Method for culturing immune cells

Abstract

The present application relates to the detection of Mycobacterium tuberculosis and / or novel coronavirus. Specifically, the invention relates to a method for culturing immune cells and a method for detecting whether mycobacterium tuberculosis and / or novel coronavirus exist in a biological sample in vitro, or detecting whether the biological sample is infected with mycobacterium tuberculosis and / or novel coronavirus. The present application also relates to a computer readable medium performing the method.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application 202311295502.7 filed on October 8, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to the detection of Mycobacterium tuberculosis and / or the novel coronavirus. Specifically, the present application relates to a method for culturing immune cells and a method for detecting in vitro whether Mycobacterium tuberculosis and / or the novel coronavirus are present in a biological sample, or detecting whether a biological sample has been infected with Mycobacterium tuberculosis and / or the novel coronavirus. The present application also relates to a computer-readable medium for executing the method. Background Art

[0004] Tuberculosis (TB) infection is characterized by a sustained immune response to Mycobacterium tuberculosis (Mtb) antigens without the typical clinical symptoms of TB. It is estimated that about one-quarter of the world's population is infected with Mtb. From a clinical management perspective, there are two recognized states of Mtb infection, latent tuberculosis infection (LTBI, also known as inactive TB infection) and active TB, but both states are on a continuum of infection and immune control. LTBI is a state of sustained immune response to Mtb antigens that is not clinically manifested as active TB due to immune control, and the tuberculosis bacilli are either absent or at a level below an undefined threshold. Most patients with LTBI never develop TB; however, approximately 5% to 15% of patients will develop TB. TB is caused by bacterial replication, including tissue damage and inflammation, which leads to clinical manifestations. Currently available interferon-gamma release assays (IGRA) and tuberculin skin tests (TST) can indirectly assess infection by measuring cell-mediated immune responses to Mycobacterium tuberculosis antigens. These tests cannot differentiate between LTBI and tuberculosis disease or monitor treatment effectiveness because the immune responses detected by these tests can persist even after the patient has been effectively treated.

[0005] Both the tuberculin skin test and the interferon-γ release test are recommended by the WHO for the diagnosis of tuberculosis infection. TST is a purified protein derivative (PPD) test, which is simple to operate and easy to interpret, so it is widely used. However, TST results are easily affected by nontuberculous mycobacteria (NTM) infection and BCG vaccination, resulting in false positive results. In contrast, IGRA has the advantages of high specificity, no interference from BCG vaccination and most NTM, and a single judgment threshold. It is often used for the diagnosis of latent tuberculosis infection and auxiliary diagnosis of active tuberculosis. IGRA is mainly divided into two types. One is to detect the release level of IFN-γ using enzyme-linked immunosorbent assay (ELISA), and the other is to detect the number of effector T cells that release IFN-γ in peripheral blood mononuclear cells using enzyme-linked immunospot assay (ELISPOT), which is the T-SPOT.TB test. The two methods of IGRA have their own advantages and disadvantages. Compared with ELISA, T-SPOT.TB is less affected by immunosuppression, but its operation is more complicated, the detection time is longer, and the experimental environment requirements are higher.

[0006] Tuberculosis infection exists on a continuum of bacterial metabolic activity and immune response, including intermediate states such as “primary” and “subclinical” tuberculosis. Biomarkers are often needed to identify two populations: those at high risk of progression or who have already progressed, and those in whom persistent Mycobacterium tuberculosis has been cleared or is under effective control. In a meta-analysis of eight studies published in 2007, Menzies et al. pointed out that IGRA has a high specificity for diagnosing LTBI in the general population, especially in those who have been vaccinated with BCG; however, like TST, the sensitivity of IGRA is unsatisfactory, and neither can distinguish LTBI from active tuberculosis. Therefore, a method is needed to improve the sensitivity of tuberculosis-positive detection in TB-IGRA. Summary of the invention

[0007] The inventors of the present application unexpectedly discovered that exposing immune cells to air during the process of culturing immune cells can significantly increase the cell secretions produced by immune cells. On this basis, the method is used to further culture immune cells and detect and analyze cell secretions, which also significantly improves the sensitivity of the test results. Moreover, in the detection of Mycobacterium tuberculosis and the detection of the new coronavirus, the detection level of positive results is improved. Thus, the applicant has completed the present invention.

[0008] Methods for culturing immune cells

[0009] Therefore, in a first aspect, the present application provides a method for culturing immune cells, the method comprising:

[0010] (1) providing a biological sample obtained from a subject and containing the immune cells;

[0011] (2) contacting the biological sample with a specific antigen;

[0012] (3) Under conditions that allow the immune cells to produce cell secretions, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can circulate and / or be replaced, or the immune cells are exposed to air outside the culture environment during the 4th to 60th hour of culturing the immune cells (for example, 4-8h, 8-16h, 16-20h, 20-24h, 24-28h, 28-32h, 32-40h, 40-48h, 48-56h or 56-60h).

[0013] In certain embodiments, in step (3), the method produces an increased amount of cell secretions at the end of the culture compared to the beginning of the culture. In some embodiments, the increase is greater than or equal to about 2 times, greater than or equal to about 5 times, greater than or equal to about 10 times, greater than or equal to about 50 times, greater than or equal to about 100 times, greater than or equal to about 200 times, greater than or equal to about 300 times, or greater than or equal to about 500 times. In some embodiments, the increase is at or about 1000 times or greater. In some embodiments, the increase is at or about 2000 times or greater, at or about 3000 times or greater, or at or about 3500 times or greater.

[0014] As used herein, "conditions that allow immune cells to produce cell secretions" have the meaning commonly understood by those skilled in the art and can be determined by conventional methods. For example, immune cells are placed in a suitable temperature, environment and nutrients.

[0015] In certain embodiments, in step (3), the condition allowing the immune cells to produce cell secretions is to culture the immune cells at 20-50°C (e.g., 20-25°C, 25-30°C, 30-35°C, 35-40°C, 40-45°C, 45-50°C). In this article, the temperature for culturing immune cells is not limited to the temperature most suitable for cell growth. In certain embodiments, a cell cryoprotectant (e.g., DMSO) may also be provided to culture immune cells.

[0016] In certain embodiments, in step (3), the conditions that allow the immune cells to produce cell secretions are culturing the immune cells under nutrients suitable for cell growth (eg, cell culture medium).

[0017] In certain embodiments, in step (3), the immune cells are contacted with air outside the culture environment 1 time, 2 times, 3 times, 4 times, or 5 times during the process of culturing the immune cells.

[0018] In certain embodiments, in step (3), during the process of culturing the immune cells, the immune cells are contacted with the air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

[0019] In certain embodiments, in step (3), the immune cells are cultured for a total of 16-60 h (e.g., 16-20 h, 20-24 h, 24-28 h, 28-32 h, 32-40 h, 40-48 h, 48-56 h or 56-60 h).

[0020] In certain embodiments, the culturing is performed for about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days or 25 days. In some of any of the foregoing embodiments, the culturing is performed for any time length between about 2 days and 25 days.

[0021] In certain embodiments, in step (3), the conditions allowing the immune cells to produce cell secretions are culturing the immune cells at 20-50° C. (eg, 25-45° C.).

[0022] In certain embodiments, in step (3), the lid of the culture tube in which the immune cells are cultured is opened so that the immune cells are exposed to air outside the culture environment during the process of culturing the immune cells.

[0023] In certain embodiments, in step (3), the immune cells are brought into contact with air outside the culture environment during the culturing of the immune cells by connecting a culture tube for the immune cells to an air valve and opening the air valve.

[0024] In certain embodiments, in step (3), the immune cells are placed in a culture tube comprising a gas-permeable membrane so that the immune cells are always in a culture environment (eg, culture tube) where air can circulate and / or be replaced during the process of culturing the immune cells.

[0025] In the method herein, in step (1), the biological sample can be collected directly from the subject, or a biological sample that has been collected can be provided. In either case, the biological sample needs to be placed for a total of more than 16 hours before contacting with the specific antigen. In certain embodiments, in step (1), the biological sample is placed for 0 hours.

[0026] In certain embodiments, in step (1), the biological sample is collected immediately from the subject (for example, the biological sample is stored for 0 hours).

[0027] Methods for collecting biological samples are known in the art, and, depending on the biological sample collected, a person skilled in the art can select a corresponding collection method. In certain embodiments, the biological sample is whole blood. In certain embodiments, in such embodiments, a vacuum blood collection tube is used to collect whole blood from a subject.

[0028] In the method herein, in step (2), the biological sample is placed for more than 16 hours and then contacted with a specific antigen. In certain embodiments, after the immune cell contacts the specific antigen, an immune response can be produced under the stimulation of the specific antigen. In certain embodiments, the immune response of the immune cell includes the production of cell secretions (e.g., cytokines). In certain embodiments, after the immune cell contacts the specific antigen, a cell secretion can be produced.

[0029] In certain embodiments, in step (2), the biological sample is left for 16-120 hours.

[0030] In certain embodiments, in step (2), the biological sample is placed for 16-108 h, 16-96 h, 16-84 h, 16-72 h, 16-60 h, 16-48 h, 16-40 h, 16-36 h or 16-24 h. In certain embodiments, in step (2), the biological sample is placed for 16-108h, 16-96h, 16-84h, 16-72h, 16-60h, 16-48h, 16-40h, 16-36h or 16-24h, 24-32h, 32-40h, 40-48h, 48-56h, 56-64h, 64-72h, 72-80h, 80-88h, 88-96h, 96-104h, 104-112h, 112-120h, 120-128h, 128-136h, 136-144h, 144-152h or 152-160h.

[0031] In certain embodiments, in step (2), the biological sample is left for 16-72 hours.

