CN112114146A

CN112114146A - Kit for diagnosing active tuberculosis

Info

Publication number: CN112114146A
Application number: CN201910531571.0A
Authority: CN
Inventors: 程小星; 杨秉芬; 安红娟; 翟斐; 蒋静; 王心静; 曹志红; 刘艳华; 王若
Original assignee: 8th Medical Center of PLA General Hospital
Current assignee: 8th Medical Center of PLA General Hospital
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2020-12-22

Abstract

The invention discloses a kit for diagnosing active tuberculosis. The kit comprises a substance for detecting the IFIT3 protein. Compared with healthy people and tuberculosis latent infected people, the expression level of the IFIT3 gene in the PBMCs of the active tuberculosis patients is obviously increased; the expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients and tuberculosis latent infection patients; the expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients from healthy people. Therefore, the IFIT3 protein and/or IFIT3 gene can be used as a marker for diagnosing active tuberculosis. The invention has great application value.

Description

Kit for diagnosing active tuberculosis

Technical Field

The invention belongs to the technical field of medical immunology diagnosis, and particularly relates to a kit for diagnosing active tuberculosis.

Background

Tuberculosis is an infectious disease caused by Mycobacterium Tuberculosis (MTB), which is transmitted mainly through the respiratory tract. After MTB infects human bodies, three different results can appear, firstly, the immunity of the organisms is better, and MTB is directly eliminated; secondly, MTB is immunosuppressed by the body but cannot be completely eliminated, and is developed into latent tuberculosis infection (LTBI); MTB proliferates rapidly in the body and develops into active tuberculosis. Tuberculosis is a serious infectious disease which needs to be mainly prevented and controlled in China.

At present, methods such as imaging diagnosis, tubercle bacillus diagnosis, immunological diagnosis and the like are mainly used for tuberculosis diagnosis, but all methods have certain defects. Imaging diagnosis makes it difficult to distinguish between tuberculosis and other pulmonary diseases. The false negative of tubercle bacillus diagnosis is high. Immunological diagnostics are mainly divided into antibody detection and cellular immunodetection (e.g., Tuberculin Skin Test (TST) and interferon gamma release test (IGRA)). Both TST and IGRA assess tuberculosis infection by detecting the main anti-tuberculosis immunity, i.e., cellular immune response. TST is used as a main detection means at present in China, and a person with strong positive or short-term conversion from negative to positive of Pure Protein Derivative (PPD) and no clinical tuberculosis evidence is generally judged as a tubercle bacillus latent infected person. TST is characterized by simple operation and low price, so that TST becomes the most common and most convenient tubercle bacillus infection diagnosis method in clinic at present. PPD is an antigen mixture which is crude extracted from mycobacterium tuberculosis and contains more than 200 proteins, and many of the PPD are common antigen components of nontuberculous mycobacteria and bacille calmette-guerin, so that the specificity of TST detection is determined to be poor, and false positive results are easily generated in bacille calmette-guerin (BCG) inoculated people and nontuberculous mycobacteria infected people. TST can only be diagnosed by the aid of the strength of skin reaction, and the sensitivity of TST is only 70-80%. In addition, TST has the defects of time-consuming detection, requirement of return visit of a subject (72h), subjective dependence of skin test operation and result explanation, and the like. IGRA is used for diagnosing tubercle bacillus infection by quantitatively detecting IFN-gamma detection release reaction of whole blood or peripheral blood mononuclear cells of a detected person to mycobacterium tuberculosis specific antigens (ESAT6, CFP10 and TB7.7) by adopting an enzyme-linked immunosorbent assay (ELISA) or an enzyme-linked immunospot (ELISPOT) method, but IGRA is difficult to distinguish active tuberculosis infection from latent tuberculosis infection. The early diagnosis of active tuberculosis can not be realized, so that on one hand, the condition is delayed, and the treatment cost and the death rate are increased; on the other hand, the infection source cannot be effectively controlled, and tuberculosis is caused to spread. Therefore, the development of specific and effective active tuberculosis diagnostic reagents has important significance for preventing and treating tuberculosis.

The IFIT3 protein (Interferon induced protein with tetratricopeptide repeats 3) is an Interferon induced protein discovered earlier, can play an antiviral role by directly inhibiting viral translation or promoting viral RNA degradation, and also participates in intracellular signal transduction regulation and induces the generation of type I Interferon, thereby enhancing the antiviral effect of cells. The IFIT3 protein can be used as a target for predicting the therapeutic effect in the treatment of liver cancer by interferon, and the IFIT3 protein is not available for diagnosing active tuberculosis at present.

