CN109187987B

CN109187987B - Application of MS4A3 protein as marker in diagnosis of active tuberculosis

Info

Publication number: CN109187987B
Application number: CN201810965702.1A
Authority: CN
Inventors: 程小星; 杨秉芬; 翟斐; 安红娟; 曹志红; 刘艳华; 王若
Original assignee: 309th Hospital of PLA
Current assignee: 309th Hospital of PLA
Priority date: 2018-08-23
Filing date: 2018-08-23
Publication date: 2021-05-11
Anticipated expiration: 2038-08-23
Also published as: CN109187987A

Abstract

The invention discloses application of MS4A3 protein as a marker in diagnosing active tuberculosis. Compared with healthy people and tuberculosis latent infected people, the expression level of the MS4A3 gene in the PBMCs of the active tuberculosis patients is obviously increased; the expression quantity of the MS4A3 gene can be used for distinguishing active tuberculosis patients and tuberculosis latent infection patients; the expression level of the MS4A3 gene can be used for distinguishing active tuberculosis patients from healthy people. Therefore, the MS4A3 protein and/or the MS4A3 gene can be used as markers for diagnosing active tuberculosis. The invention has great application value.

Description

Application of MS4A3 protein as marker in diagnosis of active tuberculosis

Technical Field

The invention belongs to the technical field of medical immunological diagnosis, and particularly relates to application of MS4A3 protein serving as a marker in diagnosis of 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 research shows that the MS4A3 protein is possibly related to the proliferation of leukemia cells; the expression of the MS4a3 protein in breast, ovarian and prostate cancers is significantly different from that in normal tissues.

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 MS4A3 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 for detecting the MS4A3 protein and the carrier recorded with the judgment standard A and/or the judgment standard C 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 judgment standard A can be as follows: if the expression level of the MS4A3 protein in the peripheral blood of the patient is higher than that of the MS4A3 protein in the peripheral blood of the control, the patient is or is suspected to be the active tuberculosis patient; if the expression level of the MS4A3 protein in the peripheral blood of the patient is lower than that of the MS4A3 protein in the peripheral blood of the control, the patient is not or is suspected not to be the active tuberculosis patient;

the judgment criterion C can be: if the concentration of the MS4A3 protein in the peripheral blood of the patient is higher than that of the MS4A3 protein in the peripheral blood of the control, the patient is or is suspected to be the active tuberculosis patient; if the concentration of the MS4A3 protein in the peripheral blood of the subject is lower than the concentration of the MS4A3 protein in the peripheral blood of the control, the subject is not or is suspected not to be the active tuberculosis patient;

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

The expression level of the MS4A3 protein in the peripheral blood can be specifically the expression level of the MS4A3 protein in PBMCs separated from the peripheral blood, the expression level of the MS4A3 protein in serum or the expression level of the MS4A3 protein in plasma.

The invention also protects the application of the substance for detecting the MS4A3 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 for detecting the MS4A3 gene and the carrier recording the judgment standard B 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 judgment criterion B can be: if the expression level of the MS4A3 gene in the peripheral blood of the patient is higher than that of the MS4A3 gene in the control peripheral blood, the patient is or is suspected to be the active tuberculosis patient; if the expression level of the MS4A3 gene in the peripheral blood of the patient is lower than that of the MS4A3 gene in the peripheral blood of the control, the patient is not or is suspected not to be the active tuberculosis patient; the control peripheral blood can be peripheral blood of a person with latent tuberculosis infection or a healthy person.

The expression level of the MS4A3 gene in the peripheral blood can be specifically the expression level of the MS4A3 gene in PBMCs separated from the peripheral blood.

The invention also protects a kit which can comprise a substance for detecting the MS4A3 protein and/or a substance for detecting the MS4A3 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 MS4A3 protein" may be a substance for detecting the expression level of MS4A3 protein (corresponding to the judgment criterion A) and/or a substance for detecting the concentration of MS4A3 protein (corresponding to the judgment criterion C).

Any of the above-mentioned "substances for detecting MS4A3 gene" may be a substance for detecting the expression level of MS4A3 gene.

The expression level of any one of the MS4A3 proteins can be the relative expression level of the MS4A3 protein and a reference internal reference protein.

The expression level of any one of the MS4A3 genes can be relative expression level of reference internal reference gene of MS4A3 gene.

Any one of the above methods for detecting the expression level of the MS4A3 protein can be specifically carried out by using a Western Blot experiment.

Any of the above described methods for determining the concentration of MS4a3 protein may be used in particular in an Elisa assay.

