Application of reagent for detecting content or expression level of TIPE2 in sample in preparation of reagent for diagnosing and / or predicting active tuberculosis

Abstract

The invention discloses application of a reagent for detecting the content or expression level of TIPE2 in a sample in preparation of a reagent for diagnosing and / or predicting active tuberculosis. In the application, the sample comprises a blood sample. According to the application, the TIPE2 in a blood sample is used as the biomarker for detecting the active tuberculosis for the first time. Results of ROC curve analysis show that the expression level of the TIPE2 in whole blood has good sensitivity and specificity on active tuberculosis diagnosis.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and further to the application of reagents for detecting the content or expression level of TIPE2 in a sample in the preparation of reagents for diagnosing and / or predicting active pulmonary tuberculosis, and even further to the application of reagents for detecting the content or expression level of TIPE2 in a sample in the preparation of products for diagnosing and / or predicting active pulmonary tuberculosis. Background Technology

[0002] Successful treatment of pulmonary tuberculosis (TB) largely depends on accurate diagnosis and timely initiation of standardized anti-TB therapy. In clinical practice, the diagnosis of TB is typically based on the patient's symptoms, abnormal chest X-ray findings, and sputum bacteriological examination results. Acid-fast bacilli can be detected in approximately 50% to 60% of TB patients on sputum smears. However, in some cases of active TB, neither bacteriological examination nor consecutive chest X-rays can clearly indicate disease activity. Sputum-negative TB with radiographic findings but lacking clinical or microbiological evidence is considered a significant risk factor for the development of active TB.

[0003] In recent years, research on biomarkers for tuberculosis diagnosis has continued to be a hot topic.

[0004] Overall, most current biomarker development is based on cross-sectional studies. While these studies can differentiate between latent tuberculosis (LTBI) and active tuberculosis (TB), they remain significantly insufficient in predicting the onset of TB. Therefore, finding novel early warning indicators with better sensitivity and specificity remains an urgent research need. Summary of the Invention

[0005] Based on this, this application provides at least one application of a reagent for detecting the content or expression level of TIPE2 in a sample in the preparation of reagents for diagnosing and / or predicting active pulmonary tuberculosis.

[0006] In a first aspect of this application, a reagent for detecting the content or expression level of TIPE2 in a sample is provided for use in the preparation of reagents for diagnosing and / or predicting active pulmonary tuberculosis; said sample includes a blood sample.

[0007] In a second aspect of this application, a kit for diagnosing and / or predicting active pulmonary tuberculosis is provided, comprising reagents for detecting the content of TIPE2 or its expression level in a sample, said sample including a blood sample.

[0008] In a third aspect of this application, a system for diagnosing and / or predicting active pulmonary tuberculosis is provided, the system comprising:

[0009] The data processing module is used to calculate the received or input data on the TIPE2 content or expression level in whole blood to obtain the calculation results; and

[0010] The judgment and output module is used to judge whether the calculation result meets the preset judgment conditions in order to diagnose and / or predict an individual's active pulmonary tuberculosis, and output the prediction result.

[0011] In a fourth aspect of this application, a computer-readable storage medium is provided that stores a computer program, which, when executed by a processor, can perform the functions of the system described in the third aspect.

[0012] In a fifth aspect of this application, a computer device is provided, comprising a memory and a processor, the memory storing a computer program and the processor executing the computer program to perform the functions of the system as described in the third aspect.

[0013] In a sixth aspect of this application, an apparatus for diagnosing and / or predicting active pulmonary tuberculosis is provided, comprising one or more of the system described in the third aspect, the computer-readable storage medium described in the fourth aspect, or the computer device described in the fifth aspect.

[0014] This application is the first to use TIPE2 from blood samples as a biomarker for the detection of active pulmonary tuberculosis. ROC curve analysis showed that the expression level of TIPE2 in whole blood has good sensitivity and specificity for the diagnosis of active pulmonary tuberculosis. This application overcomes the shortcomings of existing methods for diagnosing active pulmonary tuberculosis, such as low detection rate and long diagnostic time, and features high specificity and sensitivity, demonstrating good clinical application value for the auxiliary diagnosis of active pulmonary tuberculosis. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0016] Figure 1 This diagram illustrates the mRNA expression levels of TIPE2 in whole blood of healthy volunteers, individuals with latent tuberculosis infection, patients with active pulmonary tuberculosis, and patients with active pulmonary tuberculosis who have undergone standard anti-tuberculosis treatment for 2 months, according to one embodiment of this application. Data are the mean of two parallel trials; ns indicates no statistically significant difference. This indicates a statistically significant difference. This indicates a significant difference.