Disclosure of Invention

The object of the present invention is to diagnose active tuberculosis.

The invention firstly protects the application of a substance for detecting IFIT3 protein in preparing products; the function of the product can be at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

The invention also protects the application of the substance and the device for detecting the IFIT3 protein in the preparation of products; the function of the product can be at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis;

the device can be a device A and/or a device C;

the device A comprises data input equipment 1, a data recording module 1, a data comparison module 1 and a conclusion output module 1;

the data input device 1 is used for inputting an expression quantity value of the IFIT3 protein;

the data recording module 1 is used for storing the expression quantity value of the IFIT3 protein;

the data comparison module 1 is used for comparing the expression level of the IFIT3 protein in the peripheral blood of the person to be tested with the expression level of the IFIT3 protein in the control peripheral blood;

the conclusion output module 1 is used for displaying a conclusion, namely if the expression level of the IFIT3 protein in the peripheral blood of the testee is higher than the expression level of the IFIT3 protein in the control peripheral blood, the conclusion output module 1 displays that the testee is an active tuberculosis patient; if the expression level of the IFIT3 protein in the peripheral blood of the person to be tested is lower than that of the IFIT3 protein in the peripheral blood of a control, the conclusion output module 1 displays that the person to be tested is the inactive tuberculosis patient;

the device C comprises 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 for inputting the concentration value of the IFIT3 protein;

the data recording module 3 is used for storing the concentration value of the IFIT3 protein;

the data comparison module 3 is used for comparing the concentration of the IFIT3 protein in the peripheral blood of the person to be tested with the concentration of the IFIT3 protein in the control peripheral blood;

the conclusion output module 3 is used for displaying a conclusion, namely if the concentration of the IFIT3 protein in the peripheral blood of the testee is higher than that of the IFIT3 protein in the control peripheral blood, the conclusion output module 3 displays that the testee is an active tuberculosis patient; if the concentration of the IFIT3 protein in the peripheral blood of the testee is lower than that of the IFIT3 protein in the peripheral blood of the control, the conclusion output module 3 displays that the testee is an inactive tuberculosis patient;

the control peripheral blood can be peripheral blood of a person with latent tuberculosis infection or a healthy person.

The device A can be composed of a data input device 1, a data recording module 1, a data comparison module 1 and a conclusion output module 1.

The device can be composed of a data input device 3, a data recording module 3, a data comparison module 3 and a conclusion output module 3.

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

The invention also protects the application of the substance for detecting the IFIT3 gene in preparing products; the function of the product can be at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

The invention also protects the application of the substance and the device B for detecting the IFIT3 gene in the preparation of products; the function of the product can be at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis;

the device B comprises data input equipment 2, a data recording module 2, a data comparison module 2 and a conclusion output module 2;

the data input device 2 is used for inputting the expression quantity value of the IFIT3 gene;

the data recording module 2 is used for storing the expression quantity value of the IFIT3 gene;

the data comparison module 2 is used for comparing the expression level of the IFIT3 gene in the peripheral blood of the person to be tested with the expression level of the IFIT3 gene in the control peripheral blood;

the conclusion output module 2 is used for displaying a conclusion, namely if the expression quantity of the IFIT3 gene in the peripheral blood of the person to be tested is higher than the expression quantity of the IFIT3 gene in the control peripheral blood, the person to be tested is an active tuberculosis patient through the conclusion output module 2; if the expression level of the IFIT3 gene in the peripheral blood of the person to be tested is lower than that of the IFIT3 gene in the control peripheral blood, the conclusion output module 2 displays that the person to be tested is the inactive tuberculosis patient;

The device B can be composed of a data input device 2, a data recording module 2, a data comparison module 2 and a conclusion output module 2.

The expression level of the IFIT3 gene in the peripheral blood can be specifically the expression level of the IFIT3 gene in PBMCs separated from the peripheral blood.

The invention also protects a kit which can comprise a substance for detecting the IFIT3 protein and/or a substance for detecting the IFIT3 gene; the kit can have at least one of the following functions from a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

Any of the above-mentioned "substances for detecting IFIT3 protein" may be a substance for detecting the expression level of IFIT3 protein and/or a substance for detecting the concentration of IFIT3 protein.

