of CCL3, TNFα and CCL20 in a patient's biological sample; when the biomarker protein concentration is lower than a critical value, it indicates recent pulmonary tuberculosis infection.

6. The use as claimed in claim 5, when the TNFα protein concentration in the subject's biological sample is lower than 93.37 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL20 protein concentration in the subject's biological sample is lower than 149.55 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL3 protein concentration in the subject's biological sample is lower than 170.65 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis.

7. A kit for differential diagnosis of recent and long-term tuberculosis infection, the kit comprising reagents for detecting the content of CCL3, TNFα and CCL20 in a patient's biological sample; when the concentration of the biomarker protein is lower than the critical value, it indicates recent pulmonary tuberculosis infection.

8. The use according to claim 7, wherein when the TNFα protein concentration in the biological sample of the subject is lower than 93.37 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL20 protein concentration in the biological sample of the subject is lower than 149.55 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis; when the CCL3 protein concentration in the biological sample of the subject is lower than 170.65 pg / ml, it indicates that the subject has recently been infected with pulmonary tuberculosis.

9. The use according to claim 7, wherein the pulmonary tuberculosis is an infection caused by the pathogen Mycobacterium tuberculosis; the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

10. The use as claimed in claim 7, wherein the kit can be one or more of an ELISA detection kit, a colloidal gold detection kit, an immunohistochemistry kit, an immunofluorescence kit and / or an in situ hybridization staining kit; and the diagnostic method of the kit includes one or more of a direct method, an indirect method, a double antibody sandwich method and / or a competitive method.

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