[0032] In step (2), the biological sample is placed at a temperature below 60° C. for more than 16 hours and then contacted with a specific antigen.

[0033] In certain embodiments, in step (2), the biological sample is placed at 1-60°C, 2-50°C, 2-37°C, 2-30°C, 2-20°C or 2-8°C for more than 16 hours. In certain embodiments, the biological sample is placed at 2-8°C, 8-16°C, 16-22°C, 22-30°C, 30-38°C, 38-46°C or 46-50°C for more than 16 hours.

[0034] In certain embodiments, in step (2), the biological sample is placed at 2-37° C. for more than 16 h (eg, 16-72 h).

[0035] In certain embodiments, in step (2), the placing includes: standing, or rotating and mixing at 10 to 300 rpm, and the rotation speed of the rotating and mixing can be, for example, 10 rpm, 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm.

[0036] In certain embodiments, in step (2), the biological sample is placed at 0-150rpm, 0-140rpm, 0-130rpm, 0-120rpm, 0-110rpm, 0-100rpm, 0-90rpm, 0-80rpm, 0-70rpm, 0-60rpm, 0-50rpm, 0-40rpm, 0-30rpm, 0-20rpm or 0-10rpm for more than 16 hours and then contacted with the specific antigen.

[0037] In certain embodiments, the biological sample is placed at 0-50 rpm for more than 16 hours before contacting it with the specific antigen.

[0038] Immune cells

[0039] In this article, immune cells can be any kind. In certain embodiments, the immune cells are selected from T cells, natural killer (NK) cells, dendritic cells, macrophages, neutrophils, myeloid immune cells and B cells, or any combination thereof. In certain embodiments, the immune cells are cell populations composed of two or more immune cells, and these cells can be present in any kind of suitable culture medium or suitable carrier.

[0040] In certain embodiments, where the immune cells are NK cells and / or T cells, the NK cells and / or T cells may be derived from umbilical cord blood (including mixed umbilical cord blood units), peripheral blood, induced pluripotent stem cells, bone marrow, hematopoietic stem cells, cell lines and / or whole blood.

[0041] In certain embodiments, the immune cell is a T cell, such as a T cell derived from whole blood. In certain embodiments, the T cell may be a CD4+ T cell, or a CD8+ T cell.

[0042] Cell secretions

[0043] In some embodiments, the immune cells described above may express one or more cell secretions. In some embodiments, the cell secretions are selected from proteins, polypeptides, nucleic acids, polysaccharides, small molecules, or any combination thereof.

[0044] In certain embodiments, the cellular secretion is a cytokine.

[0045] In certain embodiments, the cell secretion is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-α, IFN-β, IFN-γ, TNF-β, SCF, G-CSF, M-CSF, GM-CSF, neuroleukin, IgA, IgG, perforin, granulin, IP10, TGF-β, or any combination thereof.

[0046] In certain embodiments, the cell secretion is a combination of IL-2 and IFN-γ. In certain embodiments, the cell secretion is IL-2. In certain embodiments, the cell secretion is IFN-γ.

[0047] Specific antigen

[0048] In this article, any substance that can stimulate immune cells to secrete cell secretions (eg, proteins, polypeptides, nucleic acids, polysaccharides, small molecule compounds) can be used as a specific antigen.

[0049] Therefore, in certain embodiments, the specific antigen is selected from antigenic proteins on the surface or inside of viruses or bacteria, nucleoproteins, proteins secreted by viruses or bacteria, or any combination thereof.

[0050] In certain embodiments, the specific antigen is derived from a bacterium capable of infecting a mammal, such as mycobacteria. For example, Mycobacterium tuberculosis. In certain embodiments, the specific antigen may be Mycobacterium tuberculosis with reduced activity (e.g., inactivated or attenuated Mycobacterium tuberculosis), or may be hydrolyzed Mycobacterium tuberculosis (e.g., a portion of Mycobacterium tuberculosis), or may be an immunogenic protein or portion thereof derived from Mycobacterium tuberculosis, or may be an immunogenic nucleic acid or portion thereof derived from Mycobacterium tuberculosis.

[0051] In certain embodiments, the specific antigen is derived from a virus capable of infecting a mammal, such as a coronavirus. For example, a novel coronavirus. In certain embodiments, the specific antigen may be a novel coronavirus with reduced activity (e.g., an inactivated or attenuated novel coronavirus), a hydrolyzed novel coronavirus (e.g., a portion of a novel coronavirus), an immunogenic protein or portion thereof derived from a novel coronavirus, or an immunogenic nucleic acid or portion thereof derived from a novel coronavirus.

[0052] In certain embodiments, the mammal is selected from the group consisting of humans, mice, rats, guinea pigs, sheep, horses, pigs, and rabbits.

[0053] In certain embodiments, the biological sample is selected from blood, isolated leukocytes, isolated peripheral blood mononuclear cells, a lung biopsy sample, a lymph node biopsy sample, isolated T cells, or any combination thereof.

[0054] In certain embodiments, the biological sample is whole blood and / or peripheral blood.

[0055] In certain embodiments, the subject is a subject already infected with Mycobacterium tuberculosis, or a subject at risk of infection with Mycobacterium tuberculosis, or a subject who has recovered from tuberculosis but is at risk of reinfection with Mycobacterium tuberculosis.

[0056] In certain embodiments, the subjects already infected with Mycobacterium tuberculosis include active tuberculosis subjects and inactive tuberculosis subjects.

[0057] In certain embodiments, the subject is a subject who has been infected with the novel coronavirus, or a subject who is at risk of being infected with the novel coronavirus, or a subject who has recovered from the novel coronavirus infection but is at risk of being infected with the novel coronavirus again.

[0058] In certain embodiments, the method further comprises: (4) detecting the cell secretions.

[0059] In certain embodiments, in step (4), the method for detecting cell secretions is selected from: ELISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation experiment, immunoagglutination experiment or microfluidics experiment, or any combination thereof.

[0060] Detection Methods

[0061] In a second aspect, the present application provides a method for detecting in vitro whether a biological sample is infected with Mycobacterium tuberculosis and / or a novel coronavirus, or improving the positive result detection rate of a biological sample infected with Mycobacterium tuberculosis and / or a novel coronavirus, comprising:

[0062] (1) providing a biological sample containing immune cells;

[0063] (2) contacting the biological sample with a specific antigen;

[0064] (3) under conditions that allow the immune cells to produce cell secretions, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can circulate and / or be replaced, or the immune cells are exposed to air outside the culture environment during the 4th to 60th hour of culturing the immune cells (for example, 4-8h, 8-16h, 16-20h, 20-24h, 24-28h, 28-32h, 32-40h, 40-48h, 48-56h or 56-60h);

[0065] (4) Detecting the cell secretions to determine whether the biological sample is infected with Mycobacterium tuberculosis and / or the new coronavirus, or to determine whether the biological sample is positive for infection with Mycobacterium tuberculosis and / or the new coronavirus.

[0066] In certain embodiments, the methods can improve the sensitivity of a test result of a biological sample (eg, improve the detection value of a positive result of a biological sample).

[0067] In certain embodiments, in step (4), the method for detecting cell secretions is selected from: ELISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation experiment, immunoagglutination experiment or microfluidics experiment, or any combination thereof.

[0068] In certain embodiments, the positive result for the biological sample infected with Mycobacterium tuberculosis includes active tuberculosis and inactive tuberculosis.

[0069] In certain embodiments, the methods are used to differentiate and / or identify a subject already infected with M. tuberculosis, a subject at risk of becoming infected with M. tuberculosis, a subject who has recovered from tuberculosis but is at risk of becoming reinfected with M. tuberculosis, or any combination thereof.

[0070] In certain embodiments, the method is used to distinguish and / or identify subjects who have been infected with the novel coronavirus, subjects who are at risk of infection with the novel coronavirus, and subjects who have recovered from novel coronavirus infection but are at risk of reinfection with the novel coronavirus.

[0071] In certain embodiments, in step (3), the immune cells are contacted with air outside the culture environment 1 time, 2 times, 3 times, 4 times, or 5 times during the process of culturing the immune cells.

[0072] In certain embodiments, in step (3), during the process of culturing the immune cells, the immune cells are contacted with the air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

[0073] In certain embodiments, in step (3), the immune cells are cultured for a total of 16-60 h (e.g., 16-20 h, 20-24 h, 24-28 h, 28-32 h, 32-40 h, 40-48 h, 48-56 h or 56-60 h).

[0074] In certain embodiments, in step (3), the conditions allowing the immune cells to produce cell secretions are culturing the immune cells at 20-50° C. (eg, 25-45° C.).

[0075] In certain embodiments, in step (3), the lid of the culture tube in which the immune cells are cultured is opened so that the immune cells are exposed to air outside the culture environment during the process of culturing the immune cells.

[0076] In certain embodiments, in step (3), the immune cells are brought into contact with air outside the culture environment during the culturing of the immune cells by connecting a culture tube for the immune cells to an air valve and opening the air valve.

[0077] In certain embodiments, in step (3), the immune cells are placed in a culture tube comprising a gas-permeable membrane so that the immune cells are always in a culture environment (eg, culture tube) where air can circulate and / or be replaced during the process of culturing the immune cells.

[0078] In certain embodiments, the immune cell is as defined above. In certain embodiments, the specific antigen is as defined above. In certain embodiments, the biological sample is as defined above.

[0079] In certain embodiments, step (1) is as defined above. In certain embodiments, step (2) is as defined above. In certain embodiments, step (3) is as defined above.

