CN116466084A - Reagent for detecting tuberculosis infection state and application thereof - Google Patents
Reagent for detecting tuberculosis infection state and application thereof Download PDFInfo
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- 201000008827 tuberculosis Diseases 0.000 title claims abstract description 102
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- 101001057048 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) ESAT-6-like protein EsxB Proteins 0.000 claims abstract description 36
- 101100374224 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) Rv2028c gene Proteins 0.000 claims abstract description 32
- 230000000638 stimulation Effects 0.000 claims abstract description 31
- 230000028327 secretion Effects 0.000 claims abstract description 28
- 102100021943 C-C motif chemokine 2 Human genes 0.000 claims abstract description 25
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- 108010012236 Chemokines Proteins 0.000 claims abstract description 20
- 102000019034 Chemokines Human genes 0.000 claims abstract description 20
- 108010074328 Interferon-gamma Proteins 0.000 claims abstract description 19
- 238000003748 differential diagnosis Methods 0.000 claims abstract description 14
- 238000001514 detection method Methods 0.000 claims abstract description 11
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- 102000007079 Peptide Fragments Human genes 0.000 claims description 20
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- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
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- 208000032420 Latent Infection Diseases 0.000 claims description 6
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/5695—Mycobacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to the technical field of biological detection, and discloses a reagent for detecting tuberculosis infection state and application thereof. The diagnosis of active tuberculosis and the differential diagnosis of latent tuberculosis infection are realized by detecting the secretion of the cytokine IFN-gamma and/or the chemokine MCP-1 after the stimulation of the antigen CFP-10 and/or the antigen Rv2028c. The method has high sensitivity, high specificity, high accuracy and high precision, and has important significance for improving cure rate, improving prognosis and reducing spreading risk.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a reagent for detecting tuberculosis infection state and application thereof.
Background
The number of tuberculosis latent infectious agents is huge, 5% -10% of tuberculosis latent infectious agents can develop into Active Tuberculosis (ATB), and differential diagnosis of ATB and tuberculosis latent infection (LTBI) has important significance for improving cure rate, improving prognosis and reducing transmission risk.
The current gamma interferon release assay (Interferon gamma release assays, IGRAs) based on ESAT-6 and CFP-10 as antigens has high accuracy in identifying tuberculosis infected and uninfected populations, with two methods widely used internationally: one is QuantiFERON-TB Gold In-Tube (QFT-GIT), which uses an Enzyme-linked immunosorbent assay (Enzyme-linked Immunosorbent Assay, ELISA) to detect IFN-gamma levels released from sensitized T cells In whole blood after re-stimulation with a Mycobacterium tuberculosis specific antigen. Another is T-SPOT.TB, the number of T cells capable of releasing IFN-gamma in peripheral blood mononuclear cells (Peripheral Blood Mononuclear Cells, PBMC) under stimulation with a Mycobacterium tuberculosis specific antigen, as determined by the Enzyme-linked immunosorbent assay (Enzyme-linked Immunospot Assay, ELISPOT).
The world health organization report shows that the proportion of the Chinese pulmonary tuberculosis etiology diagnosis is about 55 percent. In ATB patients diagnosed in integrated hospitals, the proportion of confirmed etiology is less than 30%. For comprehensive hospitals, the immunological detection means is more convenient and quicker for diagnosing tuberculosis, and when etiology evidence cannot be obtained, the IGRAs have certain auxiliary diagnosis value and popularization for active tuberculosis. However, the IGRAs disadvantage is also apparent: the diagnosis efficiency is not high, and the problems of ATB and LTBI cannot be distinguished, so that the application of the kit in tuberculosis high-prevalence areas is limited, and therefore, a better biomarker is required to be sought to assist diagnosis of ATB and differential diagnosis of ATB and LTBI.
Disclosure of Invention
Aiming at the defects of the existing method, the invention provides a reagent for detecting tuberculosis infection state and application thereof. The invention can realize diagnosis of ATB and differential diagnosis of ATB and LTBI by detecting secretion of cytokine IFN-gamma and/or chemokine MCP-1 after stimulation by antigen CFP-10 and/or antigen Rv2028c.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the present invention provides a reagent for detecting the secretion of the cytokines IFN-gamma and the chemokines MCP-1 under antigen stimulation, in particular for diagnosing active tuberculosis and/or for differential diagnosis of active tuberculosis and tuberculosis latent infection.
