CN112684184A - Kit for detecting latent infection of mycobacterium tuberculosis - Google Patents

Abstract

The invention relates to a kit for detecting latent infection of mycobacterium tuberculosis, which comprises an immunochromatographic test strip and methylated HBHA (hepatitis B HA), wherein the immunochromatographic test strip comprises a sample loading area, a cross-linking pad containing an anti-human IFN-gamma antibody II marked by quantum dot microspheres, a detection line coated with the anti-human IFN-gamma antibody I, and a detection area and a water absorption pad which are formed by a quality control line coated with the anti-human IFN-gamma antibody II, wherein the sample loading area, the cross-linking pad and the detection line are mutually overlapped along the chromatography direction. The kit provided by the invention can be used for establishing an immunochromatography detection system for latent infection of mycobacterium tuberculosis based on quantum dot labeling, and has the advantages of simple and rapid detection process, accurate and reliable detection result and low cost. The kit can be combined with the detection of the in vitro sample of the positive person in the IGRA test, so as to identify the active tuberculosis patients and the latent tuberculosis infected persons.

Description

Kit for detecting latent infection of mycobacterium tuberculosis

Technical Field

The invention relates to a kit for detecting latent infection of mycobacterium tuberculosis, belonging to the field of biotechnology application.

Background

Tuberculosis is a chronic infectious disease caused by infection with Mycobacterium Tuberculosis (MTB), which is commonly transmitted via the respiratory tract. Most newly infected individuals do not present typical clinical symptoms, but are in the state of latent tuberculosis infection (LTBI). About 17 hundred million people worldwide have reported LTBI in 2018 by WHO. Only 5% -10% of the LTBI population suffer from Active Tuberculosis (ATB), so it is necessary to pay attention to screening latent tuberculosis infection for high tuberculosis burden areas, which helps to determine active tuberculosis patients in early diagnosis and eliminates interference of other causes on the diagnosis process of active tuberculosis.

Currently, a rapid detection means for determining whether MTB infection occurs is to detect an immunological reaction stimulated by an MTB-specific antigen. For example, by the Tuberculin Skin Test (TST) or IFN-gamma release test (IGRA); however, LTBI, an immunological status that exists in the body as MTB but has no clinically clear indications of active infection, appears positive for either the TST or IGRA test as for ATB, and therefore, cannot reliably distinguish LTBI from ATB.

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA) is an important pathogenic factor discovered in recent years. HBHA is a glycoprotein expressed by Mycobacterium tuberculosis and can be present in both the bacterial cells and the culture medium. HBHA is composed of 199 amino acids, and its coding gene Rv0475 (ID: 886272) has a total length of about 600bp and a relative molecular mass of about 28 kDa. HBHA is a multifunctional binding protein which not only can induce the aggregation of Mycobacterium tuberculosis and the adhesion between Mycobacterium tuberculosis and non-phagocytic cells (such as epithelial cells), but also can regulate the phagocytosis of Mycobacterium tuberculosis by alveolar type II epithelial cells by combining with complement C3 and mediate the dissemination of Mycobacterium tuberculosis into the lung. HBHA can also inhibit autophagy of macrophage by inhibiting LC3 in autophagy process, and creates conditions for long-term stable survival of Mycobacterium tuberculosis in macrophage; moreover, under the control of a dormant regulatory system, HBHA is highly expressed in dormant mycobacterium tuberculosis and is closely related to latent infection of the mycobacterium tuberculosis.

HBHA exists in a methylated form in mycobacterium tuberculosis, which can not only protect HBHA protein from hydrolysis, but also induce an organism to generate IFN-gamma (interferon-gamma), and play an important protective immune role in the process of infecting tuberculosis by the organism. In vitro mHBHA as antigen directly stimulates human Peripheral Blood Mononuclear Cells (PBMCs), and LTBI patients find that T lymphocytes have a significantly higher IFN-gamma (gamma-interferon) producing capacity than ATB patients and healthy control populations, which makes it possible to distinguish ATB from LTBI. However, the major problems with the established methods or kits for detecting latent infection of mycobacteria based on this feature of LTBI on IFN-gamma production ability are: the IFN-gamma detection mainly adopts enzyme-linked immunosorbent assay (ELISA) method or spot count (ELISPOT) method, and has the defects of high cost, multiple operation steps, complex process, long detection time (more than 3 hours) and the like, and the wide application of the method in the tuberculosis screening process is seriously influenced.