[0080] In certain embodiments, the method comprises:

[0081] (1) providing a biological sample containing T cells obtained from a subject;

[0082] (2) placing the biological sample for 16-72 hours and then contacting it with specific antigens of Mycobacterium tuberculosis and / or the new coronavirus;

[0083] (3) under conditions that allow T cells to produce IFN-γ and / or IL-2, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can be circulated and / or replaced, or, at 4-60 hours (e.g., 4-8 hours, 8-16 hours, 16-20 hours, 20-24 hours, 24-28 hours, 28-32 hours, 32-40 hours, 40-48 hours, 48-56 hours or 56-60 hours) of culturing the immune cells, the immune cells are exposed to air outside the culture environment;

[0084] (4) Detecting IFN-γ and / or IL-2 to determine whether the biological sample is infected with Mycobacterium tuberculosis and / or the new coronavirus, or to determine whether the biological sample is positive for infection with Mycobacterium tuberculosis and / or the new coronavirus.

[0085] In certain embodiments, the method can be implemented by the following steps (a) to (f):

[0086] (a) Collection: collecting a whole blood sample from a subject (for example, using a vacuum blood collection tube to collect the whole blood from the subject);

[0087] (b) Storage treatment: After the whole blood sample is collected, it is placed at 2 to 37° C. for 16 to 72 hours;

[0088] (c) Aliquoting: Gently invert the whole blood sample to mix thoroughly and then dispense it into negative culture tubes, test culture tubes, and positive culture tubes. The aliquot volume of the whole blood sample is 0.1 to 5 mL / tube;

[0089] (d) Cultivation: After gently inverting the culture tube to mix, place the culture tube in an incubator at 25-45° C. for 2-60 hours, and during the culturing process, the whole blood sample is always in a culture environment where air can circulate and / or be replaced, or, during the 4-60 hours of culturing the whole blood sample (for example, 4-8 hours, 8-16 hours, 16-20 hours, 20-24 hours, 24-28 hours, 28-32 hours, 32-40 hours, 40-48 hours, 48-56 hours or 56-60 hours), the whole blood sample is exposed to air outside the culture environment;

[0090] (e) Centrifugation: centrifuging the culture medium for 1 to 20 minutes (e.g., centrifuging at 1000-10000 rpm), and taking plasma as a test object;

[0091] (f) Detection of IFN-γ and / or IL-2 content: ELISA was performed on plasma to detect IFN-γ and / or IL-2.

[0092] In certain embodiments, in step (d), the whole blood sample is contacted with air outside the culture environment 1 time, 2 times, 3 times, 4 times, or 5 times during the culture process.

[0093] In certain embodiments, in step (d), during the culturing of the whole blood sample, the whole blood sample is contacted with air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

[0094] In a third aspect, the present application provides the use of the aforementioned method in preparing a kit.

[0095] In a fourth aspect, the present application provides a kit for performing the method as described above, wherein the instructions of the kit contain the method as described above.

[0096] In a fifth aspect, the present application provides a computer-readable medium comprising a plurality of instructions for controlling a computing system, wherein the plurality of instructions are used to control the computing system to execute the aforementioned method, so as to determine whether a biological sample is infected with Mycobacterium tuberculosis and / or a novel coronavirus, or to determine whether the biological sample is a positive result of infection with Mycobacterium tuberculosis and / or a novel coronavirus. In certain embodiments, the aforementioned method can improve the positive result detection rate of biological samples infected with Mycobacterium tuberculosis and / or a novel coronavirus.

[0097] Definition of terms

[0098] As used herein, the term "mycobacterium" is a genus of aerobic intracellular bacterial organisms. Diseases caused by mycobacterial infection in humans include tuberculosis (caused by Mycobacterium tuberculosis), leprosy (caused by Mycobacterium leprae), Bairnsdale ulcer (caused by Mycobacterium ulcerans), etc.

[0099] The main response to mycobacterium in the organism includes the cell-mediated hypersensitivity (DTH) reaction of T cells and macrophages. The main T cell response involves the CD4+ lymphocytes that recognize mycobacterium heat shock proteins and immunodominant antigens. As used herein, the term "CD4" refers to differentiation cluster factor 4, which is a T cell surface protein that mediates interaction with class II MHC molecules. Cells expressing CD4 are typically helper T cells. As used herein, the term "CD8" refers to differentiation cluster factor 8, which is a T cell surface protein that mediates interaction with class I MHC molecules. Cells expressing CD8 are typically cytotoxic T cells." CD8+T cell-mediated immunity" is an immune response performed by presenting antigens to CD8+T cells.

[0100] As used herein, the term "Mycobacterium tuberculosis", also referred to as Mtb, is a bacterium of the genus Mycobacterium. It is the pathogen that causes tuberculosis (TB) infection. Typically, tuberculosis infection leads to tuberculosis (TB).

[0101] Without limitation, TB disease in humans and other animals may be caused by mycobacteria other than M. tuberculosis. This is because all mycobacteria belong to the same class of pathogens, some of which are virulent and some of which are not. Thus, in the text, when referring to mycobacteria, it includes, but is not limited to, the following: M. tuberculosis., M. avium, M. habana, M. abscessus, M. aurum, M. bovis, M. africanum, M. chelonae, M. gastri, M. good i), Mycobacterium gordonae, Mycobacterium paratuberculosis, Mycobacterium lufu, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium leprae, Mycobacterium mageritense, Mycobacterium porcinum, Mycobacterium septicum and Mycobacterium simiae.

[0102] As used herein, the term "tuberculosis (TB)" is a disease caused by infection with Mycobacterium tuberculosis. Tuberculosis includes pulmonary tuberculosis and extrapulmonary tuberculosis. Pulmonary tuberculosis is a lung disease caused by Mtb, and symptoms generally include coughing and may include coughing up blood or sputum, chest pain, weakness, weight loss, fever, chills, and night sweats.

[0103] Herein, tuberculosis patients are divided into two types, namely, active tuberculosis (ATB) and inactive tuberculosis (also known as latent tuberculosis infection (LTBI)). Active tuberculosis refers to patients infected with Mycobacterium tuberculosis and showing symptoms; and inactive tuberculosis refers to patients infected with Mycobacterium tuberculosis but not showing symptoms. Therefore, in certain embodiments, active tuberculosis can be clinically diagnosed, while inactive tuberculosis will not be clinically diagnosed.

[0104] In this article, as long as the subject is infected with Mycobacterium tuberculosis, regardless of whether the subject shows tuberculosis symptoms, he or she is considered "TB positive". Therefore, TB positive subjects include subjects with both active TB and inactive TB (also known as latent tuberculosis infection (LTBI)).

[0105] Furthermore, "active tuberculosis exclusion samples" refer to all subjects other than those with active tuberculosis, including subjects with inactive tuberculosis and subjects not infected with Mycobacterium tuberculosis.

[0106] As used herein, the term "TB-IGRA positive" means that the TB-IGRA test result of the subject's sample is positive. The reason for the TB-IGRA positive may be that the sample tested is tuberculosis positive, or it may be due to a false positive.

[0107] As used herein, the term "diagnosis" refers to the presence or nature of identifying a pathological condition (e.g., tuberculosis). Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of sick individuals that are tested as positive (true positive percentage). The "specificity" of a diagnostic assay is 1 minus the false positive rate, wherein the false positive rate is defined as the ratio of individuals that are tested as positive but not sick. As used herein, the term "prognosis" refers to the possibility of predicting the development (e.g., severity) of a pathological condition such as tuberculosis.

[0108] As used herein, the term "immune response" refers to the response of cells of the immune system (e.g., B cells, natural killer cells, or T cells) to a stimulus. In certain embodiments, the response is specific to a particular antigen ("antigen-specific response"). In certain embodiments, the immune response is a T cell response, such as a Th1, Th2, or Th3 response.

[0109] As used herein, the term "cytokine" refers to a protein produced by a cell that can affect the behavior of other cells (e.g., lymphocytes). Cytokines include, but are not limited to, interleukins (IL-2, IL-4, IL-6, IL-10, IL-21, etc.) and interferons (IFN-γ, etc.).

[0110] As used herein, the terms "interferon-γ" or "IFN-γ" have the same meaning and can be used interchangeably. IFN-γ is a dimeric protein whose subunits have 146 amino acids. The protein is glycosylated at two sites with a pI of 8.3-8.5. IFN-γ is synthesized as a 166 amino acid precursor protein that includes a 23 amino acid secretion signal sequence.

[0111] As used herein, the term "antigen" refers to a molecule that can stimulate an immune response, for example. Any macromolecule (including almost all proteins or peptides) can be an antigen. Antigens can also be derived from genomic recombinants or DNA. For example, any DNA encoding "antigen" comprising a nucleotide sequence or partial nucleotide sequence encoding a protein that can elicit an immune response. In embodiments, antigens do not need to be encoded only by the full-length nucleotide sequence of a gene, and antigens do not need to be encoded by a gene at all. In embodiments, antigens can be synthetic or can be derived from biological samples, for example, tissue samples, tumor samples, cells, or fluids with other biological components. As used herein, the term "specific antigen" refers to any molecule that can stimulate an immune response of an immune cell, and can stimulate immune cells to produce cell secretions.

[0112] As used herein, the term "immune cell" refers to various cells that function in the immune system. In certain embodiments, immune cells include leukocytes, for example, neutrophils, eosinophils, basophils, lymphocytes and monocytes. Innate leukocytes include phagocytes (for example, macrophages, neutrophils and dendritic cells), mast cells, eosinophils, basophils and natural killer cells. Innate leukocytes identify and eliminate pathogens by contacting larger pathogens or by engulfing and then killing microorganisms, and are mediators of adaptive immune response activation. The cells of the adaptive immune system are special types of leukocytes, referred to as lymphocytes. B cells and T cells are important types of lymphocytes, derived from hematopoietic stem cells in the bone marrow. B cells participate in humoral immune responses, and T cells participate in cell-mediated immune responses. In certain embodiments, immune cells can participate in immune responses after contacting with specific antigens.

