CN112684183B

CN112684183B - Application of multi-antigen protein combination in identification and diagnosis of pulmonary tuberculosis

Info

Publication number: CN112684183B
Application number: CN202011512557.5A
Authority: CN
Inventors: 金奇; 郑建华; 刘立国; 邓佳珩
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2020-12-20
Filing date: 2020-12-20
Publication date: 2022-09-02
Anticipated expiration: 2040-12-20
Also published as: CN112684183A

Abstract

The invention relates to the identification and diagnosis of active tuberculosis by using 5 antigen proteins, and provides a diagnostic reagent, in particular to a novel tuberculosis diagnosis method, namely, 5 antigen proteins P22352, Q9P121, P15151, Q13291 and Q8NDA2 are used as biomarkers, the content of the 5 antigen proteins in urine is detected, the value of a specific content is used as a diagnostic index, and the result is negative in the normal value range. The concentration of any 3 or more antigen proteins exceeding or falling below the normal value range is judged as a positive result.

Description

Application of multi-antigen protein combination in identification and diagnosis of pulmonary tuberculosis

Technical Field

The invention relates to a diagnostic reagent, in particular to a multi-antigen protein applied to the identification and diagnosis of active tuberculosis.

Background

Tuberculosis is one of the main infectious diseases which endanger human health, is the main killer of adult infectious diseases in the world today, and has made a serious challenge to international public health. China is one of the countries with high tuberculosis burden in the world, the tuberculosis epidemic situation is 'three high one low', namely, the prevalence rate is high, the mortality rate is high, the drug resistance rate is high, the annual degradation rate is low, and the prevention and control situation is very severe along with the occurrence of the situations of AIDS complicated infection, drug-resistant tuberculosis, increase of mobile population and the like. Since the discovery of mycobacterium tuberculosis, the early and rapid diagnosis of tuberculosis has low sensitivity and no breakthrough progress because of the characteristics of thicker cell wall, higher fatty acid content, intracellular parasitism and the like of thalli. At present, the combined application of multiple diagnostic means (sputum smear, sputum culture and molecular diagnostic techniques) can only diagnose about 50% of tuberculosis patients, and nearly half of tuberculosis patients with negative clinical etiology or sputum negative still cannot be scientifically, reasonably and quickly diagnosed, even the disease state is delayed, and the drug resistance is caused. Therefore, the tuberculosis crowd can be quickly and accurately diagnosed, so that effective treatment and intervention measures can be taken subsequently, the morbidity risk of an infected person is reduced, and the method is the most direct means for reducing the morbidity of the tuberculosis.

Given that many diseases are reflected in chemical metabolites of urine, finding one or more biomarkers specific for a disease in urine will help researchers detect many diseases with a small amount of urine or even a drop of urine. The urine has the characteristics of simple collection, convenient material taking, rich sources, non-invasiveness and the like, and the change of the type and the quantity of the urine protein carries various information of occurrence, development and prognosis of a plurality of diseases, so the urine protein has value in the aspects of disease diagnosis, monitoring, prognosis and the like, and has wide application prospect. Although the traditional sputum smear examination is a gold standard for diagnosing the pulmonary tuberculosis, is simple and easy to implement, and has high result reliability, the positive rate is low, and only about 20-30% of patients with the pulmonary tuberculosis have positive sputum smear detection clinically. The method for culturing the mycobacterium phlei is long in time consumption, often needs 1-3 weeks or even 4-6 weeks, and cannot meet the requirement of rapid diagnosis. The currently adopted molecular diagnostic techniques, such as the GeneXpert MTB/RIF method, have high sensitivity, and can often detect whether the Mycobacterium tuberculosis and the drug resistance to rifampicin exist in a short time (about 2 hours) directly from fresh sputum or cryopreserved sputum of a patient, but the whole process needs to be carried out in a closed environment, needs a professional GeneXpert detection instrument and a specimen collection box, needs professional operation, is high in cost, and is not suitable for large-scale popularization. Even if a plurality of etiological diagnoses are jointly applied (sputum smear, sputum culture and molecular diagnosis technologies) at present, only about 50 percent of tuberculosis patients can be diagnosed, and nearly half of tuberculosis patients with negative clinical etiology or sputum bacteria can not be scientifically, reasonably and quickly diagnosed. In recent years, gamma interferon release tests in blood based on cellular immune response, such as T-SPOT and QuantiFERON-TB Gold tests, are rapidly developed and widely applied, but the sensitivity of diagnosis is still to be improved, the detection result cannot be determined frequently, active tuberculosis and latent tuberculosis infection cannot be distinguished, the cost is high, and the tests are invasive. Therefore, under the current situation, development of noninvasive diagnosis products which are suitable for the national conditions of China, have independent intellectual property rights, low cost, simplicity and rapidness is urgently needed.