Any of the above-mentioned "substances for detecting the IFIT3 gene" may be a substance for detecting the expression level of the IFIT3 gene.

The expression level of any of the IFIT3 proteins can be the relative expression level of the IFIT3 protein and a reference internal reference protein.

The expression level of any one of the IFIT3 genes can be the relative expression level of the IFIT3 gene reference internal reference gene.

Any one of the above methods for detecting the expression level of the IFIT3 protein can be specifically carried out by using a Western Blot experiment.

Any of the above-described assays for detecting the concentration of IFIT3 protein may be specifically used in an Elisa assay.

The substance for detecting the expression level of the IFIT3 gene or the substance for detecting the relative expression level of the IFIT3 gene reference internal reference gene may comprise a primer pair combination consisting of a specific primer pair and an internal reference primer pair;

the specific primer pair can consist of a primer IFIT3-F and a primer IFIT 3-R; the target gene of the specific primer pair contains a DNA fragment shown by 2035-2193 th site from 5' end of a sequence 6 in a sequence table;

the internal reference primer pair can consist of a primer F and a primer R; the target gene of the internal reference primer pair can be all or part of the human internal reference gene.

The primer IFIT3-F may be a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in sequence 1 of the sequence table;

a2) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.

The primer IFIT3-R can be a3) or a4) as follows:

a3) a single-stranded DNA molecule shown in a sequence 2 of a sequence table;

a4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 2 and has the same function as the sequence 2.

The primer F can be b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in sequence 3 of the sequence table;

b2) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 3 and has the same function as the sequence 3.

The primer R can be b3) or b4) as follows:

b3) a single-stranded DNA molecule shown in a sequence 4 of the sequence table;

b4) and (b) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 4 and has the same function as the sequence 4.

The substance for detecting the expression level of the IFIT3 gene or the substance for detecting the relative expression level of the "IFIT 3 gene-referenced reference gene" may be any of the above primer pair combinations.

Any specific primer pair described above also belongs to the protection scope of the invention.

The specific primer pair is used for detecting the expression level of the IFIT3 gene or detecting the relative expression level of the IFIT3 gene reference internal reference gene, and the protection scope of the invention is also included.

The primer pair combination is used for detecting the expression quantity of the IFIT3 gene or detecting the relative expression quantity of the IFIT3 gene reference internal reference gene, and the invention also belongs to the protection scope of the invention.

In the above, the method for detecting the relative expression amount of the IFIT3 gene reference internal reference gene in the cDNA of the subject by using any one of the primer pair combinations may specifically be: taking cDNA of a to-be-detected person as a template, carrying out real-time fluorescence quantitative PCR by adopting any one of the specific primer pairs or any one of the internal reference primer pairs, and then using 2-delta^CtAnd (4) calculating. The cDNA of the testee can be cDNA of PBMCs separated from peripheral blood of the testee.

Any of the above-described reference proteins can be a GAPDH protein.

Any of the above-described reference genes may be a GAPDH gene.

The invention also protects Y1) or Y2) or Y3) or Y4).

Y1) IFIT3 protein as a marker in the development of reagents for diagnosing active tuberculosis.

Y2) IFIT3 protein as a marker for diagnosing active tuberculosis.

Y3) IFIT3 gene as a marker in the development of a reagent for diagnosing active tuberculosis.

Y4) IFIT3 gene as a marker in diagnosing active tuberculosis.

The amino acid sequence of any of the IFIT3 proteins (GeneID No.: NP-001540.2) is shown as the sequence 5 in the sequence table. The nucleotide sequence of any of the IFIT3 genes (Genebank number: NM-001549.6) is shown as a sequence 6 in a sequence table.

Above, the lower may be statistically lower. The above may be statistically above.

Experiments prove that compared with healthy people and latent tuberculosis infected people, the expression level of the IFIT3 gene in the PBMCs of the active tuberculosis patients is obviously increased; the expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients and tuberculosis latent infection patients; the expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients from healthy people. Therefore, the expression level of the IFIT3 gene has important application value in diagnosing active tuberculosis.

Drawings

FIG. 1 shows the relative expression of IFIT3 gene in PBMCs of patients with active tuberculosis, latent tuberculosis infected patients and healthy people detected by real-time fluorescence quantitative PCR.

FIG. 2 is a diagram showing the relative expression level of IFIT3 gene in PBMCs of patients with active tuberculosis and those with latent tuberculosis infection analyzed by ROC curve.