[0113] As used herein, the term "subject" generally refers to an individual with a biological sample that is being processed or analyzed. In certain embodiments, subjects include mammals, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits), livestock (e.g., cattle, sheep, goats, pigs, turkeys, and chickens), household pets (e.g., dogs, cats, and rodents), horses, and transgenic non-human animals.

[0114] In certain embodiments, the subject may be a patient, for example, suffering from or suspected of having a disease. In certain embodiments, the subject may be receiving or has been receiving treatment. In certain embodiments, the subject may be asymptomatic. In certain embodiments, the subject may be healthy but desires to prevent the individuality of the disease (for example, the individuality of the vaccine). In certain embodiments, the terms "individual", "subject" or "individual" can be used interchangeably. In certain embodiments, the subject may include people or non-human animals of any age, therefore including adults and adolescents (that is, children) and infants, and including individuals in the womb.

[0115] As used herein, the term "culture medium" refers to a matrix that provides cells with the necessary components for growth. Examples of culture media that can be used for the immune cells of the present application include, but are not limited to: DMEM / F12 (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12), RPMI-1640 (RPMI: Roswell Park Memorial Institute), MEM (Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium).

[0116] As used herein, the term "biological sample" generally refers to a sample obtained from an organism. A biological sample can be taken from an individual's tissue or cell. In some embodiments, a biological sample may include or be derived from a tissue biopsy, blood (e.g., whole blood), plasma, extracellular fluid, dried blood spots, cultured cells, or discarded tissue. A biological sample may have been separated from a source before collection. Examples of biological samples include, but are not limited to, blood, cerebrospinal fluid, pleural fluid, amniotic fluid, lymph fluid, saliva, urine, feces, tears, sweat, or mucosal secretions, and other body fluids separated from a primary source before collection. In certain embodiments, during the preparation of a biological sample, a sample is separated from its original source (cells, tissues, body fluids such as blood, environmental samples, etc.). A biological sample may be purified or otherwise concentrated from its original source, or it may not be purified or concentrated from its original source. In certain embodiments, the original source is homogenized before further processing. A biological sample may be filtered or centrifuged to remove a buffy coat, lipids, or particulate matter. A biological sample may also be purified or enriched for its nucleic acid, or it may be treated with an RNA enzyme. Biological samples may contain intact, fragmented or partially degraded tissues or cells.

[0117] The first purpose of the present invention is to provide a method for stimulating the in vitro release of cell secretions during infection detection, which can significantly improve the in vitro release and detection sensitivity of cell secretions, and thus has a significant effect on distinguishing current symptoms from past infections.

[0118] The second object of the present invention is to provide a method for detecting tuberculosis infection status.

[0119] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0120] In a first aspect, the present invention provides a method for stimulating the in vitro release of cell secretions during infection detection, wherein the infection detection comprises: collecting blood to be tested, packaging, culturing, and centrifuging in sequence, and then detecting cell secretion markers, wherein the method for stimulating the in vitro release of cell secretions comprises:

[0121] The collected blood to be tested is left to stand for a period of time before being packaged;

[0122] Alternatively, during the culture process for infection detection, the culture sample is exposed to air at certain stages.

[0123] It should be noted that the applicant has discovered two methods that can stimulate the release of cell secretions in vitro through research on blood collection, sample culture, and culture tube packaging during infection detection. Specifically, they are:

[0124] (1) Allowing the collected blood to stand for a period of time before packaging;

[0125] (2) During the culture process of infection detection, the culture sample is exposed to air in stages; this step can ensure that whole blood or other culture type samples have sufficient air (oxygen, CO2, etc.) during the culture process, allowing the cells to enhance their ability to secrete cytokines under appropriate conditions, thereby increasing the amount of cytokine secretion.

[0126] By adjusting the above steps, the in vitro release and detection sensitivity of cell secretions can be significantly improved, which in turn plays a significant role in distinguishing current symptoms from past infections.

[0127] Furthermore, the infection detection includes: identifying the infection status of bacteria through cell secretion markers; or identifying the infection status of viruses through cell secretion markers.

[0128] Furthermore, the identification of infection status includes: identifying latent tuberculosis-infected persons and active tuberculosis patients; or, identifying people with current COVID-19 infection, previous infection or vaccination.

[0129] Furthermore, the immunostimulatory antigens released in vitro by the stimulatory cell secretion body are feasible as long as they are proteins, polypeptides, nucleic acids, polysaccharides, and small molecule compounds that can stimulate immune cells, including antigenic proteins and nucleoproteins on the surface or inside of viruses or bacteria, and proteins secreted by viruses or bacteria, without special limitation.

[0130] Preferably, the immunostimulatory antigen released in vitro by the stimulatory cell secretion body is: a Mycobacterium tuberculosis-specific antigen, or a SARS-CoV-2-specific antigen.

[0131] Furthermore, the cell secretion markers include cytokines secreted by immune cells or non-immune cells upon stimulation.

[0132] The cell secretion markers include: any one of IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-α, IFN-β, TNF-β, SCF, G-CSF, M-CSF, GM-CSF, neuroleukin, IgA, IgG, perforin, granulin, IP10 or TGF-β, or a combination of at least two thereof.

[0133] Furthermore, the method for detecting the cell secretion marker includes: one of EILISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation experiment, immunoagglutination experiment or microfluidics experiment.

[0134] It should be noted that the method of stimulating the in vitro release of cell secretions during the infection detection process of the present invention is for the purpose of non-disease diagnosis, and its application scenarios include scientific research, basic technology research, basic medical research, etc.

[0135] In a second aspect, the present invention provides an application of the method for stimulating the in vitro release of cell secretions during the above-mentioned infection detection process in the detection of tuberculosis infection status.

[0136] The method of stimulating the in vitro release of cell secretions during the infection detection process of the present invention can be widely used in the detection of tuberculosis infection status, and can improve the detection rate of tuberculosis-positive samples.

[0137] In a third aspect, the present invention provides an application of the above-mentioned method for stimulating the in vitro release of cell secretions during infection detection in the preparation of a tuberculosis infection status detection product.

[0138] The detection product prepared by the method of stimulating the in vitro release of cell secretions during the above-mentioned infection detection process of the present invention can identify latent tuberculosis infected persons and active tuberculosis patients, which is of great significance for scientific research and clinical medicine.

[0139] In a fourth aspect, the present invention provides a device for stimulating the in vitro release of cell secretions during infection detection, comprising:

[0140] Pre-treatment module: After the blood to be tested is collected, it is packaged, cultured, and centrifuged in sequence;

[0141] Stimulation module: to process the collected blood to stimulate the release of cell secretions in vitro;

[0142] Detection module: detect the released cell secretions;

[0143] Wherein, the processing of the collected blood to be tested includes:

[0144] The blood to be tested is placed before being packaged;

[0145] Alternatively, during the culture process of the blood to be tested, the culture sample is brought into contact with the air in stages.

[0146] Using the device for stimulating the in vitro release of cell secretions during the above-mentioned infection detection process to treat the blood to be tested can promote the release of cell secretions and improve the sensitivity of detection, thereby achieving the effect of distinguishing latent tuberculosis infected persons from active tuberculosis patients.

[0147] In a fifth aspect, the present invention provides a method for detecting tuberculosis infection status, comprising: collecting blood from a tuberculosis infected person to be tested, packaging, culturing, centrifuging in sequence, and then detecting the IFN-γ content, the detection method further comprising:

[0148] The step of allowing the collected blood from the tuberculosis infected person to stand for a period of time before packaging;

[0149] Alternatively, during the culture process for infection detection, the culture sample is exposed to air at certain stages.

[0150] It should be noted that the method for detecting tuberculosis infection status provided in the present application can improve the detection of active tuberculosis and reduce the detection of active tuberculosis exclusion samples by introducing into the detection process: a step of statically treating the collected blood of the tuberculosis-infected person before packaging; or, during the culture process, a step of periodically contacting the culture sample with the air, which can effectively improve the sensitivity and specificity of the reagent and achieve the effect of increasing the detection rate of tuberculosis positivity.

[0151] In the present invention, active tuberculosis refers to clinical diagnosis of active tuberculosis, inactive tuberculosis (also called latent tuberculosis) refers to infection with Mycobacterium tuberculosis but exclusion of active tuberculosis; tuberculosis positive refers to infection with Mycobacterium tuberculosis but no limitation on whether tuberculosis symptoms are present. Active tuberculosis exclusion samples refer to all cases except active tuberculosis, including latent tuberculosis and non-tuberculosis infection.

[0152] Furthermore, the blood collection from the tuberculosis infected person to be tested includes: collecting whole blood from the tuberculosis infected person to be tested using a blood collection tube;

[0153] Furthermore, the packaging includes: gently inverting the whole blood specimen to mix well and then packaging it into negative culture tubes, test culture tubes and positive culture tubes, and the sample volume added to the test tube is 0.1-5mL, for example, it can be 0.1mL, 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL, 3mL, 3.5mL, 4mL, 4.5mL or 5mL, etc.

[0154] Furthermore, the culturing includes: gently inverting the culture tube to mix, and then placing it in an incubator at 25-45°C for 2-60 hours. The culture temperature can be, for example, 25°C, 30°C, 35°C, 37°C, 40°C or 45°C, and the culture time can be, for example, 2h, 5h, 10h, 12h, 20h, 24h, 30h, 36h, 40h, 48h, 50h, 55h or 60h.