[0017] Figure 2 This is a schematic diagram of ROC analysis of the relative expression level of TIPE2 in whole blood of tuberculosis patients (TB) and healthy individuals (HC) according to one embodiment of this application; the data are the mean of two parallel experiments.

[0018] Figure 3 ROC analysis of the relative expression level of TIPE2 in whole blood of tuberculosis patients (TB) and latent tuberculosis-infected individuals (LTBI) according to one embodiment of this application; data are the mean of two parallel trials. Detailed Implementation

[0019] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0021] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0022] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0023] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0024] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0025] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0026] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0027] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0028] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0029] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0030] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0031] TIPE2 (Tumor Necrosis Factor-α Induced Protein 8-Like 2) is primarily expressed in immune cells, including T cells, B cells, macrophages, and dendritic cells. Its core function is to negatively regulate the immune response, preventing overactivation of the immune system and thus maintaining immune homeostasis. TIPE2 mainly inhibits activated signaling pathways such as TLR (Toll-like receptor) and TCR (T cell receptor) by binding to and influencing key signaling molecules. It can target small G proteins such as RAC1, interfering with their activation. RAC1 is an upstream regulator of important signaling pathways such as MAPK and NF-κB. By inhibiting RAC1, TIPE2 ultimately inhibits the activity of transcription factors such as NF-κB and AP-1, thereby reducing the production of pro-inflammatory cytokines (such as TNF-α, IL-6, and IL-1β). Downregulation of TIPE2 expression leads to: autoimmune diseases (SLE, RA), excessive inflammation (sepsis), and complex effects in some cancers; in hematologic malignancies, its role may be opposite, serving as a potential disease biomarker and therapeutic target. For example, upregulating TIPE2 expression through drugs may be used to treat autoimmune diseases, while inhibiting its function may help enhance anti-tumor immunity.

[0032] During their research, the inventors of this application discovered the important immune checkpoint molecule TIPE2, which showed significantly higher expression in TB patients than in LTBI and healthy individuals (HC). More importantly, TIPE2 expression was significantly elevated in patients whose latent tuberculosis infection progressed to the active phase, and decreased after treatment improvement. Further investigation using in vitro tuberculosis infection models confirmed that Mycobacterium tuberculosis specifically induces TIPE2 expression, thereby inhibiting host anti-tuberculosis immunity and enabling its own replication. Given the host immune damage mechanism caused by tuberculosis, further research is warranted into identifying early warning biomarkers for tuberculosis development around TIPE2.

[0033] This application provides at least one diagnostic reagent and kit for detecting active pulmonary tuberculosis using TIPE2 in blood as a biomarker, which can accurately and rapidly detect active pulmonary tuberculosis and detect the stage of active pulmonary tuberculosis, facilitating clinical application.

[0034] One of the technical problems this application aims to solve is to overcome the issues of poor specificity, low sensitivity, and time-consuming detection of existing diagnostic markers for active tuberculosis. This application provides a technical concept for using TIPE2 as a diagnostic marker for active pulmonary tuberculosis, offering a new target for the diagnosis of active pulmonary tuberculosis and demonstrating good clinical application value in the diagnosis of active pulmonary tuberculosis.

[0035] In one aspect of this application, the use of a reagent for detecting the content or expression level of TIPE2 in a sample in the preparation of a reagent for diagnosing and / or predicting active pulmonary tuberculosis is provided; the sample includes a blood sample.

[0036] Unless otherwise specified, the blood sample may be whole blood, plasma, etc.

[0037] The TIPE2 content or expression level involved is found to be differentially present in biological samples obtained from subjects with active pulmonary tuberculosis compared to “normal” subjects; or there is a significant difference between subjects before and after treatment. For example, a difference in the expression level of an mRNA biomarker between samples is determined to be statistically significant if such a difference is determined to be statistically significant. Common tests for statistical significance include, but are not limited to: t-test, ANOVA, Kniskal-Wallis, Wilcoxon, Mann-Whitney, and odds ratio. Individual or combined mRNA biomarkers can be used to provide a measure of the relative risk of a subject having or not having active pulmonary tuberculosis.