FIG. 3 is a diagram showing the relative expression level of IFIT3 gene in PBMCs of active tuberculosis patients and healthy persons analyzed by ROC curve.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Ficoll-Paque PLUS is a product of GE corporation, USA. The 96-well plate is a product of Millipore corporation. AIM V^TMMedium serum-free Medium is a product of the Gibco company under product catalog number 12055091. RPMI 1640 medium was Gibco's product, catalog number 11875-093. The IFN-gamma ELISPOT detection kit is a product of Dake corporation. The IFN-gamma monoclonal capture antibody, the IFN-gamma detection antibody, the tubercle bacillus specific mixed polypeptide A, the tubercle bacillus specific mixed polypeptide B and the phytohemagglutinin are all components in an IFN-gamma ELISPOT detection kit. TRIzol^TMReagent is a product of Invitrogen corporation. PrimeScript^TMRT reagent Kit with gDNA Eraser is a product of TaKaRa Biotechnology. KAPA^TM

The rapid quantitative PCR kit is a product of Kapa Biosystems company. 2 × Green Master Mix is KAPA^TM

Components in the rapid quantitative PCR kit. Nuclease-free water is a product of Ambion, USA. Light

The 480 II fluorescent quantitative PCR instrument is a product of Roche.

Washing liquid: 0.05% (v/v) Tween 20 in PBS buffer, pH7.4, 0.01M.

Example 1 application of relative expression level of IFIT3 Gene in diagnosing active tuberculosis

First, obtaining peripheral blood specimen

1. Differentiating peripheral blood specimen of tuberculosis latent infected person from peripheral blood specimen of healthy person

The latent tuberculosis infected person and the healthy person have no signs or symptoms of tuberculosis, and are distinguished according to the following steps:

a. coating quilt

(1) A96-well plate was taken, 100. mu.L of IFN-. gamma.monoclonal capture antibody was added to each well, and the plate was coated overnight at 4 ℃.

(2) After the completion of step (1), the 96-well plate was taken out, the liquid phase was discarded, and the plate was washed twice (1 min each) with 0.01M PBS buffer solution (pH7.4), and patted dry.

(3) After completion of step (2), the 96-well plate was taken, 200. mu.L of PBS buffer pH7.4 containing 2% (v/v) BSA and 0.01M was added to each well, and incubated at 37 ℃ for 1 hour.

(4) And (4) after the step (3) is finished, taking the 96-well plate, discarding the liquid phase, and adding RPMI 1640 culture solution for rinsing once.

b. Preparation of PBMCs suspension

(1) Uniformly mixing 2mL of peripheral blood to be detected and 2mL of RPMI 1640 culture solution; then slowly adding into a sterile centrifuge tube filled with 3mL of Ficoll-Paque PLUS, centrifuging at room temperature and 2000rcf for 20min, and separating into three layers from top to bottom.

(2) After the completion of step (1), the middle layer was aspirated and transferred to a centrifuge tube containing 10mL of RPMI 1640 medium, gently pipetted and mixed, and centrifuged at 1400rpm for 7min at room temperature.

(3) After the step (2) is completed, the centrifuge tube is taken, the supernatant is discarded, 6mL of RPMI 1640 culture solution preheated to 37 ℃ is added for resuspension, and the mixture is centrifuged at 1400rpm for 7min at room temperature.

(4) After the step (3) is finished, taking the centrifuge tube, discarding the supernatant, and adding AIM V preheated to 37 DEG C^TMResuspending Medium in serum-free Medium to obtain a concentration of 2.5X 10⁶one/mL of PBMCs suspension.

c. Immunospot detection

The kit is adopted to carry out the immune spot detection by referring to the instruction of the IFN-gamma ELISPOT detection kit. The dosage of the reagent is carried out according to the instruction. The method comprises the following specific steps:

(1) taking the 96-well plate to complete step a, adding 100. mu.L of the PBMCs suspension prepared in step b (about 2.5X 10) per well⁵Individual PBMCs).

(2) After the step (1) is completed, adding the tubercle bacillus specific mixed polypeptide A or tubercle bacillus specific mixed polypeptide B into each detection hole; adding serum-free culture medium into each negative control hole; phytohemagglutinin was added to each positive control well.

(3) After the step (2) is completed, the 96-well plate is placed in an incubator at 37 ℃ and 5% CO₂Culturing for 20 h.