Disclosure of Invention

Technical problem to be solved

In order to solve the above problems of the prior art, the present invention provides a kit for detecting latent infection with Mycobacterium tuberculosis.

(II) technical scheme

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

a kit for detecting latent infection of mycobacterium tuberculosis comprises an immunochromatographic test strip and methylated HBHA, wherein the immunochromatographic test strip comprises a sample loading area, a cross-linking pad containing an anti-human IFN-gamma antibody II marked by quantum dot microspheres, a detection line coated with the anti-human IFN-gamma antibody I, and a detection area and a water absorption pad which are formed by a quality control line coated with the anti-human IFN-gamma antibody II, wherein the sample loading area, the cross-linking pad and the detection line are mutually overlapped along the chromatography direction.

Wherein, the methylated HBHA is used for stimulating IFN-gamma production of an ex vivo sample.

The kit preferably further comprises an IFN-gamma standard and mitogen as positive control substances, wherein the standard substance is dry powder or solution.

The solution can provide a standard solution with the concentration of 100pg/mL or a series of gradient concentrations, such as IFN-gamma concentration gradient standard solutions with the concentration of 5pg/mL, 12.5pg/mL, 25pg/mL, 50pg/mL and 100pg/mL, and the dosage of the mitogen in each reaction tube is 5-10 mu g/tube.

Preferably, the kit as described above, the methylated HBHA is separately contained in a reaction tube, and the methylated HBHA is coated on the inner wall of the reaction tube.

The kit preferably further comprises a blood collection tube for separating gel and anticoagulant in the reaction tube, and the dosage of the methylated HBHA is 5-10 mu g/tube.

The reaction tube added with the separation gel and the anticoagulant is simpler and easier to operate during detection without a centrifugal machine for centrifugation, and the supernatant after the reaction can be directly taken for subsequent detection after the reaction. The separation glue is an inert thixotropic polymeric glue, the specific gravity is about 1.04, the separation glue is liquefied and moved to the center of the tube during centrifugation and is between the serum or plasma and the blood forming components, a barrier is formed by solidification after centrifugation is finished, the serum and the plasma are completely separated from cells, and the anticoagulant is heparin sodium.

In the kit, preferably, the material of the sample loading area is absorbent paper, and the material of the sample loading area is the same as that of the absorbent pad; the crosslinking pad is a glass fiber membrane sprayed with an anti-human IFN-gamma antibody II marked by quantum dot microspheres, and the concentration of the anti-human IFN-gamma antibody II marked by the quantum dot microspheres is more than or equal to 1 IU/mL; the material of the detection area is a nitrocellulose membrane sprayed with a detection line of anti-human IFN-gamma antibody I with the concentration of more than or equal to 1IU/mL and an anti-human IFN-gamma antibody II with the concentration of more than or equal to 1IU/mL serving as a quality control line.

In the kit, preferably, the excitation light of the quantum dot microspheres is near-blue-violet light, preferably 365nm ultraviolet light, the fluorescence peak is located between 580 and 680nm, and the detection wavelength is 615 nm. The quantum dot microsphere excitation light wave band is wide, an excitation light source is convenient to select, the detection wavelength is located in a visible light region, the fluorescence peak wave band is narrow, the conditions are easy to realize, and the mutual interference is small.