[0038] The expression level of TIPE2, such as mRNA content or protein expression level. In some embodiments, mRNA biomarkers are used to determine active tuberculosis: they can be used in diagnostic tests to assess a subject's active tuberculosis status. Disease status includes, but is not limited to, the presence or absence of active tuberculosis. Disease status may also include monitoring the progression of active tuberculosis, for example, monitoring disease progression (including prognosis). Based on the subject's active tuberculosis status, other actions may be indicated, including, for example, other diagnostic tests or treatments.

[0039] The ability of a diagnostic test to correctly predict disease states is typically measured by its accuracy, sensitivity, specificity, or area under the curve (AUC) (e.g., the area under the receiver operating characteristic (ROC) curve). Accuracy, as used herein, is a measure of the proportion of samples that are misclassified. Accuracy can be calculated as the total number of correctly classified samples divided by the total number of samples (e.g., in the test population). Sensitivity is a measure of “true positives” predicted by the test and can be calculated as the number of correctly identified active tuberculosis samples divided by the total number of active tuberculosis samples. Specificity is a measure of “true negatives” predicted by the test and can be calculated as the number of correctly identified normal samples divided by the total number of normal samples. AUC is a measure of the area under the receiver operating characteristic curve, which is a graph of sensitivity versus the false positive rate (specificity). The higher the AUC, the more effective the test's predictive power. Other useful measures of test utility include “positive predictive value” (which is the percentage of actual positives when the test is positive) and “negative predictive value” (which is the percentage of actual negatives when the test is negative).

[0040] It should be understood that the reagents mentioned refer to any reagents capable of detecting, for example, the content or expression level of TIPE2 in whole blood samples, including but not limited to specific probes, gene chips, specific primers, and anti-TIPE2 antibodies.

[0041] Unless otherwise specified, the term "primer" in this application refers to a nucleic acid sequence capable of forming a base pair complementary to the template strand and serving as a starting point for template strand replication; for example, its length may be 7-50 bases. Primers are usually synthesized, but naturally occurring nucleic acids may also be used. The primer sequence does not necessarily need to be completely identical to the template sequence, as long as it is sufficiently complementary to the template and can hybridize.

[0042] Unless otherwise specified, the term "probe" in this application refers to a nucleic acid fragment, such as RNA or DNA, ranging from a few to hundreds of bases in length, which can specifically bind to mRNA and can determine the presence of a specific mRNA through labeling. Probes can be prepared in the form of oligonucleotide probes, single-stranded DNA probes, double-stranded DNA probes, and RNA probes.

[0043] It should be noted that other technical means can also be used to detect the expression level of biomarkers, such as RNA sequencing, immunohistochemistry, flow cytometry, and in situ hybridization.

[0044] In some embodiments, the use of a reagent for selectively detecting whole blood TIPE2 is provided in the preparation of a kit for rapid diagnosis of active pulmonary tuberculosis, the reagent being used to determine the level of TIPE2 in whole blood obtained from the subject, wherein an increase in the level of TIPE2 relative to the level in a normal subject indicates the presence of active pulmonary tuberculosis in the subject.

[0045] Unless otherwise specified, the term "diagnosis" in this application refers to the discovery, judgment, or recognition of an individual's health status or condition based on one or more symptoms, data, or other information relating to that individual. An individual's health status may be diagnosed as healthy / normal (e.g., not having active tuberculosis), or as unhealthy / abnormal (e.g., having active tuberculosis), or as a specific degree or progression of illness. The term diagnosis includes the early detection of a disease / symptom associated with a particular disease or ailment; the characteristics or classification of a disease; the discovery of disease progression, cure, or recurrence; and the discovery of an individual's response to the disease after treatment or intervention.

[0046] In some implementations, methods such as amplification, hybridization, and / or sequencing are used to detect the content of TIPE2 or its expression level (e.g., the level of TIPE2 mRNA in whole blood).

[0047] In some implementations, quantitative PCR is used to detect the level of TIPE2 mRNA in the whole blood.