Based on the prior art, the invention adopts a biomarker method to unexpectedly discover new biomarkers, namely 5 antigen proteins (P22352, Q9P121, P15151, Q13291 and Q8NDA2), and the invention discovers that the tuberculosis can be diagnosed by detecting the content of any combination of 3 or more than 5 antigen proteins, the diagnostic sensitivity can reach 82.7 percent, and tuberculosis people and latent infection people can be well distinguished. Therefore, the invention provides a new tuberculosis diagnosis method, which adopts 5 antigen proteins and the combination thereof as biomarkers, detects the content of the 5 antigen proteins in urine, takes the value of the specific content as a diagnosis index, and judges that the concentration of any 3 or more antigen proteins in the 5 proteins is lower than or exceeds the limit to be a positive result.

Disclosure of Invention

The invention aims to provide application of the 5 antigen combinations in diagnosing tuberculosis. The antigen combination can be used as a diagnostic marker for diagnosing tuberculosis or detecting latent infection of mycobacterium tuberculosis, and has good sensitivity and specificity.

The five antigen proteins are all proteins in human urine and are respectively named as: p22352, Q9P121, P15151, Q13291, Q8NDA 2.

The invention aims to provide application of the 5 antigen proteins as biomarkers in tuberculosis diagnosis. The invention discovers for the first time that 5 antigen proteins can be used as biomarkers for diagnosing active tuberculosis or latent tuberculosis infection, and further discovers that the 5 antigen proteins have good sensitivity and specificity.

The more detailed technical scheme of the invention is described as follows:

the invention provides application of 5 antigen proteins in preparing a reagent for diagnosing tuberculosis.

The invention further provides application of the 5 antigen proteins in preparation of medical instruments for tuberculosis diagnosis.

The tuberculosis diagnosis uses 5 antigen proteins as diagnosis molecular markers.

The invention provides a reagent combination, which can be used for directly or indirectly extracting and separating glutathione from urine, further detecting the content of 5 antigen proteins, and using the content of 5 antigen proteins as a diagnostic index to determine whether a urine provider has tuberculosis, namely the reagent combination provided by the invention uses 5 antigen proteins as detection targets and uses the detection targets as biomarkers for diagnosing the tuberculosis.

Therefore, the invention further provides the application of the reagent combination in the preparation of a kit for diagnosing tuberculosis.

The 5 antigen proteins of the biomarker of the invention, which are known compounds, can be extracted or detected according to any one of the existing methods, including any one of the methods for detecting antigen proteins, such as the methods reported in the existing literature.

The application of the invention comprises the application of distinguishing active tuberculosis patients, tuberculosis latent infection patients and inactive tuberculosis non-latent infection patients.

The tuberculosis of the invention is pulmonary tuberculosis or extrapulmonary tuberculosis.

The body fluid is urine.

In particular, the kit of the invention can be used for extracting and separating 5 antigen proteins from urine,

the kit of the present invention includes any one of existing kits for detecting 5 antigenic proteins.

Preferably, the kit of the present invention comprises 5 antigen protein standard reference substances, and further comprises a detection method instruction, reference values of detection standards, organic or inorganic reagents, a preparation method of a solvent, and the like.