The use method of the kit comprises the following steps:

s1, mixing the methylated HBHA with a sample to be detected, namely in-vitro whole blood, performing co-incubation, and then taking the supernatant after incubation to perform detection of an immunochromatography test strip;

s2, preparing a standard solution of IFN-gamma with a certain gradient concentration, and detecting the IFN-gamma by using an immunochromatography test strip;

s3, after the chromatographic reaction, detecting the fluorescence intensity of the detection line of the immunochromatographic test strip to obtain the fluorescence intensity of each concentration of the standard solution, and drawing a standard curve by taking the concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate;

s4, the fluorescence intensity detected by the sample to be detected corresponds to a standard curve, the concentration of the sample to be detected is obtained, and the negative and positive of the latent infection of the mycobacterium tuberculosis are judged:

if the concentration of the IFN-gamma of the sample is more than or equal to 25pg/mL (background subtraction), the sample is judged to be positive by LTBI; if the sample IFN-. gamma.concentration is < 25pg/mL (minus background), it is judged as negative for LTBI.

In the using method, preferably, in step S1, the amount of the methylated HBHA is 5 to 10 μ g/tube, the amount of the whole isolated blood is 1 to 3mL, and the incubation condition is 25 to 37 ℃ for 18 to 24 hours.

A large number of time optimization experiments prove that the short time can cause insufficient release of IFN-gamma generated by antigen stimulation, and false negative results can occur; the time is too long, which causes time waste and prolongs the time required for reporting the detection result, so the incubation time is preferably 18 to 24 hours.

Further preferably, the amount of methylated HBHA is 5 μ g/tube and the amount of whole blood ex vivo is 1mL, and the incubation is performed at 37 ℃ for 18 hours.

In step S2, the concentrations of the standard solution are 5pg/mL, 12.5pg/mL, 25pg/mL, 50pg/mL, and 100pg/mL, respectively.

In the using method of the kit, in step S3, after the chromatography reaction is carried out for 5-10 min at 20-25 ℃, a 365nm ultraviolet light source is used for detecting the fluorescence intensity at 615nm exciting light.

It should be noted that the method for detecting latent infection of Mycobacterium tuberculosis provided by the present invention is not suitable for people with cellular immune dysfunction (such as HIV infection).

(III) advantageous effects

The invention has the beneficial effects that:

the kit for detecting latent infection of mycobacterium tuberculosis comprises methylated HBHA protein and IFN-gamma immunochromatographic test paper, stimulates T lymphocytes in a latently infected peripheral blood and other in vitro samples to generate IFN-gamma by using dormancy-related antigen, namely the methylated HBHA protein, then detects the content level of the IFN-gamma by using an immunochromatographic technology, and identifies a person to be detected who is latently infected with mycobacterium tuberculosis according to the content of the IFN-gamma. The kit can be used for establishing an immunochromatography detection system for latent infection of mycobacterium tuberculosis based on quantum dot labeling, and has the advantages of simple and rapid detection process, accurate and reliable detection result and low cost.

According to the invention, by optimizing the immunochromatographic test strip, only a small amount of peripheral blood serum subjected to co-incubation needs to be loaded, and the detection time is shortened to 15-20 minutes.

The kit can be combined with the detection of the in vitro sample of the positive person in the IGRA test, so as to identify the active tuberculosis patients and the latent tuberculosis infected persons.

Drawings

FIG. 1 is a graph of the effect of concentration of methylated HBHA protein (mHBHA) on the level of IFN- γ release from stimulated whole blood samples;

FIG. 2 is a graph of the effect of the incubation time (hours) of methylated HBHA protein (mHBHA) with whole blood samples on the stimulation of IFN-. gamma.release levels;

FIG. 3 is a schematic diagram of the structure of an immunochromatographic test strip in the kit;

FIG. 4 is a standard graph of an immunochromatographic assay;

FIG. 5 shows a finished product of the immunochromatographic assay test strip;

FIG. 6 is a diagram showing the case where the test result is judged to be positive;

FIG. 7 is a diagram showing the case where the detection result is negative.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