The substance for detecting the expression level of the MS4A3 gene or the substance for detecting the relative expression level of the reference internal reference gene of the MS4A3 gene comprises 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 MS4A3-F and a primer MS4A 3-R; the target gene of the specific primer pair contains a DNA fragment shown in the 438 th to 517 th bits from the 5' end of the sequence 6 in the 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 MS4A3-F can 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 MS4A3-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 1 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 1 and has the same function as the sequence 1.

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

b3) a single-stranded DNA molecule shown in a sequence 2 of a 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 2 and has the same function as the sequence 2.

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 MS4A3 gene or detecting the relative expression level of a reference internal reference gene of the MS4A3 gene, and the invention also belongs to the protection scope of the invention.

The primer pair combination is adopted to detect the expression quantity of the MS4A3 gene or the relative expression quantity of the reference internal reference gene of the MS4A3 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 reference internal reference gene of the MS4a3 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^-ΔCtAnd (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) MS4A3 protein as a marker in the development of a reagent for diagnosing active tuberculosis;

y2) application of MS4A3 protein as a marker in diagnosing active tuberculosis;

y3) application of MS4A3 gene as a marker in developing a reagent for diagnosing active tuberculosis;

y4) MS4A3 gene as a marker in diagnosing active tuberculosis.

The amino acid sequence of any one of the MS4A3 proteins (GeneID number: NP-006129.4) is shown as a sequence 5 in a sequence table. The nucleotide sequence of any one of the MS4A3 genes (Genebank number: NM-006138.4) is shown as a sequence 6 in a sequence table.

Experiments prove that compared with healthy people and latent tuberculosis infected people, the expression level of the MS4A3 gene in the PBMCs of the active tuberculosis patients is obviously increased; the expression quantity of the MS4A3 gene can be used for distinguishing active tuberculosis patients and tuberculosis latent infection patients; the expression level of the MS4A3 gene can be used for distinguishing active tuberculosis patients from healthy people. Therefore, the expression level of the MS4A3 gene has important application value in diagnosing active tuberculosis.

Drawings

FIG. 1 shows the real-time fluorescence quantitative PCR detection of the relative expression of MS4A3 gene in PBMCs of active tuberculosis patients, tuberculosis latent infected patients and healthy people.

FIG. 2 is a diagram showing the relative expression level of MS4A3 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 MS4A3 gene in PBMCs of an active tuberculosis patient and a healthy person analyzed by a 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 corporation of America。

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 quantity of MS4A3 gene in diagnosis of 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 completion of step (1), the middle layer was aspirated and transferred to a centrifuge tube containing 2mL of RPMI 1640 medium.

(3) After the step (2) is completed, 8mL of RPMI 1640 culture solution preheated to 37 ℃ is added into the centrifuge tube, the mixture is gently blown and beaten by a dropper and is centrifuged at room temperature and 1400rpm for 7 min.

(4) After the step (3) 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.

(5) After the step (4) 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: 20 peripheral blood samples.

20 peripheral blood specimens: 2-3mL of peripheral blood of 20 patients (all patients informed consent) clinically diagnosed with active tuberculosis 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 mix the anticoagulated liquid and the peripheral blood uniformly), so that 20 peripheral blood specimens are obtained.

(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: 35 peripheral blood samples.

35 peripheral blood specimens: 2-3mL of peripheral blood of 35 healthy people (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 35 peripheral blood specimens.

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

Second, application of relative expression quantity of MS4A3 gene in diagnosing active tuberculosis

The amino acid sequence of the MS4A3 protein (GeneID number: NP-006129.4) is shown as a sequence 5 in a sequence table. The nucleotide sequence of the gene (MS 4A3 gene for short, Genebank number: NM-006138.4) for coding the MS4A3 protein is shown as a sequence 6 in the sequence table.

1. Acquisition of cDNA from 80 peripheral blood specimens

(1) Preparation of PBMCs suspension

And (b) replacing the peripheral blood to be detected in the step (1) with 80 peripheral blood samples in the step (2), and obtaining PBMCs suspension of 80 peripheral blood samples without changing other steps.

(2) RNA extraction

RNA was extracted from PBMCs suspensions of 80 peripheral blood samples. The method comprises the following specific steps:

firstly, a 96-well plate is taken and 80 PBMCs suspensions of peripheral blood samples are respectively added. 150 μ L per well.

② after finishing the step I, taking the 96-well plate, adding 50 μ L of the diluent of the mycobacterium tuberculosis H37Rv lysate (prepared by AIM V)^TMDiluting Mycobacterium tuberculosis H37Rv lysate with Medium serum-free Medium; protein concentration 10. mu.g/mL), and mixing.