[0048] In some embodiments, the reagents include upstream and downstream primers for quantitative PCR of TIPE2 mRNA in whole blood.

[0049] In some embodiments, the upstream and downstream primer sequences for the quantitative PCR of TIPE2 mRNA in whole blood are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0050] In some implementations, detecting the content or expression level of TIPE2 in the sample includes comparing the content or expression level of TIPE2 in the sample with a control.

[0051] For example, comparisons are made with a positive control and a negative control, respectively; wherein the negative control may be, for example, a normal subject (e.g., a subject who does not have active pulmonary tuberculosis or a healthy subject); and the positive control may be, for example, a patient diagnosed with active pulmonary tuberculosis.

[0052] In some implementations, the sample type is whole blood, and the content or expression level of TIPE2 is higher than that of the negative control, indicating that the subject corresponding to the sample detected by the reagent has active pulmonary tuberculosis or is at high risk of having the disease; the negative control is the content or expression level of TIPE2 in the whole blood of healthy subjects or subjects who do not have active pulmonary tuberculosis.

[0053] In some embodiments, the sample type is whole blood, and the TIPE2 content or expression level is consistent with the negative control. The subject corresponding to the sample tested by the reagent is considered to be of low risk, meaning they have no active pulmonary tuberculosis, latent tuberculosis infection, or active pulmonary tuberculosis. Based on the above, it can be understood that "diagnosis" in this application includes a judgment of prognosis. In this scenario, the TIPE2 content or expression level in the whole blood of the subject before and after treatment can be compared. If the TIPE2 content or expression level in the whole blood of the subject after treatment is lower than before treatment, it is determined that the treatment of the subject corresponding to the sample tested by the reagent has a certain effect.

[0054] In some embodiments, the reagent is used to distinguish between healthy subjects and patients with active pulmonary tuberculosis. In this scenario, the AUC of ROC analysis can reach 0.8645, with a sensitivity of 86.21% and a specificity of 71.7%.

[0055] In some embodiments, the reagent is used to differentiate between patients with latent tuberculosis infection and those with active pulmonary tuberculosis. In this scenario, the AUC of ROC analysis can reach 0.8136, with a sensitivity of 74.14% and a specificity of 70.27%.

[0056] In another aspect of this application, a kit for diagnosing and / or predicting active pulmonary tuberculosis is provided, comprising reagents for detecting the content of TIPE2 or its expression level in a sample, said sample including a blood sample.

[0057] In some embodiments, the reagents include, but are not limited to, specific probes, gene chips, specific primers, and anti-TIPE2 antibodies.

[0058] In some embodiments, the reagent is used to detect mRNA biomarkers.

[0059] Determining the level of mRNA biomarkers in a sample: The level of mRNA biomarkers in a biological sample can be determined by any suitable method. Any reliable method for measuring the level or amount of mRNA in a sample can be used. Typically, mRNA can be detected and quantified from a sample, such as a sample of RNA isolated by various methods known about mRNA, including, for example, amplification-based methods (e.g., polymerase chain reaction (PCR), real-time polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), rolling circle amplification, etc.).

[0060] In some embodiments, a kit for rapid detection of active pulmonary tuberculosis is provided, the kit comprising reagents for total RNA extraction from whole blood, reagents for TIPE2 mRNA RT-PCR, and reagents for internal reference gene assay and quantitative PCR.

[0061] For example, the quantitative PCR-related reagents include specific primers with sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0062] In some embodiments, the kit also includes a positive control and / or a negative control.

[0063] In some embodiments, the positive control includes or is whole blood, mRNA, or cDNA from a subject who has been diagnosed with active pulmonary tuberculosis.

[0064] In some embodiments, the negative control comprises whole blood, mRNA, or cDNA from a healthy subject or a subject who does not have active tuberculosis.

[0065] Another aspect of this application also provides a method for using the kit, including:

[0066] The content or expression level of TIPE2 in the sample was detected;

[0067] Compare the test results with positive and / or negative controls.

[0068] In some implementations, the method of use further includes interpreting the results of the comparison.

