A kit for diagnosing active tuberculosis

Abstract

The present invention discloses a kit for diagnosing active tuberculosis. The kit includes a substance for detecting IFIT3 protein. Compared with healthy people and latent tuberculosis infected people, the expression of IFIT3 gene in PBMCs of active tuberculosis patients is significantly increased; the expression of IFIT3 gene can be used to distinguish active tuberculosis patients from latent tuberculosis infected people; the expression of IFIT3 gene can be used to distinguish active tuberculosis patients from healthy people. Therefore, IFIT3 protein and / or IFIT3 gene can be used as a marker to diagnose active tuberculosis. The present invention has great application value.

Description

Technical Field

[0001] This invention belongs to the field of medical immunology diagnostic technology, specifically relating to a reagent kit for diagnosing active tuberculosis. Background Technology

[0002] Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB), primarily transmitted through the respiratory tract. After MTB infects a human, three different outcomes can occur: first, if the body's immunity is strong, the MTB is cleared directly; second, the MTB is suppressed by the body's immune system but cannot be completely cleared, developing into latent tuberculosis infection (LTBI); and third, the MTB proliferates rapidly in the body, developing into active tuberculosis. TB is a major infectious disease that requires focused prevention and control in my country.

[0003] Currently, tuberculosis diagnosis mainly involves imaging, Mycobacterium tuberculosis (MBTB) diagnosis, and immunological diagnosis, but each method has its limitations. Imaging diagnosis struggles to differentiate pulmonary tuberculosis from other lung diseases. MBTB diagnosis has a high false-negative rate. Immunological diagnosis primarily involves antibody testing and cellular immune testing (such as the tuberculin skin test (TST) and interferon gamma release assays (IGRA)). Both TST and IGRAS assess tuberculosis infection by detecting the body's primary anti-tuberculosis immune response, i.e., cellular immune response. In China, TST is currently the primary detection method. A strong positive result for purified protein derivatives (PPD) or a rapid shift from negative to positive without clinical evidence of tuberculosis is generally considered a latent MTB infection. Due to its simplicity and low cost, TST has become the most commonly used and convenient method for diagnosing MTB infection in clinical practice. However, PPD is a crude antigen mixture extracted from Mycobacterium tuberculosis, containing more than 200 proteins, many of which are common antigenic components of non-tuberculous mycobacteria and BCG. Therefore, TST has poor specificity and is prone to false positives in BCG-vaccinated individuals and those without tuberculous mycobacterial infection. TST can only assist in diagnosis based on the strength of the skin reaction, with a sensitivity of only 70-80%. Furthermore, TST has drawbacks such as time-consuming testing, the need for subject follow-up (72 hours), and subjective dependence on skin test procedures and result interpretation. IGRA uses enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunospot assay (ELISPOT) to quantitatively detect the IFN-γ release reaction of whole blood or peripheral blood mononuclear cells to Mycobacterium tuberculosis-specific antigens (ESAT6, CFP10, and TB7.7), thus diagnosing tuberculosis infection. However, IGRA has difficulty distinguishing between active tuberculosis and latent tuberculosis infection. The inability to diagnose active tuberculosis in its early stages leads to delayed treatment, increased costs, and higher mortality rates. Furthermore, it hinders effective control of the source of infection, resulting in the spread of tuberculosis. Therefore, developing specific and effective diagnostic reagents for active tuberculosis is of great significance for the prevention and control of tuberculosis.

[0004] IFIT3 protein (Interferon-induced protein with tetratricopeptide repeats 3) is one of the earliest discovered interferon-induced proteins. It exerts antiviral effects by directly inhibiting viral translation or promoting viral RNA degradation. It also participates in intracellular signal transduction regulation and induces the production of type I interferon, thereby enhancing cellular antiviral activity. In interferon therapy for liver cancer, IFIT3 protein can serve as a target for predicting treatment efficacy. Currently, IFIT3 protein has not been used to diagnose active pulmonary tuberculosis. Summary of the Invention

[0005] The purpose of this invention is to diagnose active tuberculosis.

[0006] This invention first protects the use of a substance for detecting IFIT3 protein in the preparation of a product; the product may have at least one of the following functions: a1) to a3) diagnosing active tuberculosis; a2) diagnosing whether the subject is a patient with active tuberculosis; a3) preventing and controlling tuberculosis.

[0007] This invention also protects the use of substances and devices for detecting IFIT3 protein in the preparation of products; said products may have at least one of the following a1) to a3): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is a patient with active tuberculosis; a3) preventing and controlling tuberculosis;

[0008] The device may be device A and / or device C;

[0009] The device A includes a data input device 1, a data recording module 1, a data comparison module 1, and a conclusion output module 1;

[0010] Data input device 1 is used to input the expression level of IFIT3 protein;

[0011] Data recording module 1 is used to store the expression level values ​​of IFIT3 protein;

[0012] Data comparison module 1 is used to compare the expression level of IFIT3 protein in the peripheral blood of the test subject with the expression level of IFIT3 protein in the peripheral blood of the control.