EXAMPLE 1 optimal kit detection conditions exploration

1. In vitro stimulation of IFN- γ released methylated HBHA protein (mHBHA) optimal concentration exploration 6mL of each of 8 individual whole blood samples of latent tuberculosis infection (LTBI) were collected, 1mL of each was taken as a blank control, 1. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 20. mu.g/mL, 50. mu.g/mL of methylated HBHA protein (protein concentration is the final concentration of mHBHA added in the mixed incubation system, methylated HBHA protein was prepared by the laboratory preliminary study, the specific preparation method is described in the same patent application) and 1mL of whole blood were incubated at 37 ℃ for 24 hours, centrifuged at 3000g for 15min, the supernatant was collected, and the IFN- γ released level of each sample after blank control was subtracted was detected by ELISA (Wuhan doctor bioscience). The results are shown in figure 1 (n-8, P <0.01, vs. 1 μ g/mL) and indicate that neither too high nor too low a concentration of methylated HBHA protein induces high levels of IFN- γ, and that at a final concentration of 5 μ g/mL, methylated HBHA protein has the strongest ability to stimulate the T lymphocytes of LTBI individuals to release IFN- γ in vitro.

2. Exploration of optimal Co-incubation time of methylated HBHA protein (mHBHA) with Whole blood

5 LTBI individual whole blood samples were collected at 5mL each, incubated with 5. mu.g/mL of methylated HBHA protein (protein concentration is the final concentration of mHBHA added to the mixed incubation system) and 1mL of whole blood at 37 ℃ for 0, 6, 12, 18 and 24 hours, followed by centrifugation at 3000g, and supernatants were collected and assayed for IFN-. gamma.release levels by ELISA. The results are shown in FIG. 2, which shows that the level of IFN-. gamma.production induced by methylated HBHA protein increases with time, reaches a maximum at about 18 hours, and remains stable.

Example 2 kit and composition for detecting latent infection with Mycobacterium tuberculosis

The kit comprises an immunochromatographic test strip, a methylated HBHA coated blood collection tube containing separation gel and a heparin anticoagulant, and a methylated HBHA protein standard substance.

The bottom of the tube wall of the blood collection tube is pre-coated with methylated HBHA protein, the amount of the coated methylated HBHA protein is 5 mug/tube, the blood collection amount of each tube is 1mL, and the specific coating method is shown in the latest biochemical guideline (the method of coating the protein solution at 4 ℃ overnight).

The immunochromatographic test strip comprises a sample loading zone (for example, a sample adding hole), a cross-linking pad, a detection zone, a water absorption pad and the like which are sequentially arranged and overlapped with each other in the chromatography direction, and the two adjacent parts are tightly combined with each other, as shown in fig. 3. Wherein the absorbent pad 1: the material is absorbent paper, and excessive liquid for chromatography is absorbed; detection zone 2: the material is a nitrocellulose membrane coated with an anti-human IFN-gamma antibody I line (a T area, a detection line) with the concentration of more than or equal to 1IU/mL and an anti-human IFN-gamma antibody II line (a C area, a quality control line) with the concentration of more than or equal to 1IU/mL, and the cross-linked pad 3: the material is a glass fiber film sprayed with anti-human IFN-gamma antibody II marked by quantum dot microspheres, and the concentration of the anti-human IFN-gamma antibody II marked by the quantum dot microspheres is more than or equal to 1 IU/mL; sample loading zone 4: the material is absorbent paper, and has the function of absorbing the sample liquid to be detected and leading the sample liquid to enter the detection area; bottom plate 5: the material is cardboard or plastic sheet, which is used for carrying the above components.

Specifically, the cross-linked pad in this embodiment contains a detection line composed of an anti-human IFN-gamma antibody I line (T region, detection line) and an anti-human IFN-gamma antibody II line (C region, quality control line) with different binding sites from the anti-human IFN-gamma antibody II (antibody concentration is 1IU/mL when the detection line is prepared; and the antibody fixing method refers to the latest version of biochemical guidelines).

Anti-human IFN-. gamma.antibody I, anti-human IFN-. gamma.antibody II, and anti-human IFN-. gamma.antibody II were purchased from Mabtech Biotech Ltd.