[0069] In some embodiments, the method of using the kit includes:

[0070] a. Measure the level of TIPE2 mRNA and / or plasma protein level obtained from the whole blood of the subject;

[0071] b. Measure the levels of housekeeping genes such as GAPDH mRNA and / or plasma protein levels in the whole blood of the subjects, and calculate the relative expression levels of TIPE2 and housekeeping genes;

[0072] c. Compare the relative expression level of TIPE2 mRNA in whole blood and / or the relative protein level of TIPE2 in plasma samples with a set of data, wherein the data are the relative expression level of TIPE2 mRNA in whole blood and / or the relative protein level of TIPE2 in plasma in normal subjects, or the data are the relative expression level of TIPE2 mRNA in whole blood and / or the relative protein level of TIPE2 in plasma in subjects before treatment.

[0073] In some embodiments, the method of use further includes step d: determining whether the subject has active pulmonary tuberculosis or assessing the prognosis of the subject based on the comparison results made in step c.

[0074] In some embodiments of the method or kit described in the application, the subject may be a mammal, such as a human.

[0075] In some embodiments of the method or kit applied for, the difference in the level of at least one mRNA relative to a suitable control can be determined by executing a software classification algorithm.

[0076] Another aspect of this application provides a system for diagnosing and / or predicting active pulmonary tuberculosis, the system comprising:

[0077] The data processing module is used to calculate the received or input data on the TIPE2 content or expression level in whole blood to obtain the calculation results; and

[0078] The judgment and output module is used to judge whether the calculation result meets the preset judgment conditions in order to diagnose and / or predict an individual's active pulmonary tuberculosis, and output the prediction result.

[0079] In some implementations, in the judgment and output module, when the calculation result meets the preset judgment condition, the output prediction result is "the subject has active pulmonary tuberculosis or the probability level of having the disease is high risk", and when the calculation result does not meet the preset judgment condition, the output prediction result is "the subject has no active pulmonary tuberculosis, latent tuberculosis infection, or the level of having active pulmonary tuberculosis is low risk".

[0080] In some implementations, the preset judgment condition is "the content of TIPE2 or its expression level is higher than that of the negative control".

[0081] Another aspect of this application provides a computer-readable storage medium storing a computer program that, when executed by a processor, can perform the functions of the system as described above.

[0082] Another aspect of this application provides a computer device including a memory and a processor. The memory stores a computer program, and the processor executes the computer program to implement the functions of the system described above. The computer device can be a terminal. The computer device includes a processor, memory, a communication interface, a display screen, and an input device connected via a system bus. The processor of the computer device provides computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and internal memory. The non-volatile storage medium stores an operating system and a computer program. The internal memory provides an environment for the operation of the operating system and computer program in the non-volatile storage medium. The communication interface of the computer device is used for wired or wireless communication with an external terminal. Wireless communication can be achieved through Wi-Fi, mobile cellular networks, NFC (Near Field Communication), or other technologies. When the computer program is executed by the processor, it implements a method for determining the battery performance of an energy storage system. The display screen of the computer device can be a liquid crystal display (LCD) or an e-ink display. The input device of the computer device can be a touch layer covering the display screen, or buttons, a trackball, or a touchpad mounted on the casing of the computer device, or an external keyboard, touchpad, or mouse, etc.

[0083] Those skilled in the art will understand that all or part of the processes in the above-described method embodiments can be implemented by a computer program instructing related hardware. The computer program can be stored in a non-volatile computer-readable storage medium. When executed, the computer program can include the processes of the embodiments described above. Any references to memory, databases, or other media used in the embodiments provided in this application can include at least one of non-volatile and volatile memory. Non-volatile memory can include read-only memory (ROM), magnetic tape, floppy disk, flash memory, optical memory, high-density embedded non-volatile memory, resistive random access memory (ReRAM), magnetic random access memory (MRAM), ferroelectric random access memory (FRAM), phase change memory (PCM), graphene memory, etc. Volatile memory can include random access memory (RAM) or external cache memory, etc. By way of illustration and not limitation, RAM can take many forms, such as Static Random Access Memory (SRAM) or Dynamic Random Access Memory (DRAM). The databases involved in the embodiments provided in this application may include at least one type of relational database and non-relational database. Non-relational databases may include, but are not limited to, blockchain-based distributed databases. The processors involved in the embodiments provided in this application may be general-purpose processors, central processing units, graphics processing units, digital signal processors, programmable logic devices, quantum computing-based data processing logic devices, etc., and are not limited to these.