[0013] The conclusion output module 1 is used to display the conclusion. If the expression level of IFIT3 protein in the peripheral blood of the test subject is higher than that in the peripheral blood of the control subject, the conclusion output module 1 will show that the test subject is an active tuberculosis patient; if the expression level of IFIT3 protein in the peripheral blood of the test subject is lower than that in the peripheral blood of the control subject, the conclusion output module 1 will show that the test subject is an inactive tuberculosis patient.

[0014] The device C includes a data input device 3, a data recording module 3, a data comparison module 3, and a conclusion output module 3;

[0015] Data input device 3 is used to input the concentration value of IFIT3 protein;

[0016] Data recording module 3 is used to store the concentration values ​​of IFIT3 protein;

[0017] Data comparison module 3 is used to compare the concentration of IFIT3 protein in the peripheral blood of the test subject with the concentration of IFIT3 protein in the peripheral blood of the control.

[0018] The conclusion output module 3 is used to display the conclusion. If the concentration of IFIT3 protein in the peripheral blood of the test subject is higher than that in the peripheral blood of the control subject, the conclusion output module 3 will show that the test subject is an active tuberculosis patient; if the concentration of IFIT3 protein in the peripheral blood of the test subject is lower than that in the peripheral blood of the control subject, the conclusion output module 3 will show that the test subject is an inactive tuberculosis patient.

[0019] The control peripheral blood can be peripheral blood from individuals with latent tuberculosis infection or healthy individuals.

[0020] The device A may specifically consist of a data input device 1, a data recording module 1, a data comparison module 1, and a conclusion output module 1.

[0021] The device C can specifically consist of a data input device 3, a data recording module 3, a data comparison module 3, and a conclusion output module 3.

[0022] The expression level of IFIT3 protein in peripheral blood can specifically be the expression level of IFIT3 protein in PBMCs isolated from peripheral blood, the expression level of IFIT3 protein in serum, or the expression level of IFIT protein in plasma.

[0023] The present invention also protects the use of a substance for detecting the IFIT3 gene in the preparation of a product; the product may have at least one of the following a1) to a3): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is a patient with active tuberculosis; a3) preventing and controlling tuberculosis.

[0024] This invention also protects the use of the substance and apparatus B for detecting the IFIT3 gene in the preparation of a product; the product may have at least one of the following a1) to a3): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is a patient with active tuberculosis; a3) preventing and controlling tuberculosis;

[0025] The device B includes a data input device 2, a data recording module 2, a data comparison module 2, and a conclusion output module 2;

[0026] Data input device 2 is used to input the expression level of the IFIT3 gene;

[0027] Data recording module 2 is used to store the expression level values ​​of the IFIT3 gene;

[0028] Data comparison module 2 is used to compare the expression level of IFIT3 gene in the peripheral blood of the test subject with the expression level of IFIT3 gene in the peripheral blood of the control.

[0029] The conclusion output module 2 is used to display the conclusion. If the expression level of the IFIT3 gene in the peripheral blood of the test subject is higher than that in the peripheral blood of the control subject, the conclusion output module 2 will show that the test subject is an active tuberculosis patient; if the expression level of the IFIT3 gene in the peripheral blood of the test subject is lower than that in the peripheral blood of the control subject, the conclusion output module 2 will show that the test subject is an inactive tuberculosis patient.

[0030] The control peripheral blood can be peripheral blood from individuals with latent tuberculosis infection or healthy individuals.

[0031] The device B may specifically consist of a data input device 2, a data recording module 2, a data comparison module 2, and a conclusion output module 2.

[0032] The expression level of the IFIT3 gene in peripheral blood can specifically refer to the expression level of the IFIT3 gene in PBMCs isolated from peripheral blood.

[0033] The present invention also protects a kit that may include substances for detecting IFIT3 protein and / or substances for detecting IFIT3 gene; the kit may have at least one of the following functions a1) to a3): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is a patient with active tuberculosis; a3) preventing and controlling tuberculosis.

[0034] The "substance for detecting IFIT3 protein" mentioned above may be a substance for detecting the expression level of IFIT3 protein and / or a substance for detecting the concentration of IFIT3 protein.

[0035] The "substance for detecting the IFIT3 gene" mentioned above can be a substance used to detect the expression level of the IFIT3 gene.

[0036] The expression level of any of the IFIT3 proteins mentioned above can be the relative expression level of the IFIT3 protein as a reference internal control protein.

[0037] The expression level of any of the IFIT3 genes mentioned above can be the relative expression level of the IFIT3 gene reference internal control gene.

[0038] The expression level of IFIT3 protein described above can be detected using a Western blotting experiment.

[0039] The above-described methods for detecting IFIT3 protein concentration can be used in ELISA experiments.

[0040] The substance described above for detecting the expression level of the IFIT3 gene or the substance described above for detecting the "relative expression level of the IFIT3 gene reference internal reference gene" may include a primer pair combination consisting of a specific primer pair and an internal reference primer pair.

[0041] The specific primer pair may consist of primer IFIT3-F and primer IFIT3-R; the target gene of the specific primer pair contains the DNA fragment shown in sequence 6 of the sequence listing from position 2035 to 2193 from the 5' end.

