CN114675036A - Marker for diagnosing T-SPOT negative tuberculosis and application thereof - Google Patents

Abstract

The invention relates to the technical field of medicines, in particular to a marker for diagnosing tuberculosis infection and application thereof. A marker for diagnosing tuberculosis infection, wherein the molecular marker is a molecular marker for diagnosing T-SPOT negative tuberculosis; the marker is a combination of IFN-gamma and IL-4. Use of a combination of IFN- γ and IL-4 as a marker in the manufacture of a kit for diagnosing or monitoring tuberculosis. The invention has the advantages that: (1) high sensitivity and specificity. (2) High diagnosis efficiency and simple operation. (3) Can improve the diagnosis efficiency of T-SPOT negative tuberculosis.

Description

Marker for diagnosing T-SPOT negative tuberculosis and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a marker for diagnosing T-SPOT negative tuberculosis and application thereof.

Background

Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis, which can affect multiple systems and organs of the whole body, wherein pulmonary tuberculosis is the most common and frequent tuberculosis in clinic. The disease is taken as a public health problem of global attention, has strong infectivity, large treatment difficulty and high recurrence rate, is easy to develop into multi-drug resistant tuberculosis, and increases the treatment difficulty of the disease. It can be seen that early diagnosis and early treatment of tuberculosis are key to controlling its spread.

At present, the clinical diagnosis method of tuberculosis still is imaging and bacteriology, the bacteriology culture period is long, the diagnosis and treatment of clinical tuberculosis are not facilitated, the smear method has the characteristics of simplicity, convenience and rapidness, but the detection sensitivity is low, the correctness of a diagnosis result is influenced, and the specificity is low depending on the imaging examination and the diagnosis of clinical manifestations.

Gamma-interferon in vitro release assay (IGRAs) is a novel T cell immune-based in vitro immunoassay method for tubercle bacillus. The detection principle is that after a human body is infected with mycobacterium tuberculosis, memory T lymphocytes are generated, when specific tuberculosis antigens invade again, the memory cells are rapidly proliferated and activated to form a large number of effector T lymphocytes, gamma-interferon (IFN-gamma) and other various cytokines are released, and whether the mycobacterium tuberculosis infection exists in the body is judged by further detecting through an enzyme linked immunosorbent assay (ELISA) or an enzyme linked immunosorbent assay (ELISPOT) method. T-SPOT is a tubercle bacillus infected T lymphocyte SPOT test (T-SPOT. TB), and the method utilizes TB specific antigen to stimulate Peripheral Blood Mononuclear Cells (PBMC), judges the TB infection state by detecting IFN-gamma secreted by the antigen specific T lymphocyte, and is a clinically common immunological diagnosis method.

In recent years, IGRAs have been widely used in the diagnosis of tuberculosis, and have good sensitivity and specificity, which can improve the diagnosis rate of diseases.

At present, IFN-gamma is mainly used in tuberculosis diagnosis based on immune response of peripheral blood cells, and the diagnosis marker has good sensitivity but also has certain limitations, and clinical research shows that: because of the difference of immune response of tuberculosis patients, T-SPOT detection of about 10-15% of tuberculosis patients is negative, and therefore, the clinical expectation is to find diagnostic markers other than IFN-gamma, supplement the defects of IFN-gamma diagnosis, or improve the diagnosis sensitivity by combining with IFN-gamma.

Chinese invention patent application CN106568971A discloses IP10 and Eotaxin combined cytokine for diagnosing tuberculosis and a kit thereof. The application improves the diagnosis capability of the pulmonary tuberculosis by detecting the cell factors in the plasma of a tuberculosis patient for initial diagnosis. However, the corresponding factor appears late in plasma and has low specificity, which has certain limitations. And (4) sex.

Disclosure of Invention

The invention aims to provide a marker which has high diagnostic efficiency, simple operation, high sensitivity and good specificity and can improve the diagnostic efficiency of T-SPOT negative tuberculosis.

In order to achieve the purpose, the invention adopts the technical scheme that:

a molecular marker for diagnosing T-SPOT negative tuberculosis is a composition of IFN-gamma and IL-4.

Further, the molecular markers IFN-gamma and IL-4 are obtained by screening through a high-throughput liquid phase chip technology.