[0084] Another aspect of this application provides an apparatus for diagnosing and / or predicting active pulmonary tuberculosis, comprising one or more of the system described above, the computer-readable storage medium described above, or the computer device described above.

[0085] Another aspect of this application also provides a method for diagnosing active pulmonary tuberculosis, including:

[0086] The content or expression level of TIPE2 in samples from subjects was detected and compared with positive and / or negative controls;

[0087] The comparison results will determine whether the subject has active pulmonary tuberculosis and / or its prognosis.

[0088] Another aspect of this application also provides a method for treating active pulmonary tuberculosis, including:

[0089] The above methods for diagnosing active pulmonary tuberculosis are used to determine whether a subject has active pulmonary tuberculosis and / or its prognosis;

[0090] Develop and implement a treatment plan.

[0091] The following are some examples.

[0092] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0093] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0094] Example 1

[0095] Nucleic acid sample preparation:

[0096] 1. Blood samples from patients with active pulmonary tuberculosis and healthy volunteers were obtained from Bao'an District People's Hospital. These included 53 healthy volunteers (HC), 37 individuals with latent tuberculosis infection (LTBI), 59 patients with active pulmonary tuberculosis (TB), and 47 patients with active pulmonary tuberculosis who had undergone standard anti-tuberculosis treatment for 2 months (TB-2M). 500 μL of the collected EDTA-anticoagulated whole blood was added to lysis buffer within 4 hours, and total RNA was extracted from the whole blood using the SIMG Blood Total RNA Extraction Kit. The concentration and purity of 1 μL of the extracted total RNA were determined using Nanodrop 2000.

[0097] 2. Using the total RNA from whole blood obtained in step 1 as a template, cDNA was synthesized using the Vazyme Hiscript YV All-in-One Ultra RT SuperMix qPCR reverse transcription kit according to the instructions.

[0098] Nucleic acid amplification and analysis:

[0099] 1. Reverse transcription

[0100] The reaction solutions (see Table 1) were prepared on ice. To ensure accurate loading, a Master Mix was prepared equal to the total number of reactions + 2, thoroughly mixed, and then dispensed 10 μL into each reaction tube. After gentle mixing, the reverse transcription reaction was immediately initiated.

[0101] Table 1

[0102]

[0103] The reaction procedure is as follows:

[0104] 50℃, 5min;

[0105] 85℃, 5 seconds;

[0106] Keep at 4℃.

[0107] 2. Using cDNA as a template, the reaction mixture was prepared according to Table 2 below using the Vazyme Taq Pro Universal SYBR qPCR MasterMix kit. Specific forward and reverse primers for the target gene TIPE2 were synthesized by Suzhou Genewiz Biotechnology Co., Ltd., and the primer sequences are: TIPE2-Forward Primer: 5'-GCTGCTAGAGTTGGTGGAACAC-3' (SEQ ID NO: 1); TIPE2-Reverse Primer: 5'-CTTGCCAAGGTGCTGAGTGAAG-3' (SEQ ID NO: 2).

[0108] Table 2

[0109]

[0110] Then, PCR amplification was performed using the reaction system shown in Table 3 below.

[0111] Table 3

[0112]

[0113] The mRNA expression level of TIPE2 in whole blood of patients with active pulmonary tuberculosis is as follows: Figure 1 As shown, the results are as follows:

[0114] Significant differences were observed in TIPE2 mRNA expression levels among healthy volunteers, individuals with latent tuberculosis infection, patients with active pulmonary tuberculosis, and patients with active pulmonary tuberculosis who had undergone standard anti-tuberculosis treatment for 2 months.

[0115] ROC analysis of the relative expression levels of TIPE2 in whole blood from tuberculosis patients (TB) (59 cases) and healthy individuals (HC) (53 cases) can be found in [link to relevant documentation]. Figure 2The results show that AUC = 0.8645. After taking the optimal cut-off value, the sensitivity % is 86.21% and the specificity % is 71.7%.

[0116] ROC analysis of the relative expression levels of TIPE2 in whole blood of patients with active pulmonary tuberculosis (TB) (59 cases) and latent tuberculosis infection (LTBI) (37 cases) can be found in [link to relevant documentation]. Figure 3 The results show that AUC=0.8136, and after taking the optimal cut-off value, Sensitivity% is 74.14% and Specificity% is 70.27%.