[0042] The internal reference primer pair may consist of primer F and primer R; the target gene of the internal reference primer pair may be all or part of the human internal reference gene.

[0043] The primer IFIT3-F can be either a1) or a2):

[0044] a1) The single-stranded DNA molecule shown in Sequence 1 of the sequence listing;

[0045] a2) A DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 1 and has the same function as sequence 1.

[0046] The primer IFIT3-R can be either a3) or a4):

[0047] a3) The single-stranded DNA molecule shown in sequence 2 of the sequence listing;

[0048] a4) A DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 2 and has the same function as sequence 2.

[0049] The primer F can be either b1) or b2):

[0050] b1) The single-stranded DNA molecule shown in sequence 3 of the sequence listing;

[0051] b2) A DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 3 and has the same function as sequence 3.

[0052] The primer R can be either b3) or b4):

[0053] b3) The single-stranded DNA molecule shown in sequence 4 of the sequence listing;

[0054] b4) A DNA molecule that has undergone substitution and / or deletion and / or addition of one or more nucleotides of sequence 4 and has the same function as sequence 4.

[0055] The substance described above for detecting the expression level of the IFIT3 gene or the substance described above for detecting the "relative expression level of the IFIT3 gene reference internal control gene" can specifically be the primer pair combination.

[0056] Any of the specific primer pairs described above also fall within the scope of protection of this invention.

[0057] Using any of the specific primer pairs described above to detect the expression level of the IFIT3 gene or to detect the relative expression level of the IFIT3 gene reference internal control gene also falls within the scope of protection of this invention.

[0058] Using any of the primer pairs described above to detect the expression level of the IFIT3 gene or to detect the relative expression level of the IFIT3 gene reference internal control gene is also within the scope of protection of this invention.

[0059] The method described above for detecting the relative expression level of the IFIT3 gene and the reference internal control gene in the cDNA of the test subject using any of the primer pairs described above can be as follows: using the cDNA of the test subject as a template, real-time quantitative PCR is performed using any of the specific primer pairs or the internal control primer pairs described above, and then 2-Δ... Ct The cDNA was obtained by calculation. The cDNA of the test subject can be the cDNA of PBMCs isolated from the peripheral blood of the test subject.

[0060] The internal reference protein mentioned above may be a GAPDH protein.

[0061] The internal reference gene mentioned above may be the GAPDH gene.

[0062] This invention also protects Y1), Y2), Y3), or Y4.

[0063] Y1) Application of IFIT3 protein as a biomarker in the development of reagents for diagnosing active tuberculosis.

[0064] Application of Y2)IFIT3 protein as a biomarker in the diagnosis of active tuberculosis.

[0065] Application of Y3)IFIT3 gene as a biomarker in the development of reagents for diagnosing active tuberculosis.

[0066] Application of Y4)IFIT3 gene as a biomarker in the diagnosis of active tuberculosis.

[0067] The amino acid sequence of any of the aforementioned IFIT3 proteins (GeneID: NP_001540.2) is shown in Sequence 5 of the sequence listing. The nucleotide sequence of any of the aforementioned IFIT3 genes (Genebank ID: NM_001549.6) is shown in Sequence 6 of the sequence listing.

[0068] In the above text, "lower than" can mean "lower than" in a statistical sense. "Higher than" can mean "higher than" in a statistical sense.

[0069] Experiments have shown that the expression level of the IFIT3 gene is significantly increased in PBMCs of patients with active tuberculosis compared to healthy individuals and those with latent tuberculosis infection. Therefore, the expression level of the IFIT3 gene has significant application value in the diagnosis of active tuberculosis. Attached Figure Description

[0070] Figure 1 To detect the relative expression level of the IFIT3 gene in PBMCs of patients with active tuberculosis, latent tuberculosis infected individuals, and healthy individuals using real-time quantitative PCR.

[0071] Figure 2 The relative expression level of the IFIT3 gene in PBMCs of patients with active tuberculosis and those with latent tuberculosis infection was analyzed using ROC curve analysis.

[0072] Figure 3 The relative expression levels of the IFIT3 gene in PBMCs of patients with active tuberculosis and healthy individuals were analyzed using ROC curve analysis. Detailed Implementation

[0073] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0074] Ficoll-Paque PLUS is a product of GE (Geological Institute of America). The 96-well plate is a product of Millipore. AIMV TM The serum-free medium used is a product of Gibco, catalog number 12055091. The RPMI 1640 medium is also a product of Gibco, catalog number 11875-093. The IFN-γ ELISPOT assay kit is a product of Dakota. The IFN-γ monoclonal capture antibody, IFN-γ detection antibody, Mycobacterium tuberculosis-specific mixed polypeptide A, Mycobacterium tuberculosis-specific mixed polypeptide B, and phytohemagglutinin are all components of the IFN-γ ELISPOT assay kit. TRIzol TM Reagent is a product of Invitrogen. PrimeScript TM The RT reagent kit with gDNA Eraser is a product of TaKaRa Biotechnology. KAPATM The rapid quantitative PCR kit is a product of Kapa Biosystems. The 2×GreenMaster Mix is ​​from Kapa. TM Components of a rapid quantitative PCR kit. Nuclease-free water is a product of Ambion, Inc. (USA). Light The 480Ⅱ real-time PCR instrument is a product of Roche.