The invention uses 5 antigen proteins as diagnostic markers, and the 5 antigen proteins are detected together as a whole, namely 5 of the 5 antigen proteins are detected, and 1, 2, 3 and 4 of the 5 antigen proteins can also be detected, preferably 3, 4 or 5 of the 5 antigen proteins are detected. 3 of them are detected with higher accuracy than 1 or 2 of them, and similarly, 4 of them are detected with higher accuracy than 1-3 of them, and 5 of them are detected with higher accuracy than 1-4 of them. The detection is multiple, optional and not limited in kind, and can be any combination of 5 antigen proteins.

The technical scheme of the invention is obtained by research, and the research method comprises the following steps:

1. technical scheme

1.1 study objects and methods

1.1.1 subjects

Study subjects and inclusion criteria

The research objects are confirmed tuberculosis inpatients from Shenzhen third national hospital, the diagnosis standard is in accordance with the standard of the national health industry Standard (WS 288-2017) tuberculosis diagnosis of China, and the pathogenic detection is positive, namely: at least 1 of the sputum smear, the culture and the nucleic acid detection is positive, the past tuberculosis history (no old tuberculosis focus in inquiry and X-ray chest examination) is absent, the primary anti-tuberculosis treatment is carried out, and the medicine consumption is less than 7 days; latently infected (with a history of clinical exposure, no clinical symptoms, and positive for IGRA) and non-latently infected with inactive tuberculosis (control). The control group 118, the tuberculosis latent infection group 118 and the active tuberculosis group 118 are selected together, the detailed information of age, sex and the like is shown in table 1, and all the study subjects are less than 70 years old or more than 18 years old, women without pregnancy or lactation and other serious chronic diseases and immunodeficiency diseases. The ethical standard customized by the etiology and biology institute of Chinese medical science/Beijing cooperative medical institute and Shenzhen third people hospital ethical committee is followed, and a notice is signed. Sample information is shown in table 1.

1.1.2 sources of specimens

And (4) keeping the middle-section urine of the group in the clean urine cup for the second morning urine, wherein about 40 ml of the middle-section urine at 7-11 am is transferred into a sterile collection pipe, tightly covering the pipe cover and placing the pipe cover in an ice box or an ice-water mixture.

1.1.3 methods of investigation

And identifying the information such as the types and the contents of all proteins in the urine sample of the sample by utilizing the gel electrophoresis/liquid chromatography and mass spectrometry combined technology. And simultaneously, performing two-dimensional gel electrophoresis, selecting differentially expressed protein gel points with concentration difference larger than 2 times, or gel points which appear in tuberculosis people but do not appear in a control sample, and performing mass spectrum identification to obtain the expressed differential proteins of the urine proteins of the tuberculosis people and the non-tuberculosis control. Potential diagnosis markers in the tuberculosis urine are found out through data mining and bioinformatics analysis. Clinical verification is carried out on the screened diagnosis marker in tuberculosis populations with different disease backgrounds by adopting a Multiple Reaction Monitoring (MRM) technology and enzyme-linked immunosorbent assay (ELISA).

1.1.3.1 preparation of urine protein

(1) Immediately, the urine sample was centrifuged at 1500g for 10 minutes in a centrifuge precooled to 4 ℃ and the supernatant carefully transferred to a fresh sterile collection tube to remove sediment.

(2) One protease inhibitor tablet (Roche, Germany, this tablet is suitable for sample collection in a volume of 40 ml) was added to the collection tube immediately per tube, and after the inhibitor tablet was sufficiently dissolved, the collection tube was frozen immediately in a freezer at-20 ℃ to avoid freezing and thawing during storage. From the collection of the urine sample to the final freezing and storing in a refrigerator, the time is controlled within 4 hours.

(3) The collected urine sample was centrifuged for 20 minutes at 4000 rpm using a 50kDa ultrafiltration tube to remove the abundant proteins so as not to interfere with the identification of low abundant proteins in the urine.

(4) The filtrate was centrifuged in a 3kDa ultrafiltration tube for 20 minutes to remove small molecular weight proteins and collect a protein solution between 3kDa and 50 kDa.

