CN111521819A - Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis - Google Patents
Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis Download PDFInfo
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Abstract
The invention provides application of mycobacterium tuberculosis protein in preparing products for diagnosing latent tuberculosis infectors and active tuberculosis. The method provided by the invention can simultaneously detect the level of serum antibody response of three different mycobacterium tuberculosis proteins, so that latent tuberculosis infectors and active tuberculosis patients can be better identified and diagnosed, the sensitivity is higher, and the specificity is better.
Description
The present application is a divisional application of an invention patent application having an application date of 2016, 12 and 30 and an application number of 2016112595642.
Technical Field
The invention relates to the field of biomedicine, in particular to application of mycobacterium tuberculosis protein in preparing a product for diagnosing latent tuberculosis infectors and/or active tuberculosis.
Background
Tuberculosis caused by mycobacterium tuberculosis is still one of diseases seriously threatening human health, according to the latest report of the world health organization, 1040 ten thousands of new tuberculosis cases are found in 2015 all the world, about 180 thousands of tuberculosis cases die from tuberculosis, 91.8 thousands of new tuberculosis cases and 3.8 thousands of tuberculosis cases are found in China, and the total number of the tuberculosis cases is the fourth position in the world. The work of preventing and treating tuberculosis is still far in the world or in China, and the infection rate of tubercle bacillus is still high especially in some underdeveloped countries with poor sanitary conditions.
After a human body is infected with mycobacterium tuberculosis, about only 10% of people develop active tuberculosis, and 90% of people do not develop the active tuberculosis, but the tubercle bacillus in the human body is not completely eliminated, but is latent in the human body, namely tuberculosis latent infection, the people have no clinical symptoms, most of people do not develop the active tuberculosis in the life, and only 5% -10% of people can cause the tuberculosis when the immunity of the human body is reduced. At present, 1/3 people all over the world infect tubercle bacillus, and the number of visible latent infected people is large, so that the latent infected people need to be monitored, and when the infection state changes, the drug therapy is timely given to achieve early discovery and early treatment, so that the disease deterioration can be prevented, the treatment time and the side effect caused by the drug therapy can be reduced, and the spreading of tuberculosis can be reduced. Therefore, the detection and identification of the two states after tubercle bacillus infection are very important for the prevention and treatment of tuberculosis.
At present, the detection methods of tubercle bacillus infection comprise a plurality of methods, such as Tuberculin Skin Test (TST), gamma-interferon release test (IGRA), traditional phlegm acid-fast bacterium smear, rapid culture of mycobacteria, amplification (quantitative and qualitative) of tuberculosis specific nucleic acid fragments and the like. TST is skin allergy, has long service time, is mainly used for outpatient screening, although the test is simple and easy to implement, the same result can be produced in BCG inoculation when the tubercle bacillus infection is detected, natural infection cannot be identified, the specificity is not strong, and in addition, latent tuberculosis infection and active tuberculosis cannot be identified. The gamma interferon release assay (IGRA) is mainly used for detecting cellular immune response, although the effect of BCG vaccination can be eliminated, latent infectors and active tuberculosis patients cannot be distinguished, and treated patients and current infectors cannot be distinguished. However, the traditional sputum acid-fast bacteria smear, the rapid culture of mycobacteria and the like are mainly used for detecting active tuberculosis patients, have no significance for judging latent infection states, and in addition, bacteriological evidence may not be found for the active tuberculosis patients with atypical clinical symptoms. Therefore, the method for accurately identifying latent infection and active tuberculosis is of great value.