Further, the antigens are tuberculosis specific virulence antigen CFP-10 and tuberculosis latency related antigen Rv2028c.
In a second aspect, the invention provides a kit for detecting the status of tuberculosis infection, the kit comprising reagents for detecting the secretion of the cytokines IFN-gamma and the chemokine MCP-1 under antigen stimulation as described above.
Further, the kit also comprises the tuberculosis specific virulence antigen CFP-10 and tuberculosis latency-related antigen Rv2028c.
Further, the tuberculosis specific virulence antigen CFP-10 is a CFP-10 peptide fragment library, and contains 18 peptide fragments with sequences shown as SEQ ID NO. 1-18;
the tuberculosis latency related antigen Rv2028c is Rv2028c peptide fragment library, and contains 19 peptide fragments with sequences shown as SEQ ID NO. 19-37.
Further, the detection of tuberculosis infection status is specifically diagnosis of ATB and/or differential diagnosis of ATB and LTBI.
In a third aspect, the present invention provides a method for detecting the secretion of the cytokine IFN-. Gamma.and/or chemokine MCP-1 under stimulation of the antigen CFP-10 and/or the antigen Rv2028c, comprising the steps of:
step 1, preparing an antigen CFP-10 and/or an antigen Rv2028 c;
step 2, stimulation is performed by using an antigen CFP-10 and/or an antigen Rv2028 c;
step 3, the secretion amount of the cytokines IFN-gamma and/or the chemokines MCP-1 is detected.
Further, the step 1 specifically includes: the antigen CFP-10 and/or the antigen Rv2028c are respectively synthesized by peptide fragments shown in the table above, each peptide fragment of the antigen CFP-10 is 15 amino acids long, 10 amino acids are overlapped at two ends, and the final purity is more than or equal to 95%; each peptide segment for preparing the antigen Rv2028c is 28 amino acids long, 14 amino acids are overlapped at two ends, and the final purity is more than or equal to 80%.
Further, the specific process of the step 2 is as follows: mixing whole blood with RPMI1640 according to the volume ratio of 1:2, adding antigen CFP-10 and/or antigen Rv2028c to make the final concentration be 10 mug/ml, transferring to a 37 ℃ incubator for 16-20 h, taking supernatant, centrifuging at 10000 rpm for 2min, and obtaining the sample to be detected after antigen stimulation.
In a fourth aspect, the invention provides the use of a reagent as hereinbefore described or an antigen as hereinbefore described in the manufacture of a kit for detecting tuberculosis infection status
Compared with the prior art, the invention has the following advantages:
the invention realizes diagnosis of ATB and differential diagnosis of ATB and LTBI by detecting secretion amounts of cytokines IFN-gamma and chemokines MCP-1 after being stimulated by antigens CFP-10 and Rv2028c. For diagnosis of ATB, the area under the working characteristic curve (AUC), sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio are 0.941, 85.0%, 95.7%, 89.5%, 93.6%, 19.55, 0.16, respectively; when used for differential diagnosis of ATB and LTBI, AUC, sensitivity, specificity, positive predictive value, negative predictive value, and negative likelihood ratio are 0.948, 85.0%, 100%, 89.3%, and 0.15, respectively. The method has high sensitivity, high specificity, high accuracy and high precision, and has important significance for improving cure rate, improving prognosis and reducing spreading risk.
Drawings
FIG. 1 shows secretion of the cytokines IFN-. Gamma.and chemokine MCP-1 in the absence of specific antigen stimulation.
FIG. 2 shows secretion of the cytokines IFN-gamma and chemokine MCP-1 under stimulation of the antigen CFP-10.
FIG. 3 shows secretion of cytokine FN-gamma and chemokine MCP-1 under stimulation of antigen Rv2028c.
Fig. 4 is a diagnostic ROC curve for active tuberculosis.
Fig. 5 is a differential diagnosis ROC curve for active tuberculosis and tuberculosis latent infection.