The preparation method of the mycobacterium tuberculosis H37Rv lysate comprises the following steps: taking thalli of mycobacterium tuberculosis H37Rv, performing irradiation inactivation by cobalt 60, and then performing heavy suspension by using PBS buffer solution to obtain a heavy suspension; crushing thallus cells of the heavy suspension by using an ultrahigh pressure cell crusher, centrifuging for 10min at 4 ℃ under 12000rcf, and collecting supernatant; the supernatant was collected and the protein concentration was determined by BCA method.

Taking the 96-well plate after the step II is finished, and carrying out the treatment at 37 ℃ and 5% CO₂Culturing for 16h, and then using TRIzol^TMReagent extracts RNA.

(3) Synthesis of cDNA

Respectively taking RNA of PBMCs suspension of 80 peripheral blood samples, and adopting PrimeScript^TMThe RT reagent Kit was reverse transcribed with gDNA Eraser to obtain cDNAs of 80 peripheral blood samples.

2. Preparation of primer pair combinations

Based on the nucleotide sequence of the MS4A3 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 detection of relative expression quantity of MS4A3 gene

And (3) respectively taking the cDNAs of 80 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 MS4A3 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 an aqueous solution of MS4A3-F (10. mu.M in concentration), 0.4. mu.L of an aqueous solution of MS4A3-R (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

The reaction systems prepared in the step (1) are respectively arranged in

And carrying out real-time quantitative PCR detection on a 480 II fluorescent quantitative PCR instrument. Use 2^-ΔCtThe relative expression level of the MS4A3 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 level of the MS4A3 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 MS4A3 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 relative expression quantity of the MS4A3 gene can be used for distinguishing active tuberculosis patients from tuberculosis latent infection patients. When the transcription level cut-off value of the MS4A3 gene is 0.0008971, the Youden's Index (YI) is the maximum, the sensitivity is 85%, and the specificity is 84%.

According to the relative expression amount of the MS4A3 gene in the PBMCs of the active tuberculosis group and the 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 relative expression quantity of the MS4A3 gene can be used for distinguishing active tuberculosis patients from healthy people. The jotan index was maximal when the transcription level cut-off of the MS4A3 gene was 0.0009417, at which time the sensitivity was 85% and the specificity was 88.57%.

The results show that the relative expression quantity of the MS4A3 gene has important application value in diagnosing active tuberculosis.

<110> third good and nine hospital of liberation army of China

Application of <120> MS4A3 protein as marker in diagnosis of active tuberculosis

<160> 6

<170> PatentIn version 3.5

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atgcagcaat gattctggct ttgggtgtct ttctgggttc cttgcaatac ccataccact 360