[0075] Washing buffer: pH 7.4 PBS buffer containing 0.05% (v / v) Tween 20, 0.01M.

[0076] Example 1: Application of the relative expression level of the IFIT3 gene in the diagnosis of active tuberculosis.

[0077] I. Obtaining Peripheral Blood Specimens

[0078] 1. Differentiate peripheral blood samples from individuals with latent tuberculosis infection from those from healthy individuals.

[0079] Latent tuberculosis infected individuals and healthy individuals show no signs or symptoms of tuberculosis. The following steps can be used to differentiate them:

[0080] a. Envelope

[0081] (1) Take a 96-well plate, add 100 μL of IFN-γ monoclonal capture antibody to each well, and coat overnight at 4°C.

[0082] (2) After completing step (1), take the 96-well plate, discard the liquid phase, add pH 7.4, 0.01M PBS buffer and wash twice (1 min each time), then pat dry.

[0083] (3) After completing step (2), take the 96-well plate, add 200 μL of 0.01 M PBS buffer containing 2% (v / v) BSA at pH 7.4 to each well, and incubate at 37°C for 1 h.

[0084] (4) After completing step (3), take the 96-well plate, discard the liquid phase, and add RPMI 1640 culture medium to rinse once.

[0085] b. Preparation of PBMCs suspension

[0086] (1) Mix 2 mL of peripheral blood to be tested and 2 mL of RPMI 1640 culture medium evenly; then slowly add it to a sterile centrifuge tube containing 3 mL of Ficoll-Paque PLUS, centrifuge at room temperature and 2000 rcf for 20 min, and separate it into three layers from top to bottom.

[0087] (2) After completing step (1), aspirate the middle layer and transfer it to a centrifuge tube containing 10 mL of RPMI 1640 culture medium. Gently mix with a dropper and centrifuge at 1400 rpm for 7 min at room temperature.

[0088] (3) After completing step (2), take the centrifuge tube, discard the supernatant, add 6 mL of RPMI 1640 culture medium preheated to 37°C to resuspend, and centrifuge at room temperature and 1400 rpm for 7 min.

[0089] (4) After completing step (3), take the centrifuge tube, discard the supernatant, and add AIM V preheated to 37°C. TM Resuspended in serum-free medium to obtain a concentration of 2.5 × 10⁻⁶. 6 PBMCs suspension per mL.

[0090] c. Immunodot detection

[0091] Refer to the instructions for the IFN-γELISPOT assay kit for immunospot detection. All reagent dosages were performed according to the instructions. The specific steps are as follows:

[0092] (1) Take the 96-well plate obtained after step a, and add 100 μL of the PBMCs suspension prepared in step b (approximately 2.5 × 10⁻⁶) to each well. 5 (One PBMC).

[0093] (2) After completing step (1), add Mycobacterium tuberculosis-specific mixed polypeptide A or Mycobacterium tuberculosis-specific mixed polypeptide B to each detection well; add serum-free culture medium to each negative control well; add phytohemagglutinin to each positive control well.

[0094] (3) After completing step (2), place the 96-well plate in an incubator and incubate at 37°C and 5% CO2 for 20 hours.

[0095] (4) After completing step (3), take the 96-well plate, discard the supernatant, add 200 μL of pre-cooled ice water, and place at 4°C for 10 min (the purpose is to lyse the cells).

[0096] (5) After completing step (4), take the 96-well plate, discard the supernatant, wash it 5 times with washing solution (add 200 μL of washing solution each time, wash for 1 min each time), and pat dry.

[0097] (6) After completing step (5), take the 96-well plate and add 100 μL of IFN-γ detection antibody (avidin-labeled) dilution buffer (made by mixing 99 volumes of pH 7.4, 0.01M PBS buffer and 1 volume of IFN-γ detection antibody) to each well, and incubate at 37°C for 1 h.

[0098] (7) After completing step (6), take the 96-well plate, discard the supernatant, wash it 5 times with washing solution (add 200 μL of washing solution each time, wash for 1 min each time), and pat dry.

[0099] (8) After completing step (7), take the 96-well plate and add 100 μL of HRP-labeled streptomycin dilution (made by mixing 99 volumes of pH 7.4, 0.01M PBS buffer and 1 volume of HRP-labeled streptomycin) to each well, and incubate at 37°C for 1 h.

[0100] (9) After completing step (8), take the 96-well plate, discard the supernatant, wash it 5 times with washing solution (add 200 μL of washing solution each time, wash for 1 min each time), and pat dry.

[0101] (10) After completing step (9), take the 96-well plate, add enzyme substrate to each well, and develop color in the dark at room temperature for 15-45 minutes.

[0102] (11) After completing step (10), take the 96-well plate, rinse it three times with distilled water (to stop the reaction), and then let the 96-well plate stand at room temperature and air dry naturally.