The invention also provides application of the molecular marker.

The combination of IFN-gamma and IL-4 is used as a marker in the preparation of a kit for diagnosing or monitoring tuberculosis. Further, the tuberculosis is negative tuberculosis detected by T-SPOT.

The invention also provides a kit containing the molecular marker.

A kit comprising reagents for detecting IFN- γ and IL-4, wherein the combination of IFN- γ and IL-4 act as a common detection marker for the kit.

Further, the kit is used for diagnosing or monitoring tuberculosis.

Furthermore, the kit is used for diagnosing tuberculosis with negative detection of T-SPOT.

The invention also provides a screening method of the molecular marker for diagnosing the tuberculosis with negative T-SPOT detection, which comprises the following steps:

s101, collecting healthy control, latent infected persons and tuberculosis 4 groups of positive and negative T-SPOT blood samples respectively;

s102, preparing a blood sample into peripheral blood mononuclear cells and culturing the peripheral blood mononuclear cells;

s102, detecting the cell factors of the sample by adopting a liquid chip technology to obtain cell factor expression data;

s103, comparing the expression levels of the cytokines in the 4 groups of samples, and obtaining the biomarker of the T-SPOT negative active tuberculosis through data analysis.

The marker for diagnosing T-SPOT negative tuberculosis provided by the invention is IFN-gamma and IL-4, can be used as a molecular marker for diagnosing T-SPOT negative active tuberculosis, has higher sensitivity and specificity, is fast to operate, is reasonable and feasible, and can improve the sensitivity of diagnosis.

The composition of the marker IFN-gamma and IL-4 for diagnosing the T-SPOT negative tuberculosis is a cytokine with differential expression in patients with the T-SPOT negative tuberculosis and healthy people, so that the sensitivity of diagnosis can be effectively improved, and the false negative diagnosis can be reduced.

Compared with the prior art, the marker for diagnosing T-SPOT negative tuberculosis and the application thereof provided by the invention have the advantages that:

(1) high sensitivity and specificity.

(2) High diagnosis efficiency and simple operation.

(3) Can improve the diagnosis efficiency of T-SPOT negative tuberculosis.

Drawings

FIG. 1 shows the cytokine expression levels of peripheral blood mononuclear cells of Healthy Controls (HC), latent infected patients (LTBI), patients with T-SPOT positive tuberculosis (TB IGRA +) and patients with T-SPOT negative tuberculosis (TB IGRA-) stimulated by tuberculosis antigen.

FIG. 2 is a diagram of ROC curve analysis of IFN-. gamma.and IL-2 and compositions.

FIG. 3 is a diagram of ROC curve analysis of IFN-. gamma.and IL-4 and compositions.

FIG. 4 is a schematic diagram of the procedure for diagnosing tuberculosis infection by IFN-gamma & IL-2 and IFN-gamma & IL-4.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not for limiting the scope of the present invention.

Example 1

A molecular marker for diagnosing tuberculosis infection is a composition of IFN-gamma & IL-2 and IFN-gamma & IL-4. The molecular markers IFN-gamma, IL-4 and IL-2 are obtained by screening through a high-throughput liquid phase chip technology.

The application of the composition of IFN-gamma & IL-2 and IFN-gamma & IL-4 as a marker in the preparation of a kit for diagnosing or monitoring tuberculosis.

The composition of IFN-gamma and IL-4 is used as a diagnostic marker, a liquid phase chip technology is applied for detection, the composition can be used for the rapid detection of tuberculosis infection, has higher diagnostic efficiency, is relatively simple and convenient to operate, and can improve the diagnostic efficiency of tuberculosis infection. Specific diagnostic flow pattern diagram (see fig. 4).

Example 2

Screening method of molecular marker for diagnosing tuberculosis infection

S101 case collection

The method comprises the steps of collecting subject specimens comprising 4 groups of Healthy Control (HC), latent infected persons (LTBI), patients with T-SPOT positive tuberculosis (TB IGRA +) and patients with T-SPOT negative tuberculosis ((TB IGRA-) active tuberculosis patients refer to patients with tuberculosis clinical symptoms, and are confirmed to be diagnosed by sputum acid-fast bacillus smear or culture positive and pathologically confirmed or sputum molecular biology confirmed, wherein the patients with tuberculosis comprise T-SPOT positive tuberculosis and tuberculosis infection, the grouping standard of latent infected persons is high risk factors of tuberculosis infection, positive detection of tuberculosis specific T-SPOT, and chest slices have no evidence of tuberculosis infection, and the healthy control is high risk factors of tuberculosis infection, and the chest slices have no obvious abnormality.