(5) Sodium deoxycholate was added to the solution to a final concentration of 0.015% and incubated for 10 minutes at room temperature, then pre-cooled trichloroacetic acid was added to a final concentration of 10%, the solution was left at-20 ℃ for 5 minutes and then at 4 ℃ overnight to precipitate the protein sufficiently.

(6) The solution was centrifuged at 15000g for 30 minutes at 4 ℃ and the centrifuged pellet was washed twice with pre-cooled acetone and the concentration of the protein was determined after the pellet was dried.

1.1.3.2 identification of urine proteome

(1) The prepared protein is respectively subjected to SDS gel electrophoresis and two-dimensional liquid chromatography separation. After SDS electrophoresis separation, the gel of each protein sample is divided into 20 equal parts, and the gel blocks are collected and then decolorized, reduced and alkylated, enzymolyzed and desalted.

(2) During two-dimensional liquid chromatography separation, an enzymolyzed protein sample is dissolved in 0.1% formic acid, the formic acid sequentially passes through a strong cation exchange column, ammonium chloride solutions (2,5,10,15,20,25,30,40,50,60,70,80, 100 and 500mM) with different concentrations are adopted for elution, and eluents of each gradient directly enter a reverse phase column for separation to sequentially obtain eluted peptide sections of the protein sample.

(3) Peptide fragments were identified using LTQ Orbitrap Velos, respectively, by tandem mass spectrometry to obtain a proteome of each urine eluted with peptide fragments. The results of gel electrophoresis and two-dimensional liquid chromatography tandem mass spectrometry are combined to form the urine proteome of tuberculosis patients.

1.1.3.3 identification of differentially expressed proteins in urine

(1) Taking 300 mu g of prepared urine protein of tuberculosis people and non-tuberculosis control people respectively, carrying out isoelectric focusing electrophoresis on one dimension by adopting an adhesive tape with an isoelectric point of 3-10, carrying out electrophoresis on two dimensions by adopting 12% SDS-PAGE, carrying out parallel electrophoresis on two pieces of gel, and fixing, dyeing and decoloring the gel simultaneously after the electrophoresis is finished to obtain two-dimensional gel electrophoresis images of the urine protein of the tuberculosis people and the non-tuberculosis control people.

(2) Comparing protein staining spots in the two gels, detecting protein with the content as low as 0.1 mu g after full decolorization, selecting differentially expressed protein spots with the concentration difference larger than 2 times, respectively digging gel spots corresponding to the differentially expressed proteins, carrying out decolorization, reduction, enzymolysis and desalination, and identifying by adopting a flight time mass spectrum MALDI-TOF/TOF to obtain the expression differential protein of the urine protein of tuberculosis patients and non-tuberculosis patients (the technical route is shown in figure 1). The identification process is repeated at least three times, so that the reliability of the result is ensured. The differentially expressed proteins can be used as potential markers for molecular diagnosis of tuberculosis human urine by combining with the subsequent quantitative proteomic identification results.

(3) By comparing the results of urine proteomes of tuberculosis patients and non-tuberculosis control groups, and through data mining and bioinformatics analysis, the significant difference of protein expressions of the urine of the tuberculosis patients and the control groups is found out in the biometrical aspect, the specific protein in the urine of the tuberculosis patients is revealed, and the potential diagnosis marker in the urine of the tuberculosis patients is preliminarily identified by combining the results of the differential expression protein identified in the two-dimensional gel electrophoresis.

1.1.3.4 clinical validation of populations of different disease backgrounds

And (3) clinically verifying the screened potential markers by respectively adopting a multi-reaction monitoring mass spectrum technology and an immunoblot in collected urine samples of tuberculosis patients with different disease backgrounds and non-tuberculosis control urine samples.