[0103] (12) After completing step (11), take the 96-well plate and use an immunospot counter (Cellular Technology Ltd, USA) to perform image and spot counting, and then make the following judgments: If the number of spots in the negative control well is less than 6, and the difference between the number of spots in the test well and the number of spots in the negative control well is 6 or more, then the test well is positive; if the difference between the number of spots in the test well and the number of spots in the negative control well is less than 6, then the test well is negative. If the number of spots in the negative control well is 6 or more, and the number of spots in the test well is more than twice the number of spots in the negative control well, then the test well is positive; if the number of spots in the test well is less than twice the number of spots in the negative control well, then the test well is negative. If the test well is positive, the peripheral blood to be tested is provided by a person with latent tuberculosis infection (i.e., a peripheral blood sample from a person with latent tuberculosis infection); if the test well is negative, the peripheral blood to be tested is provided by a healthy person (i.e., a peripheral blood sample from a healthy person).

[0104] 2. Obtaining peripheral blood samples

[0105] (1) Active tuberculosis group: 86 peripheral blood samples.

[0106] 86 peripheral blood samples: 2-3 mL of peripheral blood was drawn from each of 86 patients clinically diagnosed with active pulmonary tuberculosis (all patients gave informed consent), placed in EDTA anticoagulant blood collection tubes, and inverted 5-6 times (to mix the anticoagulant and peripheral blood) to obtain 86 peripheral blood samples.

[0107] (2) Latent tuberculosis infection group: 25 peripheral blood samples.

[0108] 25 peripheral blood samples: 2-3 mL of peripheral blood was drawn from each of 25 patients clinically diagnosed with latent tuberculosis infection (all with informed consent). The blood was placed in a blood collection tube containing EDTA anticoagulant and inverted 5-6 times (to mix the anticoagulant and peripheral blood) to obtain 25 peripheral blood samples.

[0109] (3) Healthy control group: 46 peripheral blood samples.

[0110] 46 peripheral blood samples: 2-3 mL of peripheral blood was drawn from 46 healthy individuals (all with informed consent), placed in EDTA anticoagulant blood collection tubes, and inverted 5-6 times (to mix the anticoagulant and peripheral blood) to obtain 46 peripheral blood samples.

[0111] All 157 peripheral blood samples must be kept at room temperature (do not freeze or refrigerate) for less than 6 hours.

[0112] II. Application of relative expression levels of the IFIT3 gene in the diagnosis of active tuberculosis

[0113] The amino acid sequence of the IFIT3 protein (GeneID: NP_001540.2) is shown in Sequence 5 of the sequence listing. The nucleotide sequence of the gene encoding the IFIT3 protein (abbreviated as IFIT3 gene, Genebank number: NM_001549.6) is shown in Sequence 6 of the sequence listing.

[0114] 1. Obtaining cDNA from 157 peripheral blood samples

[0115] (1) Preparation of PBMCs suspension

[0116] Replace the peripheral blood samples to be tested in step 1b with the 157 peripheral blood samples in step 12, while keeping all other steps unchanged, to obtain a suspension of PBMCs from 157 peripheral blood samples.

[0117] (2) RNA extraction

[0118] ① Take PBMC suspensions from 157 peripheral blood samples (approximately 1×10⁻⁶ per sample). 6 Centrifuge at 400 rcf for 5 min (cells), and collect the precipitate.

[0119] ② After completing step ①, take the precipitate and use TRIzol... TM Reagent extracts RNA.

[0120] (3) cDNA synthesis

[0121] RNA was collected from PBMC suspensions of 157 peripheral blood samples and analyzed using PrimeScript. TM Reverse transcription was performed using an RT reagent kit with a gDNA eraser to obtain cDNA from 157 peripheral blood samples.

[0122] 2. Preparation of primer pair combinations

[0123] Based on the nucleotide sequence of the IFIT3 gene, the specific primer pairs shown in Table 1 were designed and synthesized.

[0124] Based on the nucleotide sequence of the GAPDH gene, the internal reference primer pairs shown in Table 1 were designed and synthesized.

[0125] Primer pair combinations consist of specific primer pairs and internal reference primer pairs.

[0126] The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. (and purified by HPLC).

[0127] Table 1

[0128]

[0129] 3. Real-time quantitative PCR detection of the relative expression level of the IFIT3 gene

[0130] Using cDNA from 157 peripheral blood samples as templates, real-time quantitative PCR was performed using the specific primer pairs or internal control primer pairs prepared in step 2 to obtain the relative expression levels of the IFIT3 gene in each template. The specific steps are as follows:

[0131] (1) Prepare reaction system 1 and reaction system 2

[0132] Reaction system 1 is 20 μL, consisting of 10 μL 2× Green Master Mix, 0.4 μL IFIT3-F aqueous solution (concentration 10 μM), 0.4 μL IFIT3-R aqueous solution (concentration 10 μM), 2 μL template (5-20 ng), and 7.2 μL nuclease-free water.

[0133] Reaction system 2 was 20 μL, consisting of 10 μL 2× Green Master Mix, 0.4 μL GAPDH-F aqueous solution (concentration 10 μM), 0.4 μL GAPDH-R aqueous solution (concentration 10 μM), 2 μL template (5-20 ng), and 7.2 μL nuclease-free water.