Based on different host protective immune responses caused by tubercle bacillus infection, the method for searching the serum marker to distinguish latent infection and active tuberculosis has good application prospect. The serological diagnosis method of antigen-antibody reaction is of great interest because of its simplicity, rapidity and easy availability of specimens, and some specific antigens of mycobacterium tuberculosis have been used for serological diagnosis of tubercle bacillus infection, such as secreted protein antigens: antigen 85 complex antigens, i.e., Ag85A, Ag85B, and Ag85C, etc.; lipoprotein antigen: 16kDa, 27kDa, 38kDa antigen, etc.; glycolipid antigens: lipoarabinomannan antigens, tuberculosyl lipoid antigens, and the like; however, in actual use, the antigens have the problems of low positive detection rate, cross immunity with other mycobacteria and the like, and although the antigens have certain values in the general survey, the monitoring of treatment effect, the judgment of prognosis and the prompt of relapse of high risk groups, the antigens cannot be used for the definite diagnosis of latent mycobacterium tuberculosis infection at present.
Therefore, it is very important to find a new method and marker capable of accurately diagnosing latent tuberculosis infectors and active tuberculosis patients.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, products for identifying and diagnosing latent tuberculosis infectors and/or active tuberculosis have low positive detection rate and cannot identify and diagnose latent mycobacterium tuberculosis infection, and provides an application of mycobacterium tuberculosis protein in preparing products for diagnosing latent tuberculosis infectors and/or active tuberculosis.
In order to solve the technical problems, the technical scheme provided by the invention on one hand is as follows:
use of a product for detecting the level of an anti-Rv 0284 antibody, an anti-Rv 2002 antibody and/or an anti-Rv 2421c antibody in a sample in the manufacture of a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis.
In the technical scheme of the invention, any product for detecting the level of the anti-Rv 0284 antibody, the anti-Rv 2002 antibody and/or the anti-Rv 2421c antibody in the sample can be used for preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis.
Preferably, the product for detecting the level of the anti-Rv 0284 antibody, the anti-Rv 2002 antibody and/or the anti-Rv 2421c antibody in the sample comprises an Rv0284 protein, an Rv2002 protein and/or an Rv2421c protein.
More preferably, the Rv0284 protein is a protein shown in SEQ ID NO.1, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified to have the same function as the protein shown in SEQ ID NO. 1; the Rv2002 protein is shown in SEQ ID NO.2, or the protein which has the same function with the protein shown in SEQ ID NO.2 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified; the Rv2421c protein is shown in SEQ ID NO.3, or the protein which has the same function with the protein shown in SEQ ID NO.3 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified.
The term "modification" as used above may be any prior art modification of proteins, including, but not limited to, substitution and/or addition and/or deletion of amino acid residues to form derivatives of the original protein.
Preferably, the detection may be any suitable detection method in the prior art, but preferably, the detection method may be an enzyme-linked immunosorbent assay, an agglutination assay, a precipitation assay, an E-rosette assay, a phagocytosis assay, an immunofluorescence assay, a colloidal gold immunochromatography assay, a spotted gold immunodiafiltration assay, and/or an electrochemiluminescence assay. More preferably, in one embodiment of the present invention, the detection method is an enzyme-linked immunosorbent assay.
Preferably, the sample may be any suitable sample obtained from the subject. Preferably, however, the sample comprises serum, plasma, saliva, urine, pleural effusion and/or cerebrospinal fluid. More preferably, in one embodiment of the present invention, the sample is serum.
Another aspect of the present invention provides a marker composition for identifying, diagnosing and/or screening latent infection with tuberculosis and/or active tuberculosis, the marker composition consisting of an anti-Rv 0284 antibody, an anti-Rv 2002 antibody and/or an anti-Rv 2421c antibody;
wherein, the Rv0284 is a protein which is shown in SEQ ID NO.1 or has the same function with the protein shown in SEQ ID NO.1 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified; the Rv2002 is a protein shown in SEQ ID NO.2, or one or more amino acid residues in an amino acid sequence shown in SEQ ID NO.2 are modified to have the same function with the protein shown in SEQ ID NO. 2; the Rv2421c is a protein shown in SEQ ID NO.3, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified to have the same function with the protein shown in SEQ ID NO. 3.
In another aspect of the invention, the invention provides a use of the above marker composition in the preparation of a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis.
Another aspect of the invention provides a kit for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis, the kit comprising Rv0284 protein, Rv2002 protein and/or Rv2421c protein.