A. Multiple reaction monitoring mass spectrometry technology:

(1) preparing clinical sample protein to be verified according to the urine protein preparation method, and reducing/alkylating/enzymolyzing/desalting for later use;

(2) according to the high-resolution identification result of the mass spectrometer LTQ Orbitrap Velos on the proteomics of the tuberculosis patient urine sample, selecting corresponding peptide segments of the protein to be verified, introducing the corresponding peptide segments into Skyline software, and extracting the information (including mass-to-charge ratio, ion signal intensity and retention time) of specific parent ions corresponding to each peptide segment;

(3) these information were imported into the analysis software Analyst (version 1.7, Sciex, USA) to establish the MRM method. The method only carries out collision-induced induction (collision-induced) on the selected specific parent ions, removes the interference of other daughter ions, and carries out signal acquisition on the selected specific daughter ions on a mass spectrum AB Sciex 5500;

(4) and analyzing the data in Skyline software, and confirming the specific parent ions of the selected peptide fragments so as to finish the verification of the protein.

B. And (3) enzyme-linked immunosorbent assay (ELISA) for verification:

(1) sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeled reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged in the reagent box. The standard sample is accurately loaded by 50 mu L on the enzyme-labeled coating plate, 40 mu L of sample diluent is loaded in the sample hole to be detected, and then 10 mu L of sample to be detected is loaded (the final dilution of the sample is 5 times). Adding a sample to the bottom of an enzyme label plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

(2) and (3) incubation: sealing the plate with sealing plate film, and incubating at 37 deg.C for 30 min;

(3) preparing liquid: diluting 30 times of the concentrated washing liquid with 30 times of distilled water for later use;

(4) washing: removing the sealing template, discarding liquid, spin-drying, filling each hole with washing liquid, standing for 30s, discarding, repeating the operation for 5 times, and then drying;

(5) adding an enzyme: adding 50 mu L of enzyme-labeled reagent into each hole except for a blank hole;

(6) incubation: repeating the step 2;

(7) washing: repeating the operation of the step 4;

(8) color development: adding 50 μ L of color-developing agent A into each well, adding 50 μ L of color-developing agent B, shaking, mixing, and developing at 37 deg.C in dark for 10 min;

(9) and (4) terminating: adding 50 mu L of stop solution into each hole to stop reaction;

(10) and (3) determination: and (3) adjusting the position to zero by using a enzyme-labeled analyzer Rayto RT-6100, measuring the absorbance (OD value) of each hole at one time at the wavelength of 450nm, and performing the measurement within 15min after the reaction is stopped.

2. Results of the study

Through data mining and bioinformatics analysis, 5 protein markers which can be used for diagnosing tuberculosis in urine are found out preliminarily, and are respectively P22352(Glutathione peroxidase 3), Q9P121(Neurotrimin, neurotriamine), P15151(Poliovirus receptor, Poliovirus receptor protein), Q13291(Signaling lymphocytic activation molecule family 1), and Q8NDA2(Hemicentin-2, extracellular matrix protein 2). Subsequently, based on the results of high resolution mass spectrometric identification of these 5 proteins, clinical validation was carried out on a mass spectrometer AB Sciex 5500 using the Multiple Reaction Monitoring (MRM) technique. The validation process was performed on urine samples of 52 tuberculosis patients, 52 latently infected persons, and 52 non-tuberculosis controls during the validation phase. In addition, the 5 proteins were also verified in 16 tuberculosis patients, 16 latently infected people and 16 non-tuberculosis controls by enzyme-linked immunosorbent assay (ELISA). The research result shows that 3 of 5 urine proteins are selected for combined diagnosis, the diagnosis sensitivity is 82.7%, and the specificity is 92.3%. According to the literature report, Guo Aizhen et al identified 19 differentially expressed proteins from the urine of 45 tuberculosis patients (45 healthy human controls), and found that the expression of MBL2(mannose-binding molecule 2) and ITIH4-35k (a 35-kDa fragment of inter-alpha-trypsin inhibitor H4) were the most different. Subsequently, 11 microRNAs are identified from the constructed protein-microRNA interaction network and verified by qRT-PCR. Finally, through the analysis of a binary logistic regression model, the combined screening of the three molecules, namely miR-625-3p, MBL2 and ITIH4-35k can enable the sensitivity of tuberculosis diagnosis to reach 85.87% and the specificity to reach 87.50%, but the result is not clinically verified in urine samples of tuberculosis patients and control people, and only the theoretical feasibility is obtained through data analysis. The combined diagnostic specificity of the 5 antigenic proteins P22352, Q9P121, P15151, Q13291 and Q8NDA2 in this study was significantly higher than that of the previous human results.