[0134] (2) Real-time quantitative PCR detection

[0135] The reaction systems prepared in step (1) were placed in a Light source. Real-time quantitative PCR was performed using a 480Ⅱ real-time PCR instrument. A 2-Δ... Ct The relative expression level of the IFIT3 gene in each template was calculated.

[0136] Reaction conditions: 95℃ pre-denaturation for 3 min; 95℃ for 5 s, 60℃ for 30 sec, 40 cycles, fluorescence signal collected during the extension phase.

[0137] The experimental results are shown in Figure 1 (TB represents the active tuberculosis group, LI represents the latent tuberculosis infection group, and Nor represents the healthy control group). The results showed that the relative expression level of the IFIT3 gene in PBMCs of the active tuberculosis group was significantly increased compared with the healthy control group and the latent tuberculosis infection group.

[0138] (3) Statistical analysis

[0139] The results of step (2) were statistically analyzed using GraphPad Prism 5.

[0140] Receiver operating characteristic (ROC) curves were performed using GraphPad Prism 5 based on the relative expression levels of the IFIT3 gene in PBMCs from the active tuberculosis group and the latent tuberculosis infection group. Results are shown below. Figure 2 The results showed that the area under the ROC curve was 0.918 (p<0.0001), indicating that the relative expression level of the IFIT3 gene can be used to distinguish between patients with active tuberculosis and those with latent tuberculosis infection. The Youden's index (YI) was highest when the transcriptional cut-off value of the IFIT3 gene was 0.02157, at which point the sensitivity was 91.86% and the specificity was 84.00%.

[0141] Receiver operating characteristic (ROC) curve analysis was performed using GraphPad Prism 5 based on the relative expression levels of the IFIT3 gene in PBMCs from the active tuberculosis group and the healthy control group. Results are shown below. Figure 3 The results showed that the area under the ROC curve was 0.934 (p<0.0001), indicating that the relative expression level of the IFIT3 gene can be used to distinguish between patients with active tuberculosis and healthy individuals. The Youden index was highest when the transcriptional cut-off value of the IFIT3 gene was 0.02465, at which point the sensitivity was 89.53% and the specificity was 84.78%.