[0155] Furthermore, the centrifugation includes: centrifuging the culture medium after culture at 1000-10000rpm for 1-20min, the centrifugal speed can be, for example, 1000rpm, 2000rpm, 3000rpm, 4000rpm, 5000rpm, 6000rpm, 7000rpm, 8000rpm, 9000rpm or 10000rpm, and the centrifugal time can be, for example, 1min, 5min, 10min, 15min or 20min, etc.

[0156] Furthermore, the method for detecting the IFN-γ content includes: one of EILISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation experiment, immunoagglutination experiment or microfluidics experiment.

[0157] Furthermore, the placement treatment is: after collecting whole blood from a tuberculosis-infected patient to be tested, place it in an environment of 2 to 37°C for 16 to 72 hours. The placement temperature can be, for example, 2°C, 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 37°C, and the placement time can be, for example, 16h, 20h, 24h, 30h, 36h, 40h, 48h, 50h, 55h, 60h, 65h, 70h or 72h.

[0158] It should be noted that the applicant, through research on the blood collection steps in the infection detection process, found that: the applicant tried to conduct detection after treatment at different temperatures of 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 37°C, 45°C, and 50°C. The experiment showed that temperatures below 30°C had the best detection effect, while the results became worse after treatment above 37°C.

[0159] It should be noted that the applicant, through research on the blood collection steps in the infection detection process, found that the applicant tried different placement times of 0h, 8h, 16h, 24h, 32h, 40h, 48h, 60h, and 72h before testing, and believed that the best detection effect was achieved within the range of 16h to 40h, while the results became worse after treatment for more than 60h.

[0160] Furthermore, the placing includes: standing, or rotating and mixing at 10 to 300 rpm, and the rotation speed of the rotating and mixing can be, for example, 10 rpm, 50 rpm, 100 rpm, 150 rpm, 200 rpm, 250 rpm or 300 rpm.

[0161] It should be noted that the applicant, through research on the blood collection steps in the infection detection process, found that: the applicant tried different placement methods, including static placement, placement on a rotary mixer at 10rpm, 50rpm, 100rpm, 300rpm, and placement on a shaker at 10rpm, 50rpm, and 100rpm, and then testing, and believed that static placement and low-speed mixing had the best detection effect.

[0162] Furthermore, the detection method comprises:

[0163] (A1) Collection: Collect whole blood from the tuberculosis infected patient using a vacuum blood collection tube;

[0164] (Aa) Storage treatment: After collecting whole blood from the tuberculosis infected person to be tested, place it in an environment of 2 to 37°C for 16 to 72 hours;

[0165] (B1) Aliquoting: Gently invert the whole blood sample to mix thoroughly and then dispense it into negative culture tubes, test culture tubes, and positive culture tubes. The volume of the whole blood sample dispensed is 0.1-5 mL / tube;

[0166] (C1) Cultivation: Gently invert the culture tube to mix well, and then place in an incubator at 25-45°C for 2-60 hours;

[0167] (D1) Centrifugation: The culture medium is centrifuged at 1000-10000 rpm for 1-20 min, and plasma is collected as a test object;

[0168] (E1) IFN-γ content detection: ELISA detection was performed using plasma.

[0169] Furthermore, the step of periodically contacting the culture sample with air comprises:

[0170] The packaged culture tubes are placed in an incubator and cultured at 25-45°C for 16-60 h (the culture temperature can be, for example, 25°C, 30°C, 37°C, 40°C or 45°C, and the culture time can be, for example, 16 h, 20 h, 24 h, 28 h, 30 h, 36 h, 40 h, 48 h, 50 h, 55 h or 60 h, wherein: the culture tubes are ventilated 1 to 3 times (for example, 1 time, 2 times or 3 times) during the 8th to 16th h of culture (for example, 8 h, 10 h, 12 h, 14 h or 16 h, etc.).

[0171] It should be noted that in the sample culture process after whole blood collection and packaging in this application, ventilation operation is performed during the operation. This method can improve the detection rate of TB-IGRA positive samples without affecting negative samples. Specifically:

[0172] After whole blood is collected and packaged, the samples are cultured for 16 to 60 hours, with ventilation treatment for 8 to 16 hours in the middle, and then tested according to the method for detecting the release level of IFN-γ.

[0173] Preferably, the sample culture time is 16 to 60 hours, and the best time is 16 to 36 hours. The best time for ventilation is 8 to 16 hours, and the best number of ventilations is 1 to 3 times.

[0174] In the present invention, ventilation can be performed by opening and closing the lid.

[0175] It should be noted that, through the study of the culture steps in the infection detection process, the applicant obtained the following conclusions: the applicant tried to gently invert the culture tube for 5 times to mix, and then quickly put it into 37°C for culturing for 22±2h, and opened and closed the lid once at one of the 4th, 8th, 10th, 12th, 16th, and 20th hours of the culture process, and performed ELISA testing after the culture was completed. It is believed that opening and closing the lid once within 8 to 16 hours has the best effect.

[0176] It should be noted that, through the study of the culture steps in the infection detection process, the applicant obtained the following results: the applicant tried to gently invert the culture tube to mix it 5 times and then quickly put it into 37°C for culturing for 22±2h; in the 12th hour of the culturing process, the lid was opened and closed once, twice at 8h and 16h, three times at 8h, 12h and 16h, four times at 4h, 8h, 12h and 16h, five times at 4h, 8h, 12h, 16h and 20h, and six times at 4h, 8h, 10h, 12h, 16h and 20h; ELISA test was performed after the culturing was completed, and it was believed that opening and closing the lid 1 to 3 times had the best effect.

[0177] Preferably, in addition to the ventilation operation of opening and closing the lid, the opening and closing operation can also be replaced by a bottle cap or tube cap with a matching ventilation valve or breathable membrane to maintain the ventilation volume during the culture process.

[0178] Furthermore, the detection method comprises:

[0179] (A2) Collection: Collect whole blood from the tuberculosis infected patient using a vacuum blood collection tube;

[0180] (B2) Aliquoting: Gently invert the whole blood sample to mix thoroughly and then dispense it into negative culture tubes, test culture tubes, and positive culture tubes. The volume of the whole blood sample dispensed is 0.1 to 5 mL / tube.

[0181] (C2) Cultivation: After gently inverting the culture tube to mix, place it in an incubator at 25-45°C for 16-60 hours, wherein: the culture tube is ventilated 1-3 times during the 8th to 16th hour of cultivation;

[0182] (D2) Centrifugation: centrifuge the culture medium at 1000-10000 rpm for 1-20 min, and collect plasma as the test object;

[0183] (E2) IFN-γ content detection: ELISA detection was performed using plasma.

[0184] Furthermore, the detection method also includes: during the packaging process, changing the ratio of the amount of stimulant and sample added in the test tube, then performing the steps of culturing, centrifugation, and cell secretion marker detection in sequence, and then judging and distinguishing active tuberculosis and latent tuberculosis infection through the detection results of several test tubes under different conditions.

[0185] Furthermore, the changing of the ratio between the amount of the irritant and the amount of the sample added in the test tube includes:

[0186] Set up several test tubes with different concentrations; and / or

[0187] Set up several different sample volumes to be added to the test tubes.

[0188] Preferably, several test tubes with different concentrations are set up, including: in addition to negative culture tubes, test culture tubes and positive culture tubes, T1 to Tn test culture tubes are set up, wherein the antigen amount of T1 to Tn test culture tubes is 1 / 1000 to 10 times the antigen amount of the test culture tubes, for example, it can be 1 / 1000, 1 / 100, 1 / 10, 1 times, 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 6.5 times, 7 times, 7.5 times, 8 times, 8.5 times, 9 times, 9.5 times or 10 times, etc.

[0189] Preferably, the amount of sample added to the test tube is 0.3-10 mL, for example, it can be 0.3 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 5.5 mL, 6 mL, 6.5 mL, 7 mL, 7.5 mL, 8 mL, 8.5 mL, 9 mL, 9.5 mL or 10 mL, etc.

[0190] It should be noted that the above detection method increases the number of test culture tubes, and in the population with positive specific antigen-mediated cellular immune response detected by the in vitro release method, current infection and latent infection or past infection are distinguished according to the results of the added test culture tubes. For example, in TB-IGRA-positive patients, by adding a second test tube T2, active tuberculosis and latent tuberculosis can be distinguished according to the results of T2. The above detection method can be used in the in vitro culture of cellular immune response mediated by tuberculosis antigens for auxiliary diagnosis screening of tuberculosis patients. It can also be used in the in vitro culture of other specific antigen-mediated cellular responses to distinguish current infection, past infection and latent infection.

[0191] Furthermore, the detection method comprises:

[0192] (A3) Collection: Collect whole blood from the tuberculosis infected patient using a vacuum blood collection tube;

[0193] (B3) Packaging: After gently inverting and mixing the whole blood sample, the sample is dispensed into negative culture tubes, test culture tubes, positive culture tubes and T1 to Tn test culture tubes, wherein the antigen amount of the T1 to Tn test culture tubes is 1 / 1000 to 10 times the antigen amount of the test culture tubes, and / or the sample amount added to the test tube is 0.3-10 mL;

[0194] (C3) Cultivation: Gently invert the culture tube to mix thoroughly, and then place in an incubator at 25-45°C for 2-60 hours.

[0195] (D3) Centrifugation: centrifuge the culture medium at 1000-10000 rpm for 1-20 min, and collect plasma as the test object;

[0196] (E3) IFN-γ content detection: ELISA test is performed using plasma, and the test results of T1~Tn test culture tubes are used to distinguish between active tuberculosis and latent tuberculosis infection.