3. And (4) conclusion:

the combination of P22352, Q9P121, P15151, Q13291 and Q8NDA2 in urine can clearly distinguish active tuberculosis patients from tuberculosis latent infection and inactive tuberculosis non-latent infection, has higher diagnostic sensitivity and specificity, and can be used as a candidate diagnostic marker for diagnosing active tuberculosis.

According to the research, the invention obtains the conclusion that 5 antigen proteins can be used as biomarkers of tuberculosis, but one or two of the antigen proteins are not enough to be used as diagnostic markers, and the combination of three or more than three of the antigen proteins has diagnostic sensitivity of more than 82.7% and specificity of more than 92.3%, and can be used as candidate diagnostic markers for diagnosing active tuberculosis. The test results obtained by the relevant test methods can be used to diagnose and evaluate tuberculosis and its severity.

According to the research, the invention obtains a judgment standard, and preliminarily determines that the concentration value of each protein of 5 proteins of P22352, Q9P121, P15151, Q13291 and Q8NDA2 in urine is in the range of 19.4-53.3 micrograms/milliliter, and the concentration of any 3 or more than 3 antigen proteins is lower than or exceeds the limit, so that the result is positive. Protein concentrations were negative within this normal range.

The noun interpretation:

p22352 (glutaminone peroxidase 3, GSH-Px 3): glutathione peroxidase 3 is an important peroxidase, which is widely present in the body. It can catalyze GSH to GSSG, reduce toxic peroxide to non-toxic hydroxy compound, and promote decomposition of H2O2, so as to protect the structure and function of cell membrane from interference and damage of peroxide. The active center of GSH-Px3 is selenocysteine, the activity of which reflects the level of selenium in the body.

The literature is presented: eschothy RS, Chu FF, Paxton RJ, Akman S, Doroshow JH. Characterisation and partial amino acid sequence of human plasma one peroxidase. Arch Biochem Biophys.1991; 286(2):330-336.

Amino acid sequence:

Q9P121 (neotrimin): neurotriamines are a family of five cell surface neuronal proteins involved in a variety of human diseases, interacting as homodimers and heterodimers, and may interact trans across different cells or across synaptic gaps. Genetic variation of the NTRI gene is associated with the aggressiveness of children with attention deficit/hyperactivity disorder and developmental delay.

The literature is presented: clark HF, Gurney AL, Abaya E, et AL, the Segmented Protein Discovery Initiative (SPDI), a large-scale effect to identification non-human segmented and transmbrane proteins, a Biochemical association. genome Res. 2003; 13(10):2265-2270.

Amino acid sequence:

p15151(Poliovirus receiver, NECL-5): poliovirus receptor proteins are members of the immunoglobulin superfamily, contain three Ig-like domains, and were originally identified as poliovirus receptors, and are usually upregulated in cancer cells. The cytoplasmic domain of Necl-5 may bind to Tctex-1, possibly binding to microtubules and involved in regulating cell motility. The activation of Cdc42 and Rac small G protein induced by the growth factor is enhanced by the Necl-5, so that filopodia and layered lipoprotein are formed respectively, and finally, the cell movement is enhanced; enhance activation of Ras-Raf-MEK-ERK signals, induce up-regulation and down-regulation of cell cycle regulators, and shorten the time of G1 phase of cells.

The literature is presented: mendelsohn CL, Wimmer E, Ranniello VR. cellular receiver for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin perfect. cell.1989, 56(5):855-65.