[0142] The above results indicate that the relative expression level of the IFIT3 gene has important application value in the diagnosis of active tuberculosis. <110> The Eighth Medical Center of the General Hospital of the Chinese People's Liberation Army <120> A kit for diagnosing active tuberculosis <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 21 <212> DNA <213> Artificial sequence <400> 1 tacagttaca tagtccgaag g 21 <210> 2 <211> 18 <212> DNA <213> Artificial sequence <400> 2 tgttaggcag tcaccatt 18 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <400> 3 tgttgccatc aatgacccct 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <400> 4 tcgccccact tgattttgga 20 <210> 5 <211> 490 <212> PRT <213> Artificial sequence <400> 5 Met Ser Glu Val Thr Lys Asn Ser Leu Glu Lys Ile Leu Pro Gln Leu 1 5 10 15 Lys Cys His Phe Thr Trp Asn Leu Phe Lys Glu Asp Ser Val Ser Arg 20 25 30 Asp Leu Glu Asp Arg Val Cys Asn Gln Ile Glu Phe Leu Asn Thr Glu 35 40 45 Phe Lys Ala Thr Met Tyr Asn Leu Leu Ala Tyr Ile Lys His Leu Asp 50 55 60 Gly Asn Asn Glu Ala Ala Leu Glu Cys Leu Arg Gln Ala Glu Glu Leu 65 70 75 80 Ile Gln Gln Glu His Ala Asp Gln Ala Glu Ile Arg Ser Leu Val Thr 85 90 95 Trp Gly Asn Tyr Ala Trp Val Tyr Tyr His Leu Gly Arg Leu Ser Asp 100 105 110 Ala Gln Ile Tyr Val Asp Lys Val Lys Gln Thr Cys Lys Lys Phe Ser 115 120 125 Asn Pro Tyr Ser Ile Glu Tyr Ser Glu Leu Asp Cys Glu Glu Gly Trp 130 135 140 Thr Gln Leu Lys Cys Gly Arg Asn Glu Arg Ala Lys Val Cys Phe Glu 145 150 155 160 Lys Ala Leu Glu Glu Lys Pro Asn Asn Pro Glu Phe Ser Ser Gly Leu 165 170 175 Ala Ile Ala Met Tyr His Leu Asp Asn His Pro Glu Lys Gln Phe Ser 180 185 190 Thr Asp Val Leu Lys Gln Ala Ile Glu Leu Ser Pro Asp Asn Gln Tyr 195 200 205 Val Lys Val Leu Leu Gly Leu Lys Leu Gln Lys Met Asn Lys Glu Ala 210 215 220 Glu Gly Glu Gln Phe Val Glu Glu Ala Leu Glu Lys Ser Pro Cys Gln 225 230 235 240 Thr Asp Val Leu Arg Ser Ala Ala Lys Phe Tyr Arg Arg Lys Gly Asp 245 250 255 Leu Asp Lys Ala Ile Glu Leu Phe Gln Arg Val Leu Glu Ser Thr Pro 260 265 270 Asn Asn Gly Tyr Leu Tyr His Gln Ile Gly Cys Cys Tyr Lys Ala Lys 275 280 285 Val Arg Gln Met Gln Asn Thr Gly Glu Ser Glu Ala Ser Gly Asn Lys 290 295 300 Glu Met Ile Glu Ala Leu Lys Gln Tyr Ala Met Asp Tyr Ser Asn Lys 305 310 315 320 Ala Leu Glu Lys Gly Leu Asn Pro Leu Asn Ala Tyr Ser Asp Leu Ala 325 330 335 Glu Phe Leu Glu Thr Glu Cys Tyr Gln Thr Pro Phe Asn Lys Glu Val 340 345 350 Pro Asp Ala Glu Lys Gln Gln Ser His Gln Arg Tyr Cys Asn Leu Gln 355 360 365 Lys Tyr Asn Gly Lys Ser Glu Asp Thr Ala Val Gln His Gly Leu Glu 370 375 380 Gly Leu Ser Ile Ser Lys Lys Ser Thr Asp Lys Glu Glu Ile Lys Asp 385 390 395 400 Gln Pro Gln Asn Val Ser Glu Asn Leu Leu Pro Gln Asn Ala Pro Asn 405 410 415 Tyr Trp Tyr Leu Gln Gly Leu Ile His Lys Gln Asn Gly Asp Leu Leu 420 425 430 Gln Ala Ala Lys Cys Tyr Glu Lys Glu Leu Gly Arg Leu Leu Arg Asp 435 440 445 Ala Pro Ser Gly Ile Gly Ser Ile Phe Leu Ser Ala Ser Glu Leu Glu 450 455 460 Asp Gly Ser Glu Glu Met Gly Gln Gly Ala Val Ser Ser Ser Pro Arg 465 470 475 480 Glu Leu Leu Ser Asn Ser Glu Gln Leu Asn 485 490 <210> 6 <211> 2390 <212> DNA <213> Artificial sequence <400> 6 gcacacagga agacttctga agaacaaatc agcctggtca ccagcttttc ggaacagcag 60 agacacagag ggcagtcatg agtgaggtca ccaagaattc cctggagaaa atccttccac 120 agctgaaatg ccatttcacc tggaacttat tcaaggaaga cagtgtctca agggatctag 180 aagatagagt gtgtaaccag attgaatttt taaacactga gttcaaagct acaatgtaca 240 acttgttggc ctacataaaa cacctagatg gtaacaacga ggcagccctg gaatgcttac 300 ggcaagctga agagttaatc cagcaagaac atgctgacca agcagaaatc agaagtctag 360 tcacttgggg aaactacgcc tgggtctact atcacttggg cagactctca gatgctcaga 420 tttatgtaga taaggtgaaa caaacctgca agaaattttc aaatccatac agtattgagt 480 attctgaact tgactgtgag gaagggtgga cacaactgaa gtgtggaaga aatgaaaggg 540 cgaaggtgtg ttttgagaag gctctggaag aaaagcccaa caacccagaa ttctcctctg 600 gactggcaat tgcgatgtac catctggata atcacccaga gaaacagttc tctactgatg 660 ttttgaagca ggccattgag ctgagtcctg ataaccaata cgtcaaggtt ctcttgggcc 720 tgaaactgca gaagatgaat aaagaagctg aaggagagca gtttgttgaa gaagccttgg 780 aaaagtctcc ttgccaaaca gatgtcctcc gcagtgcagc caaattttac agaagaaag 840 gtgacctaga caaagctatt gaactgtttc aacgggtgtt ggaatccaca ccaaacaatg 900 gctacctcta tcaccagatt gggtgctgct acaaggcaaa agtaagacaa atgcagaata 960 1020 tggactattc gaataaagct cttgagaagg gactgaatcc tctgaatgca tactccgatc 1080 1140 ctgaaaagca acaatcccat cagcgctact gcaaccttca gaataat gggaagtctg 1200 aagacactgc tgtgcaacat ggtttagagg gtttgtccat aagcaaaaaa tcaactgaca 1260 aggagagat caaagaccaa ccacagaatg tatctgaaaa tctgcttcca caaaatgcac 1320 1380 ccaaatgtta tgagaaggaa ctgggccgcc tgctaaggga tgccccttca ggcataggca 1440 gtattttcct gtcagcatct gagcttgagg atggtagtga ggaaatgggc cagggcgcag 1500 tcagctccag tcccagagag ctcctctcta actcagagca actgaactga gacagaggag 1560 gaaaacagag catcagaagc ctgcagtggt ggttgtgacg ggtaggacga taggaagaca 1620 gggggcccca acctgggatt gctgagcagg gaagctttgc atgttgctct aaggtacatt 1680 tttaaagagt tgttttttgg ccgggcgcag tggctcatgc ctgtaatccc agcactttgg 1740 gaggccgagg tgggcggatc acgaggtctg gagtttgaga ccatcctggc taacacagtg 1800 aaatcccgtc tctactaaaa atacaaaaaa ttagccaggc gtggtggctg gcacctgtag 1860 tcccagctac ttgggaggct gaggcaggag aatggcgtga acctggaagg aagaggttgc 1920 agtgagccaa gattgcgccc ctgcactcca gcctgggcaa cagagcaaga ctccatctca 1980 aaaaaaaaaa aaaaaaaaa aaagagttgt tttctcatgt tcattatagt tcattacagt 2040 tacatagtcc gaaggtctta caactaatca ctggtagcaa taaatgcttc aggcccacat 2100 gatgctgatt agttctcagt tttcattcag ttcacaatat aaccaccatt cctgccctcc 2160 ctgccaaggg tcataaatgg tgactgccta acaaaaaat ttgcagtctc atctcatttt 2220 catccagact tctggaactc aaagattaac ttttgactaa ccctggaata tctcttatct 2280 cacttatagc ttcaggcatg tatttatatg tattcttgat agcaatacca taatcaatgt 2340 gtattcctga tagtaatgct acaataaatc caaacatttc aactctgtta 2390