All the people can eliminate tumor, take immune regulation medicine to treat, HIV infection and other autoimmune diseases.

Preparation of S102 Peripheral Blood Mononuclear Cells (PBMCs)

(1) In the morning, 6mL of peripheral blood of each subject was collected on an empty stomach in a blood collection tube containing heparin sodium anticoagulation, and nucleated cells were prepared within 6 hours.

(2) 4mL of lymphocyte separation liquid is added into a 15mL centrifuge tube, a blood sample is slowly added along the tube wall at a constant speed by using a Pasteur pipette, the blood sample is prevented from being mutually dissolved with the lymphocyte separation liquid, and the blood sample is centrifuged at the room temperature of 1000g for 20 minutes.

(3) Sucking the clouded PBMCs cell layer (the PBMCs cell layer is positioned between the plasma layer and the separation liquid layer) into a new 15mL centrifuge tube, adding 1640 culture medium to 10mL, uniformly mixing, and centrifuging at 600g room temperature for 7 minutes.

(4) The supernatant was discarded, the precipitate was mixed well, 1640 medium was added thereto to 10mL, the mixture was mixed well, and centrifuged at 350g at room temperature for 7 minutes.

(5) The supernatant was discarded, 500. mu.L of serum-free medium was added, the cells were resuspended, and the cell suspension was prepared at a concentration of 2.5X 106/ml after cell counting.

S103 PBMCs cell culture

(1) 100. mu.L of 2.5X 106/ml resuspended PBMC cells were added to a 96-well cell culture plate, and tuberculosis-specific antigens (ESAT-6 and CFP-10) were added to each well, and the mixture was incubated at 37 ℃ in a 5% CO2 incubator for 16 to 20 hours.

(2) Cell culture supernatant was aspirated, and 130. mu.L of the supernatant was aspirated from each well and transferred to 1.5ml EP tubes.

S104 liquid phase chip technology detection

The detection principle is as follows: 12 kinds of fluorescent encoding microspheres are respectively coupled with monoclonal antibodies of 12 kinds of human cell factors (IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17, IFN-gamma, IFN-alpha and TNF-alpha) and mixed in certain quantity. In the incubation process, the capture antibody microspheres can capture twelve cytokines in a sample, and a detection complex of capture antibody microspheres-antigen-biotin labeled detection antibody-SA-PE can be formed after a biotin labeled paired detection antibody and streptavidin-phycoerythrin (SA-PE) are added. The intensity of the PE fluorescence is positively correlated with the concentration of the cytokine marker in the calibrator or sample. The concentration of the calibrator and the PE fluorescence signal intensity can be used by a detector to form a concentration-fluorescence intensity standard curve by fitting, and then the cytokine concentration in the sample is respectively calculated by using a curve equation.

The research carries out the detection of the cell factors on cell culture liquid supernatants after the specific antigen stimulation of four groups of healthy controls, latent infected patients, T-SPOT positive tuberculosis patients and T-SPOT negative tuberculosis patients, and comprises the following specific operation steps:

1) all reagents, standards and samples are placed at room temperature for 20min for balance

2) Preparing standard substance, resuspending the standard substance with 500 μ L specimen diluent, shaking for 5-10s, incubating for 20min on ice, and diluting the standard substance according to the instruction

3) Adding capture microsphere mixed solution 25 μ L and washing buffer solution 150 μ L into reaction well, shaking for 20s, placing reaction well plate on magnetic separator for 1min, and discarding supernatant

4) Adding 25 mu L of biotin-labeled detection antibody, 25 mu L of phosphate buffer solution and 25 mu L of sample or standard substance into the reaction hole in sequence

5) Vibrating at room temperature for 60min after pasting a membrane sealing plate, washing for 3 times, and adding 50 μ L phycoerythrin-labeled streptavidin into each hole