Amino acid sequence:

q13291 (signalling systemic activation motion family 1, SLAM): the signaling lymphocyte activating molecule family 1 belongs to the immunoglobulin gene superfamily and is involved in T cell stimulation. SLAM is constitutively expressed on peripheral blood CD45R hypermemory T cells, T cell clones, immature thymocytes and a proportion of B cells, and is rapidly induced on naive T cells after activation. The involvement of SLAM enhances antigen-specific proliferation and cytokine production of T cells carrying CD4 antigen (CD4 +). The expansion of T cells was enhanced and the production of Th0/Th1 cytokines was induced in a CD28 independent manner.

The literature is presented: r é thi B, Gogol a k P, Szatmura I, et al SLAM/SLAM interaction inhibition CD40-induced production of inhibition cytokines in monoclonal-derived dendritic cells, blood.2006; 107(7):2821-2829.

Amino acid sequence:

q8NDA2(Hemicentin-2, HMCN 2): extracellular matrix protein 2 is a conserved extracellular protein involved in calcium ion binding. The mutations in HMCN2 are associated with age-related familial macular degeneration.

The literature is presented: humphray SJ, Oliver K, Hunt AR, et al DNA sequence and analysis of human chromosome 9. Nature.2004; 429(6990):369-374.

Amino acid sequence:

TB: tuberculosis, Tuberculosis

M.tb: mycobacterium tuberculosis

TST: tuberculin skin test

IGRA: interferon gamma release assays, gamma interferon release assay

ATB: active Tuberculosis, Active Tuberculosis

LTBI: latent Tuberculosis Infection in late Tuberculosis

HC: health Control, non-tuberculosis infection Control group

2-DE two-dimensional gel electrophoresis, two-dimensional gel electrophoresis

ESI-MS electrophoresis on mass spectrometry, electrospray ion trap Mass Spectrometry

high-Performance liquid chromatography (HPLC)

Multiple reaction monitoring of MRM

Reference documents:

1.WHO.Global tuberculosis report 2019.

2.Cui X,Gao L,Cao B:Management of latent tuberculosis infection in China: Exploring solutions suitable for high-burden countries.Int J Infect Dis 2020 Mar；92S:S37-S40.

3.Kerkhoff AD,Lawn SD:A breakthrough urine-based diagnostic test for HIV-associated tuberculosis.Lancet 2016,387(10024):1139-1141.

4.Qi X,Ng KT,Lian Q,Li CX,Geng W,Ling CC,Yeung WH,Ma YY,Liu XB,Liu H et al:Glutathione Peroxidase 3Delivered by hiPSC-MSCs Ameliorated Hepatic IR Injury via Inhibition of Hepatic Senescence.Theranostics 2018,8(1):212-222.

5.Wang J,Zhu X,Xiong X,Ge P,Liu H,Ren N,Khan FA,Zhou X,Zhang L,Yuan X et al:Identification of potential urine proteins and microRNA biomarkers for the diagnosis of pulmonary tuberculosis patients.Emerg Microbes Infect 2018, 7(1):63.

drawings

FIG. 1. identification of expression differential protein in urine of tuberculosis by two-dimensional gel electrophoresis

FIG. 2 differential expression of tuberculous human urokinase protein

FIG. 3 Mass Spectroscopy Multiple Reaction Monitoring (MRM) validation of proteins Q9P121 and P22352 in tuberculosis human urine samples

The specific implementation mode is as follows:

the invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.

Example 1

Taking the second morning urine of the Wu family name subject, and transferring about 40 ml of the midnight urine at 7-11 am into a clean urine cup, and transferring into a sterile collection tube for treatment by the following method:

(2) One protease inhibitor tablet (Roche, Germany, which is suitable for sample collection in a volume of 40 ml) was added to the collection tube immediately per tube, and after the inhibitor tablets were sufficiently dissolved, the collection tube was frozen in a freezer at-20 ℃ to avoid freezing and thawing during storage. From the collection of the urine sample to the final freezing and storing in a refrigerator, the time is controlled within 4 hours.

(6) The solution was centrifuged at 15000g for 30min at 4 ℃ and the centrifuged precipitate was washed twice with pre-cooled acetone and the precipitate was dried to obtain the protein.