[0197] It should also be noted that the matrix liquid in the culture tube is a phosphate buffer, the test culture tube (T) contains tuberculosis-specific antigens, and the test culture tubes (T1-Tn) contain the same tuberculosis-specific antigens, and the antigen amount is 1 / 1000-10 times that of the T tube; the positive control culture tube (P) contains tuberculosis nonspecific stimulating antigens, and the background control culture tube (N) contains matrix liquid. The newly added culture tubes with different concentrations contain different amounts of stimulating antigens from the original T tubes, so the status of tuberculosis infection can be further inferred from the results of the T2 tube in the samples screened for TB-IGRA positive.

[0198] It should be noted that the applicant has found through research that the amount of stimulating antigen contained in the newly added culture tubes is 1 / 1000, 1 / 100, 1 / 50, 1 / 10, 1 / 5, 1 / 4, 1 / 2, 3 / 4, 1, 5 / 4, 3 / 2, 2 times, 5 times, and 10 times that of culture tube T. The same volume of whole blood sample is added to the "N", "T", "T2", and "P" tubes, and culture tests are carried out at the same time. The results are calculated and analyzed, and it is believed that the optimal amount of stimulating antigen in the newly added culture tubes is 1 / 10 to 3 / 4 of that in culture tube T.

[0199] It should be noted that the applicant has found through research that when the content of the irritant in the newly added culture tube is consistent with that in culture tube T, 0.3mL, 0.5mL, 1mL, 1.5mL, 2mL, 4mL, 6mL, 8mL, and 10mL of whole blood samples are added, and cultured and tested simultaneously with the "N", "T", and "P" tubes. The results are calculated and analyzed, and it is believed that the addition amount of 0.5-6mL is optimal, which can play a role in distinguishing active tuberculosis from latent tuberculosis.

[0200] It should be noted that the above-mentioned method for detecting tuberculosis infection status of the present invention is for the purpose of non-disease diagnosis, and its application scenarios include scientific research, basic technology research, basic medical research, etc.

[0201] In a sixth aspect, the present invention provides a detection device for tuberculosis infection, comprising:

[0202] Processing module: After the blood to be tested is collected, it is packaged, cultured, and centrifuged in sequence;

[0203] Detection module: detect the content of released IFN-γ.

[0204] Furthermore, the processing module also includes: placing processing before packaging.

[0205] Furthermore, the processing module also includes: during the culturing process, the culture sample is brought into contact with the air in stages.

[0206] Furthermore, the processing module further comprises: during the packaging process, changing the ratio between the amount of the stimulant and the amount of the sample added in the test tube.

[0207] In the present invention, the use of the above-mentioned tuberculosis infection detection device can achieve the following technical effects:

[0208] (1) Through the processing module, the sample is placed before packaging to improve the detection rate of active tuberculosis and reduce the detection rate of active tuberculosis exclusion samples;

[0209] (2) Through the processing module, the culture sample is exposed to air in stages during the culture process, which can improve the detection rate of tuberculosis positivity;

[0210] (3) By using the processing module, the ratio of the amount of irritant and the amount of sample added to the test tube is changed during the packaging process, so that active tuberculosis and latent tuberculosis can be distinguished.

[0211] As a preferred embodiment, the present invention provides a detection device for improving the detection rate of active tuberculosis, comprising:

[0212] Processing module: After the blood to be tested is collected, it is placed in an environment of 2-37°C for 16-72 hours, and then the operations of packaging, culturing, and centrifugation are carried out in sequence;

[0213] Detection module: detect the content of released IFN-γ.

[0214] As a preferred embodiment, the present invention provides a detection device for improving the positive detection rate of tuberculosis, comprising:

[0215] Processing module: After the blood to be tested is collected, the operations of packaging, culturing and centrifugation are carried out in sequence, wherein: the culture tube is ventilated 1 to 3 times during the 8th to 16th hour of culturing;

[0216] Detection module: detect the content of released IFN-γ.

[0217] As a preferred embodiment, the present invention provides a detection device for distinguishing active tuberculosis from inactive tuberculosis, comprising:

[0218] Processing module: after collecting the blood to be tested, the blood is packaged, cultured and centrifuged in sequence, wherein: T1 to Tn test culture tubes are set during the packaging, and the antigen amount of the T1 to Tn test culture tubes is 1 / 1000 to 10 times the antigen amount of the test culture tubes;

[0219] Detection module: detect the content of released IFN-γ.

[0220] Compared with the prior art, the present invention has the following beneficial effects:

[0221] The applicant has discovered two methods to stimulate the release of cell secretions in vitro through research on the steps of blood collection, sample culture, and culture tube packaging during infection detection, namely: (1) letting the collected blood stand before packaging; (2) during the culture process of infection detection, the culture sample is exposed to air in stages. Adjustment of the above steps can significantly improve the in vitro release and detection sensitivity of cell secretions, which in turn has a significant effect on distinguishing between current and past infections.

[0222] The method for stimulating the in vitro release of cell secretions during the above-mentioned infection detection process provided in the present application can be widely used in the detection of tuberculosis infection status to achieve the identification of latent tuberculosis infected persons and active tuberculosis patients.

[0223] The method for detecting tuberculosis infection status provided in the present application can improve the detection rate of active tuberculosis and reduce the detection of active tuberculosis exclusion samples by introducing into the detection process: the step of subjecting the collected blood of the tuberculosis-infected person to be tested to a static state before packaging; the step of subjecting the cultured sample to periodic contact with air during the culture process can further improve the detection rate of tuberculosis positivity; and the effect of distinguishing active tuberculosis from latent tuberculosis is produced by changing the proportional relationship between the amount of irritant and the amount of sample added in the test tube during the packaging process.

[0224] Advantageous Effects of the Invention

[0225] Compared with the prior art, the present application allows immune cells to contact air during the process of culturing immune cells, which can significantly increase the cell secretions produced by immune cells. Furthermore, the method of the present application also significantly improves the sensitivity of the test results (for example, the sensitivity of tuberculosis positivity is increased to 90%), thereby increasing the detection rate of tuberculosis positivity. Moreover, in the detection of Mycobacterium tuberculosis and the detection of the new coronavirus, the detection level of positive results is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0226] Figure 1 Results of comparative analysis of tuberculosis detection methods (IL-2).

[0227] Figure 2 The results of comparison and analysis of the detection methods for whether the new coronavirus is ventilated are presented. DETAILED DESCRIPTION

[0228] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0229] Unless otherwise specified, the experiments and methods described in the examples are basically carried out according to conventional methods well known in the art and described in various references.

[0230] In addition, if the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified in the reagents or instruments used, they are all conventional products that can be obtained commercially. It is known to those skilled in the art that the embodiments describe the present invention by way of example and are not intended to limit the scope of the present invention. All public cases and other references mentioned herein are incorporated herein by reference in their entirety.

[0231] Example 1.

[0232] Experimental Materials:

[0233] Reagents: Beijing Wantai Biopharmaceutical Co., Ltd. Tuberculosis Infection T Cell Detection Kit (in vitro release ELISA). QIAGEN GmbH QuantiFERON-TB Kit (ELISA).

[0234] Samples: Whole blood samples from 80 subjects who were clinically identified as tuberculosis positive and 50 subjects who were tuberculosis negative (non-tuberculosis infected) were obtained through cooperative units (including hospitals and tuberculosis prevention and control institutes, etc.). The sample background was determined by clinical diagnosis.

[0235] Instruments: Incubator or water bath, refrigerator, ELISA reader.

[0236] Experimental methods:

[0237] 1. Collection: Use venipuncture and anticoagulant vacuum blood collection tubes to collect whole blood specimens with a volume not less than the test amount.

[0238] 2. Packaging: within 48 hours after collection, gently invert and mix the whole blood specimen 3 to 5 times, and then pack it into the three culture tubes "N", "T" and "P" provided by the original kit, 0.1 to 5 mL / tube. Among them, the N tube is the background control culture tube, the T tube is the test culture tube containing tuberculosis-specific stimulating antigens, and the P tube is the positive control culture tube containing non-specific stimulating antigens.

[0239] 3. Cultivation: Gently invert the culture tube to mix well, then culture at 25-45℃ for 16-60h. Open the lid once between 8-16h during the culture process, then close the lid and continue culturing. Keep the culture tube upright during the culture process.

[0240] 4. Centrifugation: The culture tubes are centrifuged at 1000-10000 rpm for 1-20 min, and plasma is collected for ELISA testing of interferon-γ.

[0241] In this example, the control group 1, group 2 and experimental group were all carried out according to the above steps, with the only difference being that the cover was not opened for ventilation during the culture process in the control group 1 and group 2. Among them, the control group 1 and this example were both tested using the kit of Beijing Wantai Biopharmaceutical Co., Ltd., and the control group 2 was tested using the QuantiFERON-TB kit of QIAGEN.

[0242] The experimental results are shown in the ELISA test results in Table 1. Compared with the control groups 1 and 2, which were sealed throughout the culture process, the sensitivity of the tuberculosis-positive sample detection results in the experimental group was increased, the tuberculosis-positive detection rate was increased, the tuberculosis-negative sample results were unchanged, and the specificity was not affected.

[0243] Table 1

[0244]

[0245] Sensitivity = number of positive results detected by the reagent / number of background results of clinical tuberculosis positive samples × 100%

[0246] Specificity = (1-number of positive samples detected by the reagent / number of negative samples) × 100%

[0247] From the above results, it can be seen that the sensitivity of control 1 in detecting tuberculosis-positive samples was 82.5% (66 / 80), and the specificity in detecting non-tuberculosis infected samples was 84.0% (1-8 / 50);

[0248] The sensitivity of control 2 for detecting tuberculosis-positive samples was 82.5% (66 / 80), and the specificity for detecting non-tuberculosis infected samples was 84.0% (1-8 / 50).