6) Shaking at room temperature for 5min after pasting a membrane sealing plate, washing for 3 times, adding 150 μ L of washing buffer solution into each hole, and detecting on a machine

7) The data analysis is carried out by adopting the analysis software atansys corresponding to the Luminex system, and the concentration of the finally obtained cytokine is expressed by pg/ml

S105 screening biomarkers

Based on the detected expression levels of 12 cytokines in these 4 populations, cytokines with differential expression in healthy controls and tuberculosis-infected patients were selected: the expression of IL-2, IL-4 and IFN-gamma after the stimulation of the tuberculosis specific antigen is obviously different between the two groups and can be used as a diagnostic biomarker (figure 1).

Example 3

Diagnostic value of IFN-gamma & IL-2 and IFN-gamma & IL-4 compositions

To further analyze the diagnostic value of these cytokines, first, a receiver-operating-characterization (ROC) analysis was performed to evaluate the ability of individual cytokines to distinguish between the two groups using the area under the curve (AUCs) in the ROC analysis. The greater the AUCs value, the better the diagnostic effect of the cytokine. In order to improve the sensitivity of diagnosis, the diagnosis model is divided into two steps: the first step adopts the cytokines IFN-gamma and IL-2 to identify and diagnose the AUC of the tuberculosis infection respectively as 0.967 and 0.957, adopts Logistic stepwise regression model to calculate the AUC of the tuberculosis infection identified by the compositions of the cytokines IFN-gamma and IL-2 as 0.969 (see figure 2), the diagnostic sensitivity is 80.8 percent, the specificity is 97.4 percent, the second step adopts the cytokines IFN-gamma and IL-4 to identify and diagnose the AUC of the tuberculosis infection respectively as 0.758 and 0.676, adopts the Logistic stepwise regression model to calculate the AUC of the tuberculosis infection identified by the compositions of the cytokines IFN-gamma and IL-4 as 0.774 (see figure 3), the overall diagnostic sensitivity is 89.9 percent, the specificity is 92.3 percent, and has higher diagnostic efficiency.

The cytokines differentially expressed in PBMC cells between healthy controls and tuberculosis-infected patients were screened as described. The screened cell factor is used as a diagnosis marker, a liquid phase chip technology is applied for detection, the method can be used for rapid detection of tuberculosis infection, has high diagnosis efficiency, is relatively simple and convenient to operate, and can improve the detection rate of T-SPOT negative active tuberculosis. Specific diagnostic flow pattern diagram (see fig. 4).

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various changes may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are included in the protective scope of the present invention.

It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.

In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.

Claims (8)

1. A marker for diagnosing tuberculosis infection, characterized by: the molecular marker is a molecular marker for diagnosing T-SPOT negative tuberculosis; the marker is a combination of IFN-gamma and IL-4.

2. The molecular marker of claim 1, wherein: the molecular markers IFN-gamma and IL-4 are obtained by screening through a high-throughput liquid phase chip technology.

Use of a combination of IFN- γ and IL-4 as a marker in the preparation of a kit for diagnosing or monitoring tuberculosis.

4. Use according to claim 3, characterized in that: the tuberculosis is negative tuberculosis detected by T-SPOT.

5. A kit, characterized in that: it contains reagents for detecting IFN-gamma and IL-4, wherein the combination of IFN-gamma and IL-4 is used as a joint detection marker of the kit.

6. The kit of claim 4, wherein: the kit is used for diagnosing or monitoring tuberculosis.

7. The kit according to claim 4 or 5, characterized in that: the kit is used for diagnosing tuberculosis with negative detection of T-SPOT.

8. A screening method of a molecular marker for diagnosing T-SPOT negative tuberculosis is characterized by comprising the following steps:

s101, collecting healthy control, latent infected persons and tuberculosis 4 groups of positive and negative T-SPOT blood samples respectively;

s102, preparing a blood sample into peripheral blood mononuclear cells and culturing the peripheral blood mononuclear cells;

s103, detecting the cell factors of the sample by adopting a liquid chip technology to obtain cell factor expression data;

s104, comparing the expression levels of the cytokines in the 4 groups of samples, and obtaining the biomarkers of the T-SPOT negative active tuberculosis through data analysis.

Images