tccaaaagca cttctttttc ttcaccttct acacaggcta cccgatttgg ggtgctgtgt 420

ttttctgtag ttcaggaacc ttgtctgttg tagcagggat aaaacccaca agaacatgga 480

tacagaacag ttttggaatg aacattgcca gtgctacaat tgcactagtg gggactgctt 540

ttctctcact aaatatagca gttaatatcc agtcattaag gagttgtcac tcttcatcag 600

agtcaccgga cctatgcaat tacatgggct ccatatcaaa tggcatggtg tctctactgc 660

tgattctcac cttgctggaa ttatgcgtaa ccatctctac catagccatg tggtgcaatg 720

caaactgctg taattcaaga gaggaaattt cctcacctcc caattctgtg taatcaagaa 780

tacctcctta attctgagag catgaatatt tgaccttaaa tctccagtga ctcagagctt 840

cacccacaaa ctcaggagaa cataagcctg ctcgtaaagc tcaatccttc tatcatggca 900

ccaatcacaa gaaccttgga cgtttgactg actctatcct ttctctccta actataaatc 960

ctatttgtgt gtcgtgggta tggaaggaca gatatatttc tttaggcatt cttggatatc 1020

tgtaacttct atgatcatta ctccaaagtt gtttccagaa attggttcta tttcttctta 1080

tccacctact ccattgcttt atgaggttta aggaaggaag gcggtataat ccctattcaa 1140

tatatttttt ctaaaatcca acttctgacc gcccagtagg aagaaaaatg agacattttt 1200

tccattacag agaaatgctt cttgacttta acatcagcat tataaaaagt gtcaaataaa 1260

aaattaccat cattatcatt aaaataaatt ttcactgtat ttgagatggg agggttaagg 1320

ctcagggatt ttatttcagt gaactgctgg aactcacaca tgccctgata tgtaaatgat 1380

gatttatgtt ggcgagtctg agagcaagcc caaatgtgtt cttcaaagga caatgggaaa 1440

ctgtaaagta gagaactaaa gaataaggcc tttagaatct gacacatctg ggttcaaatt 1500

ctgaaactgt cacttattac ctgtatgaac atgggcaaat tatctaatct ctctgatcta 1560

tttttcctca tctgtaaaat aggtgtaata ataacaacta ctttgtcggt tgctctgagg 1620

gttaaatgaa aataaaaaga aaatgtgaaa cagcaaaaaa aaaaa 1665

Claims

1. The application of a substance for detecting MS4A3 protein in preparing products; 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. The use of claim 1, wherein: the substance for detecting the MS4A3 protein is a substance for detecting the expression quantity of the MS4A3 protein and/or a substance for detecting the concentration of the MS4A3 protein.

3. Use according to claim 2, characterized in that: the expression level of the MS4A3 protein is the relative expression level of the MS4A3 protein and reference internal reference protein.

4. The application of a substance for detecting MS4A3 protein and a carrier recorded with a judgment standard A and/or a judgment standard C in preparing 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 judgment standard A is as follows: if the expression level of the MS4A3 protein in the peripheral blood of the patient is higher than that of the MS4A3 protein in the peripheral blood of the control, the patient is or is suspected to be the active tuberculosis patient; if the expression level of the MS4A3 protein in the peripheral blood of the patient is lower than that of the MS4A3 protein in the peripheral blood of the control, the patient is not or is suspected not to be the active tuberculosis patient;

the judgment standard C is as follows: if the concentration of the MS4A3 protein in the peripheral blood of the patient is higher than that of the MS4A3 protein in the peripheral blood of the control, the patient is or is suspected to be the active tuberculosis patient; if the concentration of the MS4A3 protein in the peripheral blood of the subject is lower than the concentration of the MS4A3 protein in the peripheral blood of the control, the subject is not or is suspected not to be the active tuberculosis patient;

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

5. The use of claim 4, wherein: the substance for detecting the MS4A3 protein is a substance for detecting the expression quantity of the MS4A3 protein and/or a substance for detecting the concentration of the MS4A3 protein.

6. The use of claim 5, wherein: the expression level of the MS4A3 protein is the relative expression level of the MS4A3 protein and reference internal reference protein.

7. The application of the substance for detecting the MS4A3 gene in preparing products; 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.

8. The use of claim 7, wherein: the substance for detecting the MS4A3 gene is a substance for detecting the expression level of the MS4A3 gene.

9. The use of claim 8, wherein: the expression level of the MS4A3 gene is the relative expression level of the MS4A3 gene reference internal reference gene.

10. The use of claim 8, wherein: the substance for detecting the expression quantity of the MS4A3 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 MS4A3-F and a primer MS4A 3-R; the primer MS4A3-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table; the primer MS4A3-R is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the internal reference primer pair consists of a primer F and a primer R; the primer F is a single-stranded DNA molecule shown in a sequence 3 of the sequence table; the primer R is a single-stranded DNA molecule shown in a sequence 4 of the sequence table.

11. The application of the substance for detecting the MS4A3 gene and the carrier recorded with the judgment standard B in preparing products; 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 judgment standard B is as follows: if the expression level of the MS4A3 gene in the peripheral blood of the patient is higher than that of the MS4A3 gene in the control peripheral blood, the patient is or is suspected to be the active tuberculosis patient; if the expression level of the MS4A3 gene in the peripheral blood of the patient is lower than that of the MS4A3 gene in the peripheral blood of the control, the patient is not or is suspected not to be the active tuberculosis patient; the control peripheral blood is peripheral blood of a tuberculosis latent infected person or a healthy person.

12. The use of claim 11, wherein: the substance for detecting the MS4A3 gene is a substance for detecting the expression level of the MS4A3 gene.

13. The use of claim 12, wherein: the expression level of the MS4A3 gene is the relative expression level of the MS4A3 gene reference internal reference gene.

14. The use of claim 12, wherein: the substance for detecting the expression quantity of the MS4A3 gene comprises a primer pair combination consisting of a specific primer pair and an internal reference primer pair;

15. The specific primer pair consists of a primer MS4A3-F and a primer MS4A 3-R; the primer MS4A3-F is a single-stranded DNA molecule shown in a sequence 1 in a sequence table; the primer MS4A3-R is a single-stranded DNA molecule shown in a sequence 2 in a sequence table.

The application of MS4A3 protein as a marker in the development of a reagent for diagnosing active tuberculosis.

Application of MS4A3 gene as a marker in developing a reagent for diagnosing active tuberculosis.