[0249] In the experimental group (ventilation was performed by opening and closing the lid during culture), the same samples had a sensitivity of 90.0% (72 / 80) for tuberculosis-positive samples and a specificity of 84.0% (1-8 / 50) for non-tuberculosis-infected samples. The increased sensitivity and no difference in specificity can better detect tuberculosis-positive patients and improve the detection rate of the reagent.

[0250] Example 2.

[0251] This example is intended to explore the preferred conditions for the culturing step of Example 1.

[0252] 1) Explore different opening times

[0253] Cultivation steps: Gently invert the culture tube to mix, then culture it at 25-45℃ for 16 to 60 hours. Open and close the lid once at 4h, 8h, 16h, and 20h during the culture process. After the culture is completed, perform ELISA test and use the unopened lid as a control. The test results are shown in the table below.

[0254] Table 2 Comparison of opening time results

[0255]

[0256]

[0257] The above results show that opening the lid for ventilation during the culture process can increase the detection rate of tuberculosis-positive samples, and the best effect is to open and close the lid once within 8 to 16 hours. The results of 8 to 16 hours not only have a high compliance rate of 100%, but also a higher TN value for tuberculosis-positive samples (greater difference from the background control culture tube).

[0258] 2) Explore different opening times

[0259] Cultivation steps: Gently invert the culture tube to mix, then culture it at 25-45℃ for 16 to 60 hours. Open and close the lid 1, 2, 3, 4, or 5 times within 0 to 24 hours of the culture process. After the culture is completed, perform ELISA test and use 0 times of opening the lid as a control. The test results are shown in the table below.

[0260] Table 3 Comparison of opening times

[0261]

[0262] From the above results, it can be seen that the best effect is to open the lid for ventilation 1 to 3 times during the culture process. The performance is not further improved when the ventilation frequency is greater than 3 times, but the risk of contamination and false positive may be increased due to improper operation. Therefore, it is preferred to open the lid for ventilation 1 to 3 times.

[0263] 3) Explore different ventilation modes

[0264] Gently invert the culture tube to mix well and place it in different culture containers. Culture at 25-45℃ for 16 to 60 hours. Perform the following operations on different culture containers:

[0265] 3.1) Open the lid for ventilation: Open the lid of the culture tube for routine reagents 1 to 3 times;

[0266] 3.2) Breathable membrane mode: The culture container is replaced with a culture tube with a breathable membrane on the upper cover, and no additional cover opening and closing operation is required;

[0267] 3.3) Ventilation valve mode: The culture container is replaced with a culture tube with a vent valve on the upper cover, and the vent valve is adjusted for each ventilation;

[0268] 3.4) Control: Conventional reagent detection mode was used as the control condition (no ventilation during the culture process).

[0269] After the culture was completed, ELISA test was performed and the test results are shown in the table below.

[0270] Table 4 Comparison of experimental results of different ventilation modes

[0271]

[0272]

[0273] From the above results, it can be seen that the difference in the positive and negative results of different ventilation modes is not large, and all of them can improve the detection level of tuberculosis positive results. When analyzing the deviation of the specific results of several ventilation modes, it was found that the TN values ​​of several modes in the detection of tuberculosis positive samples were not much different, and they were all about 30% higher than the control. In particular, in the breathable membrane mode, the TN value of the negative sample (non-tuberculosis infection) was closest to the control, and the TN values ​​of the open-cover ventilation and ventilation valve modes increased by about 45%, while the specific increase of the breathable membrane mode was the smallest (only 5%).

[0274] Example 3.

[0275] Experimental Materials:

[0276] Reagents: Beijing Wantai Biological Pharmaceutical Co., Ltd. Tuberculosis Infection T Cell Detection Kit (in vitro release enzyme-linked immunosorbent assay), Beijing Wantai Biological Pharmaceutical Co., Ltd. Human Interleukin 2 (IL-2) Detection Kit (ELISA method).

[0277] Samples: Whole blood samples from 5 subjects who were clinically identified as tuberculosis positive and 5 subjects who were tuberculosis negative (non-tuberculosis infected) were selected through cooperative units (including hospitals and tuberculosis prevention and control institutes, etc.). The sample background was determined by clinical diagnosis.

[0278] Instruments: Incubator or water bath, refrigerator, ELISA reader.

[0279] Experimental method: Steps 1 and 2 are the same as those in Example 1. Steps 3 and 4 are specifically as follows.

[0280] 3. Cultivation: After gently inverting the culture tube to mix, install the ventilation valve on the culture tube cap for cultivation, and then culture it at 25-45℃ for 16-60h. Keep the culture tube upright during the cultivation process.

[0281] 4. Centrifugation: After incubation, the culture tube is centrifuged at 1000-10000 rpm for 1-20 min, and plasma is collected for detection using a human interleukin 2 (IL-2) detection kit (ELISA method).

[0282] The experimental results are as follows Figure 1 shown.

[0283] Compared with the samples cultured in conventional non-ventilated culture tubes (L1-L5 are tuberculosis positive samples, L6-L10 are non-tuberculosis infected samples), the test results after culture with the tube cap equipped with a ventilation valve showed that the tuberculosis positive IL-2 test result value increased, while the tuberculosis negative had no significant change. Based on the above data, it can be concluded that ventilation treatment during the culture process can improve the detection level of positive results.

[0284] Example 4.

[0285] Experimental Materials:

[0286] Reagents: Beijing Wantai Biopharmaceutical Co., Ltd. Novel Coronavirus (2019-nCoV) Infected T Cell Detection Kit (in vitro release enzyme-linked immunosorbent assay).

[0287] Samples: 10 COVID-19 positive whole blood samples and 5 negative whole blood samples. Positive samples were confirmed as COVID-19 positive through COVID-19 nucleic acid testing, and samples collected from subjects who had not been infected with COVID-19 were confirmed as COVID-19 negative samples.

[0288] Instruments: Incubator or water bath, refrigerator, ELISA reader.

[0289] Experimental method: Steps 1 and 2 are the same as those in Example 1. Steps 3 and 4 are specifically as follows.

[0290] 3. Cultivation: Gently invert the culture tube to mix well, then culture at 25-45℃ for 16-60h. Open the lid once in situ at 8-16h during the culture process, then close the lid and continue culturing. Keep the culture tube upright during the culture process.

[0291] 4. Centrifugation: After incubation, centrifuge the culture tube at 1000-10000 rpm for 1-20 min, and take the plasma for detection using the novel coronavirus (2019-nCoV) infected T cell detection kit (in vitro release enzyme-linked immunosorbent assay).

[0292] The experimental results are as follows Figure 2 shown.

[0293] Compared with the results of conventional closed culture, the test results of ventilation during culture (S1-S10 are COVID-19 positive samples, S11-S15 are COVID-19 negative samples) showed that the COVID-19 positive IFN-γ test result value increased, while the negative one showed no significant change. Based on the above data, it can be concluded that ventilation during culture can improve the detection level of positive results.

[0294] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The entire invention is given by the attached claims and any equivalents thereof.

Claims

1. A method for culturing immune cells, the method comprising: include: (1) providing a biological sample obtained from a subject and containing the immune cells; (2) contacting the biological sample with a specific antigen; (3) Under conditions that allow the immune cells to produce cell secretions, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can circulate and / or be replaced, or the immune cells are exposed to air outside the culture environment during the 4th to 60th hour of culturing the immune cells (for example, 4-8h, 8-16h, 16-20h, 20-24h, 24-28h, 28-32h, 32-40h, 40-48h, 48-56h or 56-60h).

2. The method according to claim 1, wherein in step (3), the immune cells are contacted with air outside the culture environment once, twice, three times, four times or five times during the process of culturing the immune cells; Preferably, in step (3), during the process of culturing the immune cells, the immune cells are contacted with the air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

3. The method of claim 1 or 2, wherein in step (3), the immune cells are cultured for a total of 16-60 h (e.g., 16-20 h, 20-24 h, 24-28 h, 28-32 h, 32-40 h, 40-48 h, 48-56 h or 56-60 h); Preferably, in step (3), the conditions allowing the immune cells to produce cell secretions are culturing the immune cells at 20-50° C. (eg, 25-45° C.).

4. The method according to any one of claims 1 to 3, wherein the method has one or more characteristics selected from the following: (1) in step (3), opening the lid of the culture tube in which the immune cells are cultured so that the immune cells are in contact with air outside the culture environment during the process of culturing the immune cells; (2) in step (3), the immune cell culture tube is connected to an air valve, and the air valve is opened to allow the immune cells to contact with air outside the culture environment during the process of culturing the immune cells; (3) In step (3), the immune cells are placed in a culture tube comprising a gas-permeable membrane so that the immune cells are always in a culture environment (eg, a culture tube) where air can circulate and / or be replaced during the process of culturing the immune cells.

5. The method according to any one of claims 1 to 4, wherein in step (1), the biological sample is obtained by immediate collection from the subject (for example, the biological sample is placed for 0 hours in this case); Preferably, in step (2), the biological sample is placed for more than 16 hours and then contacted with the specific antigen; Preferably, in step (2), the biological sample is placed for 16-120 hours; Preferably, in step (2), the biological sample is placed for 16-108 h, 16-96 h, 16-84 h, 16-72 h, 16-60 h, 16-48 h, 16-40 h, 16-36 h or 16-24 h; Preferably, in step (2), the biological sample is placed for 16-72 hours.

6. The method according to any one of claims 1 to 5, in, In step (2), the biological sample is placed at a temperature below 60° C. for more than 16 hours and then contacted with a specific antigen; Preferably, in step (2), the biological sample is placed at 1-60°C, 2-50°C, 2-37°C, 2-30°C, 2-20°C or 2-8°C for more than 16 hours; Preferably, in step (2), the biological sample is placed at 2-37°C for more than 16 hours (eg, 16-72 hours).