The excitation light of the quantum dot microspheres is near-blue-violet light, preferably 365nm ultraviolet light, the fluorescence peak is located between 580-680 nm, and the detection wavelength is 615 nm. The quantum dot microsphere excitation light wave band is wide, an excitation light source is convenient to select, the detection wavelength is located in a visible light region, the fluorescence peak wave band is narrow, the conditions are easy to realize, and the mutual interference is small. The preparation of quantum dot microspheres and the method for labeling human IFN-gamma are completed by the research team and Shenzhen Jinzhen quasi-biomedical engineering Co., Ltd, which are developed and researched in cooperation, and refer to Zhen, Rong, Qiong, et al.

1. Principle for detecting latent infection of mycobacterium tuberculosis

According to the condition that the methylated HBHA protein stimulates IFN-gamma in vitro, the invention adopts a double-antibody sandwich method, namely two anti-IFN-gamma antibodies are utilized to realize the identification, fixation and marking of the IFN-gamma reaching a certain concentration, thereby determining whether the content level of the IFN-gamma generated by stimulation reaches a judgment standard by a certain marking color development means and obtaining the detection result.

2. Establishment of IFN-gamma concentration-fluorescence intensity standard curve

Weighing several grams of IFN-gamma standard (dry powder), preparing IFN-gamma concentration gradient standard solutions of 5pg/mL, 12.5pg/mL, 25pg/mL, 50pg/mL and 100pg/mL by using distilled water, and combining blank control (0pg/mL) for detection to establish an IFN-gamma concentration-fluorescence intensity standard curve. Detection conditions are as follows: taking 100 mu L of each gradient standard solution, respectively adding the gradient standard solution into a corresponding sample loading area of the immunochromatographic test strip, and performing chromatography at room temperature for 5 min; the detection wavelength of the detection line is 615 nm.

The standard curve obtained by detection is shown in figure 4.

According to a large number of previous experimental research results, the linear range of IFN-gamma concentration and fluorescence intensity is 1-10000 pg/ml. The cut-off value of the judgment result is obtained by clinical diagnostic pre-experimental research on 22 ATB and 22 LTBI infectors in the early stage, the specific method is to use the detection method to detect two groups of people, collect IFN-gamma concentration values of samples after blank control deduction to draw a working curve (ROC curve) of a subject, find out a corresponding discrimination threshold value according to a curve Youden index, namely the cut-off value is 25pg/mL, and the concentration interval corresponding to the positive LTBI is as follows: after blank control is deducted, the IFN-gamma concentration of the sample is more than or equal to 25pg/mL, and the concentration interval corresponding to LTBI negativity is as follows: the IFN- γ concentration of the sample after blank subtraction was < 25 pg/mL.

3. Detection step

Firstly, the sample requirement is as follows: 3mL of peripheral venous blood of a subject is taken, wherein 1mL is used as a blank control (no antigen is added), 1mL is reacted with mHBHA, 1mL is used as a positive control (the reaction is carried out with 5 mu g of mitogen), and the incubation reaction is carried out immediately after collection.

Collecting 1mL of whole blood (peripheral venous blood) and 5 mu g of mHBHA, fully and uniformly mixing in a blood collection tube, and then incubating, wherein the incubation conditions are as follows: standing in a water bath at 37 ℃ for 18 hours (humidity is not required);

meanwhile, the blank control and a blood collection tube added with 5 mug of mitogen and 1mL of whole blood (peripheral venous blood) are used as positive controls to be incubated under the same conditions; ③ centrifuging for 15min at 3000g after incubation, and sucking 100 mu L of supernatant;

adding the supernatant sample sucked in the previous step into a sample adding hole (shown in figure 5) of the prepared immunochromatography test strip, and performing room-temperature chromatography for 5 min;

simultaneously, adding solutions with different IFN-gamma concentration gradients into the sample adding holes of the prepared immunochromatography test strip, detecting, and drawing a standard curve;