(4) After the step (3) is completed, the 96-well plate is taken out, the supernatant is discarded, 200 mu L of precooled ice water is added, and the mixture is placed at 4 ℃ for 10min (the aim is to crack cells).

(5) After the step (4) is completed, the 96-well plate is taken out, the supernatant is discarded, the plate is washed for 5 times by using the washing solution (200 mu L of the washing solution is added each time, and the washing is carried out for 1min each time), and the plate is dried.

(6) After the completion of step (5), the 96-well plate was taken, 100. mu.L of IFN-. gamma.detection antibody (avidin-labeled) dilution (composed of 99 parts by volume of PBS buffer solution of pH7.4 and 0.01M and 1 part by volume of IFN-. gamma.detection antibody) was added to each well, and incubated at 37 ℃ for 1 hour.

(7) After the step (6) is completed, the 96-well plate is taken out, the supernatant is discarded, the plate is washed for 5 times by using the washing solution (200 mu L of the washing solution is added each time, and the washing is carried out for 1min each time), and the plate is dried.

(8) After completion of step (7), the 96-well plate was taken, and 100. mu. LHRP-labeled streptomycin diluted solution (composed of 99 parts by volume of pH7.4, 0.01M PBS buffer and 1 part by volume of HRP-labeled streptomycin) was added to each well, followed by incubation at 37 ℃ for 1 hour.

(9) After the step (8) is completed, the 96-well plate is taken out, the supernatant is discarded, the plate is washed 5 times by the washing solution (200 mu L of the washing solution is added each time, and the washing is carried out for 1min each time), and the plate is dried.

(10) And (4) after the step (9) is finished, taking the 96-well plate, adding an enzyme substrate into each well, and performing light-blocking color development for 15-45min at room temperature.

(11) And (3) after the step (10) is finished, taking the 96-well plate, washing the 96-well plate with distilled water for 3 times (for stopping the reaction), standing the 96-well plate at room temperature, and naturally drying the 96-well plate.

(12) After completion of step (11), the 96-well plate was taken, image and spot counting was performed using an immunospot counter (Cellular Technology Ltd, USA), and then the following judgment was made: when the number of the spots of the negative control hole is less than 6, if the number of the spots of the detection hole minus the number of the spots of the negative control hole is more than 6, the detection hole is positive, and if the number of the spots of the detection hole minus the number of the spots of the negative control hole is less than 6, the detection hole is negative; when the number of spots in the negative control well is 6 or more, the detection well is positive if the number of spots in the detection well is 2 times or more the number of spots in the negative control well, and the detection well is negative if the number of spots in the detection well is less than 2 times the number of spots in the negative control well. If the detection hole is positive, the peripheral blood to be detected is provided by the tuberculosis latent infected person (namely the peripheral blood sample of the tuberculosis latent infected person); if the test hole is negative, the peripheral blood to be tested is provided by the healthy person (i.e. the peripheral blood sample of the healthy person).

2. Obtaining peripheral blood samples

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

86 peripheral blood specimens: 2-3mL of peripheral blood of 86 patients (all patients informed consent) clinically diagnosed with active tuberculosis was extracted, placed in an EDTA-containing anticoagulated blood collection tube, and turned upside down 5-6 times (for uniformly mixing anticoagulated solution and peripheral blood), to obtain 86 peripheral blood specimens.

(2) Tuberculosis latent infection group: 25 peripheral blood samples.

25 peripheral blood specimens: 2-3mL of peripheral blood of 25 clinically diagnosed latent tuberculosis infected patients (informed consent) is respectively extracted and placed in an EDTA-containing anticoagulated blood collection tube, and the upper part and the lower part are reversed for 5-6 times (the aim is to uniformly mix anticoagulated liquid and peripheral blood), so that 25 peripheral blood samples are obtained.

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

46 peripheral blood specimens: 2-3mL of peripheral blood of 46 healthy persons (informed consent) was collected, placed in an EDTA-containing anticoagulated blood collection tube, and turned upside down 5-6 times (for uniformly mixing anticoagulated solution and peripheral blood) to obtain 46 peripheral blood specimens.

157 peripheral blood specimens were kept at room temperature (without freezing or refrigeration) for less than 6 h.

Second, application of relative expression quantity of IFIT3 gene in diagnosing active tuberculosis

The amino acid sequence of the IFIT3 protein (GeneID number: NP-001540.2) is shown as a sequence 5 in a sequence table. The nucleotide sequence of the gene (IFIT 3 gene for short, Genebank number: NM-001549.6) encoding IFIT3 protein is shown as sequence 6 in the sequence table.