[0117] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0118] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. Use of a reagent for detecting the content or expression level of TIPE2 in a sample in the preparation of a reagent for diagnosing and / or predicting active tuberculosis; wherein the sample comprises a blood sample.

2. Use according to claim 1, wherein The reagent is selected from one or more of a specific probe, a gene chip, a specific primer, and an anti-TIPE2 antibody.

3. Use according to claim 2, wherein the compound is ###0002### The content or expression level of TIPE2 in the sample is detected by amplification, hybridization, and / or sequencing using the specific primer.

4. The use according to claim 3, wherein the compound is ###0002### The sequence of the specific primer is shown in SEQ ID NO: 1 and SEQ ID NO:

2.

5. The use according to any one of claims 1 to 4, wherein the compound is of formula (I) ###0001### (I) or a pharmaceutically acceptable salt thereof. The detection of the content or expression level of TIPE2 in the sample comprises comparing the content or expression level of TIPE2 in the sample with a control.

6. Use according to claim 5, wherein The detection of the content or expression level of TIPE2 in the sample meets one or more of the following items: The sample type is whole blood, and the content or expression level of TIPE2 is higher than that of a negative control, which is the whole blood of a healthy subject or a subject who does not have active tuberculosis, and it is determined that the subject corresponding to the sample detected by the reagent has active tuberculosis or a high risk of disease; The sample type is whole blood, and the content or expression level of TIPE2 is consistent with that of a negative control, which is determined to be a subject without active tuberculosis, latent tuberculosis infection, or a low risk of active tuberculosis; When the reagent is used for prognosis evaluation, the content or expression level of TIPE2 after treatment is lower than that before treatment, and it is determined that the subject corresponding to the sample detected by the reagent has a certain effect on treatment.

7. A kit for diagnosing and / or predicting active tuberculosis, characterized in that, It comprises a reagent for detecting the content or expression level of TIPE2 in a sample; wherein the sample comprises a blood sample. The reagent comprises a specific primer with a sequence as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

8. The kit of claim 7, wherein The kit further comprises a positive control and / or a negative control; wherein the positive control comprises or is the whole blood, mRNA, or cDNA of a subject with active tuberculosis, and the negative control comprises or is the whole blood, mRNA, or cDNA of a healthy subject or a subject who does not have active tuberculosis.

9. The kit of claim 7 or 8, wherein The kit further comprises primers for detecting the content or expression level of a reference gene.

10. A system for diagnosing and / or predicting active tuberculosis, characterized in that, The system comprises: a data processing module for calculating the content or expression level of TIPE2 and a reference in whole blood received or inputted, to obtain a calculation result; and a judgment and output module for judging whether the calculation result meets a preset judgment condition to diagnose and / or predict active tuberculosis in an individual, and outputting a prediction result. In the judging and outputting module, when the calculation result satisfies the preset judging condition, the prediction result is output as "the subject has active tuberculosis or the possibility level is high risk", and when the calculation result does not satisfy the preset judging condition, the prediction result is output as "the subject has no active tuberculosis, latent tuberculosis infection or the level of having active tuberculosis is low risk"; The preset judging condition is "the content or expression level of TIPE2 is higher than that of the negative control"; The reference data set comprises the content or expression level of TIPE2 in whole blood from a plurality of subjects with active tuberculosis and the content or expression level of TIPE2 in whole blood from a plurality of subjects without active tuberculosis.

11. The system for diagnosing and / or predicting active tuberculosis according to claim 10, wherein, The judging and outputting module comprises a machine learning model for performing the calculation; The machine learning model is selected from one or more of a generalized linear model, a random forest and a support vector machine.

12. A computer-readable storage medium storing a computer program, characterized in that, The computer program, when executed by a processor, can implement the functions of the system of claim 10 or 11.

13. A computer device comprising a memory and a processor, the memory storing a computer program, characterized in that, The processor is configured to execute the computer program to implement the functions of the system of claim 10 or 11.

14. An apparatus for diagnosing and / or predicting active tuberculosis, comprising one or more of the system of claim 10 or 11, the computer readable storage medium of claim 12 or the computer device of claim 13.

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