acquiring the fluorescence intensity of the detection line by using a quantum dot detection system (615nm monochromatic fluorescence detector), then calculating the specific concentration of IFN-gamma by referring to a standard curve, and judging whether the person to be detected is LTBI positive (shown in figure 6) or LTBI negative (shown in figure 7) according to a Cut-off value;

when the mitogen is used as a positive control, the purpose is to detect whether the immune function of a subject is normal, if the IFN-gamma concentration obtained by the detection of the positive control is more than 200pg/mL, the detection result is valid, and if the IFN-gamma concentration obtained by the detection of the positive control is less than 200pg/mL, the detection result is invalid, which indicates that the immune function of the blood individual is abnormal, and the tubercle bacillus infection state of the subject cannot be identified.

A large number of experiments also prove that the provided method for detecting latent infection of mycobacterium tuberculosis is not suitable for people with cellular immune dysfunction (such as HIV infection).

In order to further facilitate the operation, the methylated HBHA protein can be coated on the tube wall of a reaction tube by spraying, the reaction tube contains separation gel and anticoagulant, wherein the separation gel is used for accurately separating serum after centrifugation, and the anticoagulant is used for preventing blood from coagulating so as to ensure that cells and the methylated HBHA fully react. The separation glue adopts thixotropic polymerized glue, and the anticoagulant is heparin sodium. Wherein, the used separation gel is about 0.5 mL/tube, the dosage of the blood heparin sodium is (15 +/-2.5) IU/mL), when the detection operation is carried out, whole blood is directly added into the test tube, a centrifugal machine is not needed for centrifugation, the supernatant after the reaction can be directly taken for subsequent detection, and the preparation method of the mitogen 5 mug/blood collection tube as the positive control is the same as the method of coating the methylated HBHA protein in the reaction tube.

Example 3

The kit prepared in the embodiment 2 is adopted to carry out a pre-experimental detection method on 40 patients clinically involved in active tuberculosis, 22 latent tuberculosis infected patients and 22 healthy contrast persons not infected with mycobacterium tuberculosis in the embodiment 2, and the result shows that the accuracy of differential diagnosis of the detection method on different tuberculosis infection state patients can reach more than 85%, and the detection sensitivity and specificity can respectively reach 86.4% and 82.5%. Moreover, the standard deviation of the result is small after more than two repeated detections on different individuals, which indicates that the reproducibility is good.

Example 4

The detection results of the kit prepared in example 2 of the present invention and the conventional IGRA kit (tuberculosis infection QFT detection kit purchased from QIAGEN) were positive when the whole blood (peripheral venous blood) of a patient clinically diagnosed as latent infection was subjected to detection, and the detection results of the kit prepared in example 2 of the present invention and the conventional IGRA kit were negative when the whole blood (peripheral venous blood) of a patient clinically diagnosed as healthy was subjected to detection, and the repetition rate was 100%. The kit prepared in example 2 of the present invention and the conventional IGRA kit are used for detecting whole blood (peripheral venous blood) of a patient clinically diagnosed as active infection (ATB), the detection result of the kit of the present invention is negative, and the detection result of the conventional IGRA kit is positive, because methylated HBHA protein serving as tuberculosis antigen related to latent infection cannot induce high-level IFN-gamma in the whole blood of the active tuberculosis patient, the kit of the present invention can be used in combination with the conventional IGRA detection to judge whether the patient is latent infection, active infection or healthy person.

The specific judgment result is as follows:

on the basis that a test subject is positive in the result of a traditional IGRA test (for example, ESAT-6 and CFP-10 stimulation IFN-gamma release test (tuberculosis infection QFT test kit is purchased from QIAGEN), the test subject is detected by using the kit provided by the invention, or a test subject is detected by using the kit and a traditional IGRA kit.