7. The method according to any one of claims 1 to 5, in, In step (2), the biological sample is placed at 0-150rpm, 0-140rpm, 0-130rpm, 0-120rpm, 0-110rpm, 0-100rpm, 0-90rpm, 0-80rpm, 0-70rpm, 0-60rpm, 0-50rpm, 0-40rpm, 0-30rpm, 0-20rpm or 0-10rpm for more than 16 hours, and then contacted with the specific antigen.

8. The method according to any one of claims 1 to 7, in, The immune cells are selected from T cells, natural killer (NK) cells, dendritic cells, macrophages, neutrophils, myeloid immune cells and B cells, or any combination thereof; Preferably, the cell secretion is selected from proteins, polypeptides, nucleic acids, polysaccharides, small molecule compounds, or any combination thereof; Preferably, the cell secretion is a cytokine; Preferably, the cell secretion is selected from IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-17, IFN-α, IFN-β, IFN-γ, TNF-β, SCF, G-CSF, M-CSF, GM-CSF, neuroleukin, IgA, IgG, perforin, granulin, IP10, TGF-β, or any combination thereof; Preferably, the cell secretion is IL-2 and / or IFN-γ.

9. The method according to any one of claims 1 to 8, in, The specific antigen is selected from antigenic proteins on the surface or inside of viruses or bacteria, nucleoproteins, proteins secreted by viruses or bacteria, or any combination thereof; Preferably, the specific antigen is derived from a bacterium capable of infecting a mammal, such as a mycobacterium; for example, Mycobacterium tuberculosis; Preferably, the specific antigen is derived from a virus capable of infecting mammals, such as a coronavirus; for example, a novel coronavirus; Preferably, the mammal is selected from the group consisting of humans, mice, rats, guinea pigs, sheep, horses, pigs and rabbits.

10. The method according to any one of claims 1 to 9, in, The biological sample is selected from blood, isolated leukocytes, isolated peripheral blood mononuclear cells, lung biopsy samples, lymph node biopsy samples, isolated T cells, or any combination thereof; Preferably, the biological sample is whole blood and / or peripheral blood; Preferably, the subject is a subject already infected with Mycobacterium tuberculosis, or a subject at risk of infection with Mycobacterium tuberculosis, or a subject who has recovered from tuberculosis but is at risk of reinfection with Mycobacterium tuberculosis; Preferably, the subjects already infected with Mycobacterium tuberculosis include subjects with active tuberculosis and subjects with inactive tuberculosis; Preferably, the subject is a subject who has been infected with the novel coronavirus, or a subject who is at risk of being infected with the novel coronavirus, or a subject who has recovered from the novel coronavirus infection but is at risk of being infected with the novel coronavirus again.

11. The method according to any one of claims 1 to 10, further comprising: include: (4) detecting the cell secretions; Preferably, in step (4), the method for detecting cell secretions is selected from: ELISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation assay, immunoagglutination assay or microfluidics assay, or any combination thereof.

12. A method for detecting in vitro whether a biological sample is infected with Mycobacterium tuberculosis and / or a novel coronavirus, or increasing the positive result detection rate of a biological sample infected with Mycobacterium tuberculosis and / or a novel coronavirus, comprising: (1) providing a biological sample containing immune cells; (2) contacting the biological sample with a specific antigen; (3) under conditions that allow the immune cells to produce cell secretions, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can circulate and / or be replaced, or the immune cells are exposed to air outside the culture environment during the 4th to 60th hour of culturing the immune cells (for example, 4-8h, 8-16h, 16-20h, 20-24h, 24-28h, 28-32h, 32-40h, 40-48h, 48-56h or 56-60h); (4) detecting the cell secretions to determine whether the biological sample is infected with Mycobacterium tuberculosis and / or the novel coronavirus, or to determine whether the biological sample is positive for infection with Mycobacterium tuberculosis and / or the novel coronavirus; Preferably, the method can improve the sensitivity of the detection results of the biological sample (e.g., improve the detection value of the positive result of the biological sample); Preferably, in step (4), the method for detecting cell secretions is selected from: ELISA, chemiluminescence, immunofluorescence, colloidal gold, biochemical immunoassay, immunoprecipitation assay, immunoagglutination assay or microfluidics assay, or any combination thereof; Preferably, the positive result of the biological sample infected with Mycobacterium tuberculosis includes active tuberculosis and inactive tuberculosis; Preferably, the method is used to differentiate and / or identify a subject already infected with Mycobacterium tuberculosis, a subject at risk of infection with Mycobacterium tuberculosis, a subject who has recovered from tuberculosis but is at risk of reinfection with Mycobacterium tuberculosis, or any combination thereof; Preferably, the method is used to distinguish and / or identify subjects who have been infected with the novel coronavirus, subjects who are at risk of being infected with the novel coronavirus, and subjects who have recovered from the novel coronavirus infection but are at risk of being infected with the novel coronavirus again.

13. The method of claim 12, wherein in step (3), the immune cells are contacted with air outside the culture environment once, twice, three times, four times or five times during the process of culturing the immune cells; Preferably, in step (3), during the process of culturing the immune cells, the immune cells are contacted with air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h); Preferably, in step (3), the immune cells are cultured for a total of 16-60 h (e.g., 16-20 h, 20-24 h, 24-28 h, 28-32 h, 32-40 h, 40-48 h, 48-56 h or 56-60 h); Preferably, in step (3), the conditions allowing the immune cells to produce cell secretions are culturing the immune cells at 20-50° C. (e.g., 25-45° C.); Preferably, the method has one or more characteristics selected from the following: (1) in step (3), opening the lid of the culture tube in which the immune cells are cultured so that the immune cells are in contact with air outside the culture environment during the process of culturing the immune cells; (2) in step (3), the immune cell culture tube is connected to an air valve, and the air valve is opened to allow the immune cells to contact with air outside the culture environment during the process of culturing the immune cells; (3) In step (3), the immune cells are placed in a culture tube comprising a gas-permeable membrane so that the immune cells are always in a culture environment (e.g., a culture tube) where air can circulate and / or be replaced during the process of culturing the immune cells; Preferably, the immune cell is as defined in any one of claim 8; Preferably, the specific antigen is as defined in any one of claim 9; Preferably, the biological sample is as defined in any one of claims 10; Preferably, step (1) and step (2) are defined as in any one of claims 5-7.

14. The method of claim 12 or 13, comprising: (1) providing a biological sample containing T cells obtained from a subject; (2) placing the biological sample for 16-72 hours and then contacting it with specific antigens of Mycobacterium tuberculosis and / or the new coronavirus; (3) under conditions that allow T cells to produce IFN-γ and / or IL-2, during the process of culturing the immune cells, the immune cells are always kept in a culture environment where air can be circulated and / or replaced, or, at 4-60 hours (e.g., 4-8 hours, 8-16 hours, 16-20 hours, 20-24 hours, 24-28 hours, 28-32 hours, 32-40 hours, 40-48 hours, 48-56 hours or 56-60 hours) of culturing the immune cells, the immune cells are exposed to air outside the culture environment; (4) Detecting IFN-γ and / or IL-2 to determine whether the biological sample is infected with Mycobacterium tuberculosis and / or the new coronavirus, or to determine whether the biological sample is positive for infection with Mycobacterium tuberculosis and / or the new coronavirus.

15. The method according to any one of claims 12 to 14, wherein the method can be implemented by the following steps (a) to (f): (a) Collection: collecting a whole blood sample from a subject (for example, using a vacuum blood collection tube to collect the whole blood from the subject); (b) Storage treatment: After the whole blood sample is collected, it is placed at 2 to 37° C. for 16 to 72 hours; (c) Aliquoting: Gently invert the whole blood sample to mix thoroughly and then dispense it into negative culture tubes, test culture tubes, and positive culture tubes. The aliquot volume of the whole blood sample is 0.1 to 5 mL / tube; (d) Cultivation: After gently inverting the culture tube to mix, place the culture tube in an incubator at 25-45° C. for 2-60 hours, and during the culturing process, the whole blood sample is always in a culture environment where air can circulate and / or be replaced, or, during the 4-60 hours of culturing the whole blood sample (for example, 4-8 hours, 8-16 hours, 16-20 hours, 20-24 hours, 24-28 hours, 28-32 hours, 32-40 hours, 40-48 hours, 48-56 hours or 56-60 hours), the whole blood sample is exposed to air outside the culture environment; (e) Centrifugation: centrifuging the culture medium for 1 to 20 minutes (e.g., centrifuging at 1000-10000 rpm), and taking plasma as a test object; (f) Detection of IFN-γ and / or IL-2 content: ELISA detection of IFN-γ and / or IL-2 using plasma; Preferably, in step (d), the whole blood sample is contacted with air outside the culture environment 1 time, 2 times, 3 times, 4 times or 5 times during the culture process; Preferably, in step (d), during the culturing of the whole blood sample, the whole blood sample is contacted with the air outside the culture environment once, twice or three times at 8-16h (8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h).

16. Use of the method according to any one of claims 1 to 15 in preparing a kit.

17. A kit for performing the method according to any one of claims 1 to 15, in, The instructions of the kit include the method described in any one of claims 1 to 15.

18. A computer-readable medium comprising a plurality of instructions for controlling a computing system, wherein the plurality of instructions are used to control the computing system to execute the method according to any one of claims 1 to 15, so as to perform an operation of determining whether a biological sample is infected with Mycobacterium tuberculosis and / or a novel coronavirus, or determining whether the biological sample is a positive result of infection with Mycobacterium tuberculosis and / or a novel coronavirus; Preferably, the method can improve the detection rate of positive results of biological samples infected with Mycobacterium tuberculosis and / or the new coronavirus.

Images