1. Obtaining of cDNA from 157 peripheral blood specimens

(1) Preparation of PBMCs suspension

And (b) replacing the peripheral blood to be detected in the step (1) with 157 peripheral blood samples in the step (2) respectively, and obtaining PBMCs suspension of the 157 peripheral blood samples without changing other steps.

(2) RNA extraction

(ii) separately collecting a suspension of PBMCs from 157 peripheral blood specimens (approximately 1X 10 per specimen)⁶Individual cells), centrifuged at 400rcf for 5min and the pellet collected.

② after finishing the first step, taking the precipitate and adopting TRIzol^TMReagent extracts RNA.

(3) Synthesis of cDNA

Taking RNA of PBMCs suspension of 157 peripheral blood samples respectively, and adopting PrimeScript^TMThe RT reagent Kit was reverse transcribed with gDNA Eraser to obtain the cDNAs of 157 peripheral blood samples.

2. Preparation of primer pair combinations

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

Based on the nucleotide sequence of GAPDH gene, reference primer pairs shown in table 1 were designed and synthesized.

The primer pair combination consists of a specific primer pair and an internal reference primer pair.

Each primer was synthesized by Shanghai Biotech Co., Ltd. (HPLC purification).

TABLE 1

3. Real-time fluorescent quantitative PCR (polymerase chain reaction) detection of relative expression quantity of IFIT3 gene

And (3) respectively taking the cDNA of 157 peripheral blood samples as templates, and carrying out real-time quantitative PCR by adopting the specific primer pair or the internal reference primer pair prepared in the step (2) so as to obtain the relative expression quantity of the IFIT3 gene in each template. The method comprises the following specific steps:

(1) preparation of reaction System 1 and reaction System 2

The reaction system 1 was 20. mu.L, consisting of 10. mu.L of 2 XGreen Master Mix, 0.4. mu.L of aqueous LIFIT3-F solution (10. mu.M in concentration), 0.4. mu.L of aqueous IFIT3-R solution (10. mu.M in concentration), 2. mu.L of template (5-20ng) and 7.2. mu.L of nuclease-free water.

Reaction 2 was 20. mu.L, consisting of 10. mu.L of 2 × Green Master Mix, 0.4. mu.L of aqueous GAPDH-F (10. mu.M), 0.4. mu.L of aqueous GAPDH-R (10. mu.M), 2. mu.L of template (5-20ng) and 7.2. mu.L of nuclease-free water.

(2) Real-time quantitative PCR detection

Putting each reaction system prepared in the step (1) in Light

And carrying out real-time quantitative PCR detection on a 480 II fluorescent quantitative PCR instrument. Using 2-Delta^CtThe relative expression level of IFIT3 gene in each template was calculated.

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; at 95 ℃ for 5s, 60 ℃ for 30sec, 40 cycles, the fluorescence signal was collected during the extension phase.

The results are shown in FIG. 1(TB is active tuberculosis group, LI is latent tuberculosis infection group, and Nor is healthy control group). The results show that the relative expression quantity of the IFIT3 gene in the PBMCs of the active tuberculosis group is obviously increased compared with the healthy control group and the tuberculosis latent infection group.

(3) Statistical analysis

Statistical analysis of the results of step (2) was performed using GraphPad Prism 5.

According to the relative expression quantity of the IFIT3 gene in the PBMCs of the active tuberculosis group and the tuberculosis latent infection group, GraphPad Prism 5 is used for analyzing the working characteristic curve of the subject. The results are shown in FIG. 2. The result shows that the area under the ROC curve is 0.918(p is less than 0.0001), which indicates that the relative expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients from tuberculosis latent infection patients. When the transcription level cut-off value of IFIT3 gene is 0.02157, the Youden's Index (YI) is the maximum, the sensitivity is 91.86%, and the specificity is 84.00%.

According to the relative expression amount of IFIT3 gene in PBMCs of an active tuberculosis group and a healthy control group, GraphPad Prism 5 is used for analyzing the working characteristic curve of the subjects. The results are shown in FIG. 3. The result shows that the area under the ROC curve is 0.934(p is less than 0.0001), which indicates that the relative expression quantity of the IFIT3 gene can be used for distinguishing active tuberculosis patients from healthy people. When the transcription level cut-off value of the IFIT3 gene is 0.02465, the jotan index is the maximum, the sensitivity is 89.53 percent, and the specificity is 84.78 percent.