In conclusion, compared with the traditional ESAT-6 and CFP-10 stimulated IFN-gamma release test, the invention establishes a mycobacterium tuberculosis latent infection immunochromatography detection system by using the anti-human IFN-gamma antibody marked by the quantum dots, can quickly and accurately identify the mycobacterium tuberculosis latent infection and active infection on the basis of the traditional IGRA positive result, and has definite clinical application value.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in other forms, and any person skilled in the art can change or modify the technical content disclosed above into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.

Claims (10)

1. A kit for detecting latent infection of mycobacterium tuberculosis is characterized by comprising an immunochromatographic test strip and methylated HBHA, wherein the immunochromatographic test strip comprises a sample loading area, a cross-linking pad containing an anti-human IFN-gamma antibody II marked by quantum dot microspheres, a detection line coated with the anti-human IFN-gamma antibody I, and a detection area and a water absorption pad which are formed by a quality control line coated with the anti-human IFN-gamma antibody II, which are mutually overlapped along the chromatography direction.

2. The kit of claim 1, further comprising an IFN- γ standard and mitogen as positive controls, the standard being a dry powder or a solution.

3. The kit of claim 1, wherein the methylated HBHA is separately contained in a reaction tube, and the methylated HBHA is coated on the inner wall of the reaction tube.

4. The kit according to claim 3, wherein the reaction tube further comprises a blood collection tube containing a separation gel and an anticoagulant, and the amount of the methylated HBHA is 5-10 μ g/tube.

5. The kit of claim 1, wherein the material of the sample loading zone and absorbent pad is absorbent paper; the material of the crosslinking pad is a glass fiber membrane sprayed with an anti-human IFN-gamma antibody II marked by quantum dot microspheres, and the concentration of the anti-human IFN-gamma antibody II marked by the quantum dot microspheres is more than or equal to 1 IU/mL; the material of the detection area is a nitrocellulose membrane sprayed with an anti-human IFN-gamma antibody I detection line with the concentration of more than or equal to 1IU/mL and an anti-human IFN-gamma antibody II detection line with the concentration of more than or equal to 1IU/mL serving as a quality control line.

6. The kit of claim 1, wherein the excitation light of the quantum dot microspheres is near-blue-violet light, namely 365nm ultraviolet light, the fluorescence peak is located between 580 and 680nm, and the detection wavelength is 615 nm.

7. The method of using the kit of claim 1, comprising the steps of:

s1, mixing the methylated HBHA with a sample to be detected, namely in-vitro whole blood, performing co-incubation, and then taking the supernatant after incubation to perform detection of an immunochromatography test strip;

s2, preparing a standard solution of IFN-gamma with a certain gradient concentration, and detecting the IFN-gamma by using an immunochromatography test strip;

s3, after the chromatographic reaction, detecting the fluorescence intensity of the detection line of the immunochromatographic test strip to obtain the fluorescence intensity of each concentration of the standard solution, and drawing a standard curve by taking the concentration as a horizontal coordinate and the fluorescence intensity as a vertical coordinate;

s4, the fluorescence intensity detected by the sample to be detected corresponds to a standard curve, the concentration of the sample to be detected is obtained, and the negative and positive of the latent infection of the mycobacterium tuberculosis are judged:

if the concentration of the IFN-gamma of the sample is more than or equal to 25pg/mL, the sample is judged to be positive by LTBI; if the IFN-gamma concentration of the sample is less than 25pg/mL, the sample is judged to be negative for LTBI.

8. The use according to claim 7, wherein in step S1, the amount of the methylated HBHA is 5-10 μ g/tube, the amount of whole blood ex vivo is 1-3 mL, and the incubation is performed under the condition of co-incubation at 25-37 ℃ for 18-24 hours.

9. The use according to claim 7, wherein in step S2, the concentration of the standard solution is 5pg/mL, 12.5pg/mL, 25pg/mL, 50pg/mL, 100pg/mL, respectively.

10. The use of claim 7, wherein in step S3, after chromatography at 20-25 ℃ for 5-10 min, 365nm ultraviolet light source is used to detect fluorescence intensity at 615nm excitation light.

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