Detailed Description
The technical scheme of the invention is specifically and specifically described below with reference to the embodiment of the invention and the attached drawings. It should be noted that the following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
To evaluate the diagnostic efficacy of the invention for ATB and differential diagnosis of ATB and LTBI, 66 subjects in beijing co-hospital and beijing thoracic hospital between 2022 and 2 months were selected, including 20 ATB patients (confirmed by etiology), 25 LTBI medical staff with a history of intimate contact with tuberculosis, 11 patients with fever in contemporaneous hospitalization, and 10 healthy newborns from beijing co-hospital (tuberculosis infection has been excluded by tspan.tb and chest film).
The specific operation steps are as follows:
1. peptide fragment library synthesis
The virulence factor CFP-10 peptide fragment library contains 18 peptide fragments, each peptide fragment is 15 amino acids long, 10 amino acids are overlapped at two ends, and the purity is more than or equal to 95%; the tuberculosis latency related antigen Rv2028c peptide fragment library contains 19 peptide fragments, each peptide fragment is 28 amino acids long, two ends are overlapped by 14 amino acids, and the purity is more than or equal to 80%. The detailed peptide sequences are shown in Table 1 below.
Table 1 peptide fragment sequences of CFP-10 and Rv2028c
2. Antigen stimulation
1. 6ml of fresh heparin anticoagulated venous blood of a patient to be tested is collected, and 0.5 ml/well of 5-to 24-well cell culture plates are sub-packaged.
2. RPMI1640 (10% fbs, 2% penicillin-streptomycin) was added per well to 1:2 and whole blood are mixed uniformly, CFP-10 and Rv2028c (the final concentration is 10 mug/ml) are added for stimulation respectively, an equal volume of RPMI1640 is added as a non-antigen stimulation control, and the mixture is transferred to a 37 ℃ incubator for 16-20 h.
3. The supernatant in the cell culture plate was pipetted into a 1.5ml centrifuge tube and centrifuged at 10000 rpm for 2min.
4. Centrifuging, collecting supernatant, and freezing at-20deg.C.
3. Luminex platform chemokine detection
1. Preparation before the experiment:
all reagents were returned to room temperature.
Preparation of a washing solution: 25 XWash Buffer was diluted to 1X with deionized water.
Preparation of a standard: hybrid standard (a\b\c): ensure that the standard powder is at the bottom of the bottle, and according to the instruction book of the kit, the standard A is diluted to 0.275ml by RD21 diluent, the standard B is diluted to 0.225ml, and the standard C is diluted to 0.225ml. After dilution, the standard vials were gently turned upside down and turned to ensure no powder residue, left to stand for 15-20min, 100 μl of each standard was mixed with calibration diluent RD6-52 to form standard 1 tubes, and the final volume of standard 1 tubes would be 1000 μl (3 standards+700 μ lRD 6-52).
Standard substance dilution: 200 μl of calibrator diluent RD6-52 is injected into 6 tubes labeled 2-7. Standard 1 was diluted 3-fold to tube 2 and tubes 3-7 were as above. The individual tubes were thoroughly mixed prior to the next transfer. Standard 1 is used as the high standard. Calibrator diluent RD6-52 served as a blank.
2. Experimental procedure
(1) The black opaque 96-well plate was washed once with 1 XWash Buffer, 200 μl/well, and shaken on a shaker for 10 minutes before loading.
(2) Pouring out the liquid in the hole, and reversely buckling the hole on the absorbent paper to tap, so as to absorb the redundant liquid.
(3) Mu.l of RD6-52 serum matrix was added to the blank wells.
(4) 50 μl of the prepared standard was added to the wells of the standard from low to high concentration.
(5) Mu.l of sample was added to the sample well.
(6) The mixed microspheres were resuspended in 50 μl per well on a shaker. Each time a few wells are added, the microspheres are resuspended once again to avoid sinking.
(7) The 96-well plate was sealed with a sealing plate film, covered with aluminum foil paper to avoid light, and incubated for 2 hours on a horizontal shaker at 800 rpm.
(8) The 96-well plate was placed on a magnetic separation plate and allowed to stand for 2 minutes so that the magnetic microspheres were adsorbed to the bottom of the well.
(9) The liquid in the wells was discarded and the magnetic separation plate was removed.
(10) 1x Wash Buffer 100 μl of each well of the 96-well plate is added, and after standing for 1min, the plates are placed on a magnetic rack for 2min, and then the magnetic separation plates are removed.