Claims

1. Application of substances for detecting IFIT3 protein in the preparation of products; The IFIT3 protein alone is used as a marker; The functions of the product are at least one of the following a1) to a2): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is an active tuberculosis patient.

2. Use of a substance and device for detecting IFIT3 protein in preparing a product; the IFIT3 protein is used alone as a marker; The functions of the product are at least one of the following a1) to a2): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is an active tuberculosis patient; the device is device A and / or device C; The device A comprises a data input device 1, a data recording module 1, a data comparison module 1 and a conclusion output module 1; The data input device 1 is used to input the expression value of IFIT3 protein; The data recording module 1 is used to store the expression value of IFIT3 protein; The data comparison module 1 is used to compare the expression level of IFIT3 protein in the peripheral blood of the test subject with the expression level of IFIT3 protein in the peripheral blood of the control; The conclusion output module 1 is used to display the conclusion, that is, if the expression level of IFIT3 protein in the peripheral blood of the subject is higher than the expression level of IFIT3 protein in the peripheral blood of the control, the conclusion output module 1 displays that the subject is an active tuberculosis patient; if the expression level of IFIT3 protein in the peripheral blood of the subject is lower than the expression level of IFIT3 protein in the peripheral blood of the control, the conclusion output module 1 displays that the subject is an inactive tuberculosis patient; The device C includes a data input device 3, a data recording module 3, a data comparison module 3 and a conclusion output module 3; The data input device 3 is used to input the concentration value of IFIT3 protein; The data recording module 3 is used to store the concentration value of the IFIT3 protein; The data comparison module 3 is used to compare the concentration of IFIT3 protein in the peripheral blood of the test subject with the concentration of IFIT3 protein in the peripheral blood of the control; The conclusion output module 3 is used to display the conclusion, that is, if the concentration of IFIT3 protein in the peripheral blood of the subject to be tested is higher than the concentration of IFIT3 protein in the peripheral blood of the control, the conclusion output module 3 displays that the subject to be tested is an active tuberculosis patient; if the concentration of IFIT3 protein in the peripheral blood of the subject to be tested is lower than the concentration of IFIT3 protein in the peripheral blood of the control, the conclusion output module 3 displays that the subject to be tested is an inactive tuberculosis patient; The control peripheral blood is peripheral blood from a latent tuberculosis infected person or a healthy person.

3. The use according to claim 1 or 2, characterized in that: The substance for detecting IFIT3 protein is a substance for detecting the expression amount of IFIT3 protein and / or a substance for detecting the concentration of IFIT3 protein.

4. For detection iFit3 The use of genetic material in the preparation of products; iFit3 Genes alone serve as markers; The functions of the product are at least one of the following a1) to a2): a1) diagnosing active tuberculosis; a2) diagnosing whether the subject is an active tuberculosis patient.

5. Used for detection iFit3 Use of genetic material and device B in the preparation of products; Said iFit3 Genes alone serve as markers; The functions of the product are at least one of the following a1) to a2): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; The device B comprises a data input device 2, a data recording module 2, a data comparison module 2 and a conclusion output module 2; Data input device 2 is used to input iFit3 Gene expression value; Data recording module 2 is used to store iFit3 Gene expression value; The data comparison module 2 is used to compare the peripheral blood iFit3 Gene expression levels and control peripheral blood iFit3 The expression levels of genes were compared; The conclusion output module 2 is used to display the conclusion, that is, if the peripheral blood of the subject is iFit3 The gene expression level was higher than that in the control peripheral blood iFit3 If the expression level of the gene is higher than that of the test subject, the conclusion output module 2 shows that the test subject is an active tuberculosis patient; iFit3 The gene expression level was lower than that in the control peripheral blood iFit3 If the expression level of the gene is higher than that of the test subject, the conclusion output module 2 shows that the test subject is an inactive tuberculosis patient; The control peripheral blood is peripheral blood from a latent tuberculosis infected person or a healthy person.

6. The use according to claim 4 or 5, characterized in that: The detection iFit3 Genetic material for detection iFit3 A substance that increases the amount of gene expression.

7. Application of IFIT3 protein alone as a marker in the development of reagents for diagnosing active tuberculosis.

8. iFit3 Use of genes alone as markers in the development of diagnostic reagents for active tuberculosis.

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