The results show that the relative expression quantity of the IFIT3 gene has important application value in diagnosing active tuberculosis.

<110> eighth medical center of general hospital of people liberation force of China

<120> a kit for diagnosing active tuberculosis

<160> 6

<170> PatentIn version 3.5

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Met Ser Glu Val Thr Lys Asn Ser Leu Glu Lys Ile Leu Pro Gln Leu

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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

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Phe Lys Ala Thr Met Tyr Asn Leu Leu Ala Tyr Ile Lys His Leu Asp

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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

gcagacagga 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 agaagaaaag 840

gtgacctaga caaagctatt gaactgtttc aacgggtgtt ggaatccaca ccaaacaatg 900

gctacctcta tcaccagatt gggtgctgct acaaggcaaa agtaagacaa atgcagaata 960

caggagaatc tgaagctagt ggaaataaag agatgattga agcactaaag caatatgcta 1020

tggactattc gaataaagct cttgagaagg gactgaatcc tctgaatgca tactccgatc 1080

tcgctgagtt cctggagacg gaatgttatc agacaccatt caataaggaa gtccctgatg 1140

ctgaaaagca acaatcccat cagcgctact gcaaccttca gaaatataat gggaagtctg 1200

aagacactgc tgtgcaacat ggtttagagg gtttgtccat aagcaaaaaa tcaactgaca 1260

aggaagagat caaagaccaa ccacagaatg tatctgaaaa tctgcttcca caaaatgcac 1320

caaattattg gtatcttcaa ggattaattc ataagcagaa tggagatctg ctgcaagcag 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 aaaaaaaaaa aaagagttgt tttctcatgt tcattatagt tcattacagt 2040

tacatagtcc gaaggtctta caactaatca ctggtagcaa taaatgcttc aggcccacat 2100

gatgctgatt agttctcagt tttcattcag ttcacaatat aaccaccatt cctgccctcc 2160

ctgccaaggg tcataaatgg tgactgccta acaacaaaat 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. Use of a substance for detecting IFIT3 protein in the manufacture of a product; the function of the product is at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

2. Use of a substance and device for detecting IFIT3 protein in the manufacture of a product; the function of the product is at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis;

the device is a device A and/or a device C;

the control peripheral blood is peripheral blood of a tuberculosis latent infected person or a healthy person.

3. Use of a substance for detecting the IFIT3 gene in the manufacture of a product; the function of the product is at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

4. Use of substance and device b for detecting IFIT3 gene in the manufacture of a product; the function of the product is at least one of the following a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis;

5. A kit comprising a substance for detecting IFIT3 protein and/or a substance for detecting IFIT3 gene; the kit has at least one function of a1) to a 3): a1) diagnosing active tuberculosis; a2) diagnosing whether the person to be tested is an active tuberculosis patient; a3) preventing and controlling tuberculosis.

6. The use according to any one of claims 1 to 4 or the kit according to claim 5, wherein:

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;

the "substance for detecting the IFIT3 gene" is a substance for detecting the expression level of the IFIT3 gene.

7. The use or kit of claim 6, wherein:

the expression amount of the IFIT3 protein is the relative expression amount of the IFIT3 protein and a reference internal reference protein;

the expression level of the IFIT3 gene is the relative expression level of the IFIT3 gene reference internal reference gene.

8. The use or kit of claim 6 or 7, wherein:

the substance for detecting the expression quantity of the IFIT3 gene or the substance for detecting the relative expression quantity of the IFIT3 gene reference internal reference gene comprises a primer pair combination consisting of a specific primer pair and an internal reference primer pair;

the specific primer pair consists of a primer IFIT3-F and a primer IFIT 3-R; the target gene of the specific primer pair contains a DNA fragment shown by 2035-2193 th site from 5' end of a sequence 6 in a sequence table;

the internal reference primer pair consists of a primer F and a primer R; the target gene of the internal reference primer pair is all or part of the human internal reference gene.

9. The specific primer pair of claim 8.

10, Y1) or Y2) or Y3) or Y4):

y1) IFIT3 protein as a marker in the development of a reagent for diagnosing active tuberculosis;

y2) IFIT3 protein as a marker for diagnosing active tuberculosis;

y3) IFIT3 gene as a marker in the development of a reagent for diagnosing active tuberculosis;

y4) IFIT3 gene as a marker in diagnosing active tuberculosis.