(11) Repeating the steps 9 and 10 for 3 times.
(12) 50 μl/well of Biotin-labeled detection antibody was added and incubated for 1 hour at room temperature with a horizontal shaker at 800 rpm.
(13) Repeating the 8-10 steps for 3 times.
(14) PE-labeled streptavidin was added at 50. Mu.l/well and incubated for 30min with shaking on a horizontal shaker at 800rpm at room temperature.
(15) Repeating the 8-10 steps for 3 times.
(16) 100 μl/well of buffer was added and the microspheres were resuspended by shaking incubation for 2 minutes.
(17) The detection was performed on a Luminex 200 instrument.
4. Analysis of results
FIG. 1 shows the secretion of the cytokines IFN-. Gamma.and chemokine MCP-1 without stimulation of the antigen, as can be seen: under the stimulation of no specific antigen, the secretion amount of IFN-gamma can distinguish tuberculosis infection from normal people, but ATB patients and febrile people cannot be distinguished, and ATB and LTBI cannot be distinguished; the secretion amount of MCP-1 can distinguish ATB from LTBI, but it cannot distinguish tuberculosis infected people (ATB and LTBI) from healthy people, and tuberculosis infected people from febrile patients, and it can be seen that the two cytokines cannot distinguish ATB and LTBI without stimulation.
FIG. 2 shows the secretion of the cytokines IFN-. Gamma.and chemokine MCP-1 under CFP-10 stimulation, as follows: under the stimulation of CFP-10, the secretion amount of IFN-gamma has obvious difference between tuberculosis infection and healthy peoplep< 0.05), while there is also a significant difference between ATB and febrile patients, suggesting that IFN- γ has the ability to diagnose ATB under CFP-10 stimulation, but cannot differentiate between ATB and LTBI. The secretion of MCP-1 is obviously different from ATB, healthy people and febrile peoplep< 0.05) and also there is a significant difference in ATB and LTBIp＜0.05）。
FIG. 3 shows secretion of the cytokines IFN-. Gamma.and chemokine MCP-1 under Rv2028c stimulation, as follows: under the stimulation of Rv2028c, IFN-gamma secretion is significantly different from that of LTBI (p < 0.05) and LTBI patients, and also from that of ATB and LTBI patientsp< 0.05), extractIFN-gamma is shown to have the potential to distinguish LTBI and to discriminate ATB from LTBI simultaneously under Rv2028c stimulation, but ATB cannot be accurately discriminated from febrile populations. The detection of the secretion of MCP-1 under the stimulation of Rv2028c can accurately identify ATB with healthy people and febrile peoplep< 0.05), but is not yet able to diagnose and identify ATB and LTBIp＞0.05）。
The above results are combined to obtain: by combining two tuberculosis specific antigens of CFP-10 and Rv2028c, the secretion amounts of cytokine IFN-gamma and chemokine MCP-1 are detected, and diagnosis of ATB and differential diagnosis of ATB and LTBI can be realized.
5. Diagnostic efficacy evaluation
To further demonstrate the conclusion, the present invention also carried out diagnostic efficacy evaluation
1. Diagnostic efficacy evaluation of ATB
The total number of ATB patients, 20 persons but not ATB 46 persons, and peripheral blood of patients stimulated by CFP-10 and Rv2028c antigens, and secretion amounts of IFN-gamma and MCP-1 were detected, and the results show that when the test subjects were used for diagnosing ATB, the area under the working characteristic curve (AUC), sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio and negative likelihood ratio were respectively 0.941, 85.0%, 95.7%, 89.5%, 93.6%, 19.55 and 0.16. Has high sensitivity, high specificity, high accuracy and high precision.
2. Differential diagnostic efficacy evaluation of ATB and LTBI
The results of stimulation of peripheral blood of patients with CFP-10 and Rv2028c antigens in common with 20 ATB patients and 25 LTBI patients showed that the secretion amounts of IFN-gamma and MCP-1 were 0.948, 85.0%, 100%, 89.3% and 0.15 in terms of AUC, sensitivity, specificity, positive predictive value, negative predictive value and negative likelihood ratio, respectively, when used for distinguishing ATB from LTBI. Also has high sensitivity, high specificity, high accuracy and high precision.