The resulting proteins were then qualitatively and quantitatively determined using an enzyme linked immunosorbent ELISA:

(6) and (3) incubation: repeating the operation of the step 2;

(7) washing: repeating the operation of the step 4;

According to the analysis result of the instrument, 5 qualitative and quantitative results of the antigen protein are obtained, and the detection results are as follows:

p22352 (glutaminone peroxidase 3, Glutathione peroxidase 3): 86.3. mu.g/ml

Q9P121 (neotrimin, neurotriamine): 5.3. mu.g/ml

P15151(Poliovirus receptor, Poliovirus receptor protein): 21.4. mu.g/ml

Q13291(Signaling lymphocyte activation molecule family 1): 35.5. mu.g/ml

Q8NDA2(Hemicentin-2, extracellular matrix protein 2): 342.2 μ g/ml protein concentration normal: 19.4-53.3 microgram/ml, 3 proteins in the detection result exceed or are lower than the limit (the concentration of P22352 and Q8NDA2 is higher than the normal value range, the concentration of Q9P121 is lower than the normal value range), the detection results of the other 2 proteins are in the limit range, and the detection standard that the concentration of any 3 antigen proteins is out of (lower than or exceeds) the normal value is reached, so the detection result is a positive result.

Example 2

Taking the second morning urine of a maiden subject, taking about 40 ml of the middle urine at 7-11 am into a clean urine cup, transferring into a sterile collection tube, and treating by the following method:

The resulting proteins were then qualitatively and quantitatively determined using an enzyme-linked immunosorbent assay:

(1) sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeled reagent, and the rest steps are operated in the same way), standard holes and sample holes to be detected are respectively arranged in the reagent box. The standard sample is accurately added with 50 mu L of sample on an enzyme labeling coating plate, 40 mu L of sample diluent is added into a sample hole to be detected, and then 10 mu L of sample to be detected is added (the final dilution of the sample is 5 times). Adding a sample to the bottom of an enzyme label plate hole, keeping the sample from touching the hole wall as much as possible, and slightly shaking and uniformly mixing the sample and the hole wall;

(4) washing: removing the sealing template, discarding liquid, spin-drying, filling each hole with cleaning solution, standing for 30s, discarding, repeating the operation for 5 times, and then patting to dry;

(5) adding an enzyme: adding 50 mu L of enzyme-labeled reagent into each hole except for blank holes;

(6) and (3) incubation: repeating the operation of the step 2;

(7) washing: repeating the operation of the step 4;

The detection results are as follows:

p22352 (glutaminone peroxidase 3, Glutathione peroxidase 3): 25.2. mu.g/ml

Q9P121 (neotrimin, neurotriamine): 46.4. mu.g/ml

P15151(Poliovirus receptor, Poliovirus receptor protein): 76.6. mu.g/ml

Q13291(Signaling lymphocyte activation molecule family 1): 32.5. mu.g/ml

Q8NDA2(Hemicentin-2, extracellular matrix protein 2): 57.3 μ g/ml antigen protein concentration normal: 19.4-53.3 microgram/ml, the detection result of 3 proteins in the detection result is within the limit range of normal value, 2 proteins (P15151 and Q8NDA2) exceed the limit, and the requirement that the concentration of 3 or more than 3 antigen proteins is out of the normal value is not met, so the detection result is a negative result.

Claims

The application of any three or more than three antigenic protein combinations of 1.5 antigenic proteins P22352, Q9P121, P15151, Q13291 and Q8NDA2 in the preparation of a reagent for diagnosing tuberculosis.
2. The use according to claim 1, wherein the tuberculosis diagnosis is performed by using 5 antigen proteins of P22352, Q9P121, P15151, Q13291 and Q8NDA2 as diagnostic molecular markers.
3. Use according to claim 1, comprising a method for differentiating between patients with active tuberculosis, latent tuberculosis infection and non-latent inactive tuberculosis infection.
4. The use according to claim 1, wherein the tuberculosis is pulmonary tuberculosis or extrapulmonary tuberculosis.