In conclusion, the cytokine IFN-gamma and the chemokine MCP-1 secretion after being stimulated by the antigen CFP-10 and the antigen Rv2028c are detected, so that the kit has higher diagnosis efficacy on diagnosis of ATB and identification of ATB and LTBI.
While the invention has been described in detail in terms of the general description and the specific embodiments, it will be apparent to those skilled in the art that various modifications and improvements can be made thereto without departing from the spirit of the invention.
Claims (11)
1. A reagent for detecting tuberculosis infection status, characterized in that: the reagent is a reagent for detecting the secretion of cytokines IFN-gamma and chemotactic factor MCP-1 under antigen stimulation, and the detection of tuberculosis infection state is specifically used for diagnosing active tuberculosis and/or differential diagnosis of active tuberculosis and tuberculosis latent infection.
2. The reagent for detecting tuberculosis infection status as described in claim 1, wherein: the antigens are tuberculosis specific virulence antigen CFP-10 and tuberculosis latency related antigen Rv2028c.
3. A kit for detecting the status of tuberculosis infection, characterized in that: the kit comprises the reagent of claim 1.
4. A kit for detecting a tuberculosis infection status as described in claim 3, wherein: the kit further comprises the antigen of claim 2.
5. The kit for detecting a tuberculosis infection status as described in claim 4, wherein: the tuberculosis specific virulence antigen CFP-10 is a CFP-10 peptide fragment library and contains 18 peptide fragments with sequences shown as SEQ ID NO. 1-18;
；
the tuberculosis latency related antigen Rv2028c is Rv2028c peptide fragment library, and contains 19 peptide fragments with sequences shown as SEQ ID NO. 19-37.
6. The reagent according to any one of claims 1-2, wherein: the detection of tuberculosis infection state is specifically to diagnose active tuberculosis and/or differential diagnosis of active tuberculosis and tuberculosis latent infection.
7. The kit of any one of claims 3-5, wherein: the detection of tuberculosis infection state is specifically to diagnose active tuberculosis and/or differential diagnosis of active tuberculosis and tuberculosis latent infection.
8. A method for detecting the secretion of the cytokines IFN- γ and/or chemokine MCP-1 under stimulation by the antigen CFP-10 and/or the antigen Rv2028c, comprising the steps of:
step 1, preparing an antigen CFP-10 and/or an antigen Rv2028 c;
step 2, stimulation is performed by using an antigen CFP-10 and/or an antigen Rv2028 c;
step 3, detecting secretion amount of chemokines IFN-gamma and/or MCP-1.
9. The method for detecting cytokine IFN-. Gamma.and/or chemokine MCP-1 secretion under antigen CFP-10 and/or antigen Rv2028c stimulation according to claim 7, wherein the step 1 is specifically:
antigen CFP-10 and antigen Rv2028c are respectively synthesized by 18 peptide fragments with the sequences shown in SEQ ID NO.1-18 and 19 peptide fragments with the sequences shown in SEQ ID NO.19-37,
each peptide segment for preparing antigen CFP-10 is 15 amino acids long, 10 amino acids are overlapped at two ends, and the final purity is more than or equal to 95%; each peptide segment for preparing the antigen Rv2028c is 28 amino acids long, 14 amino acids are overlapped at two ends, and the final purity is more than or equal to 80%.
10. The method for detecting cytokine IFN-. Gamma.and/or chemokine MCP-1 secretion under antigen CFP-10 and/or antigen Rv2028c stimulation according to claim 7, wherein the specific procedure of step 2 is as follows: mixing whole blood with RPMI1640 according to the volume ratio of 1:2, adding antigen CFP-10 and/or antigen Rv2028c to make the final concentration be 10 mug/ml, transferring to a 37 ℃ incubator for 16-20 h, taking supernatant, centrifuging at 10000 rpm for 2min, and obtaining the sample to be detected after antigen stimulation.
11. Use of the reagent of claim 1 or the antigen of claim 2 for the preparation of a kit for detecting tuberculosis infection status.
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| CN202310705666.6A CN116466084A (en) | 2023-06-15 | 2023-06-15 | Reagent for detecting tuberculosis infection state and application thereof |
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