Preferably, the Rv0284 protein is a protein shown in SEQ ID NO.1, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified to have the same function as the protein shown in SEQ ID NO. 1; the Rv2002 protein is shown in SEQ ID NO.2, or the protein which has the same function with the protein shown in SEQ ID NO.2 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified; the Rv2421c protein is shown in SEQ ID NO.3, or the protein which has the same function with the protein shown in SEQ ID NO.3 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified.
The Rv0284 protein, Rv2002 protein and/or Rv2421c protein contained in the kit can be in any form, including but not limited to direct protein samples, and the form of immobilizing the protein on a carrier, such as a detection chip.
The kit comprises at least kit instructions in addition to Rv0284 protein, Rv2002 protein and/or Rv2421c protein.
The specification describes criteria for determining the results: and (3) using the three proteins for joint detection, if at least 2 antibodies in the anti-Rv 0284 antibody, the anti-Rv 2002 antibody and/or the anti-Rv 2421c antibody are positive in the detection result of the sample to be detected, judging that the sample to be detected is positive in active tuberculosis, otherwise, judging that the sample to be detected is latent tuberculosis infection.
In another aspect, the invention provides the use of the above-mentioned kit in the preparation of a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis.
The invention has the beneficial effects that:
the kit can simultaneously detect the level of the serum antibody response of three different mycobacterium tuberculosis proteins, thereby better identifying and diagnosing latent tuberculosis infectors and active tuberculosis patients, and having higher sensitivity and better specificity.
Drawings
Fig. 1 is a statistical graph of ELISA for detecting the expression levels of anti-Rv 0284 antibody, anti-Rv 2002 antibody and anti-Rv 2421c antibody in sera of tuberculosis latent infected patients (n-93) and active tuberculosis patients (n-92);
FIG. 2 is a ROC graph of antibodies corresponding to the joint detection of three antigens;
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of the Rv0284 protein in the embodiment of the invention;
SEQ ID NO.2 is the amino acid sequence of the Rv2002 protein in the embodiment of the invention;
SEQ ID NO.3 is an amino acid sequence of the Rv2421c protein in the embodiment of the invention.
Detailed Description
The invention discloses application of a product for detecting the level of an anti-Rv 0284 antibody, an anti-Rv 2002 antibody and/or an anti-Rv 2421c antibody in a sample in preparing a product for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1: collection and processing of samples
The sample to be detected is serum, the collection method comprises the steps of collecting blood of a subject in an empty stomach, using a common biochemical test tube without anticoagulant, standing at room temperature, centrifuging at the normal temperature of 3000 r/min for 10 minutes after natural coagulation, collecting the serum, subpackaging in a freezing tube, and placing in a low-temperature refrigerator at the temperature of minus 80 ℃ for storage for use in experiments.
The samples used in the present invention are divided into two groups: a latent infection group and an active tuberculosis patient group; wherein, the latent infection group refers to the patients without clinical chief complaints, positive TST experiment and T-SPOT experiment, and negative chest X-ray; the active tuberculosis patient group refers to confirmed tuberculosis patients which are positive through smear or culture and do not start or just start systemic chemical drug treatment; both groups excluded diabetes, HIV infection and other autoimmune diseases.
The specimens of the latent infection group used by the invention are from epidemiological research projects of epidemiological research rooms of tuberculosis breast tumor research institute of Beijing city, the specimens of the active tuberculosis patient group are from inpatients of each disease area of tuberculosis department of Beijing thoracic hospital affiliated to capital medical university, the subject is approved by the research ethics committee of Beijing thoracic hospital affiliated to capital medical university, and all research objects are informed.
Example 2: detection of samples using ELISA assays
1. Establishing various buffers, diluents, reaction solutions and stop solutions required by an ELSIA experiment:
(1) coating buffer (pH9.6, 0.05mol/L carbonate buffer):
Na2CO31.59g，NaHCO3adding double distilled water to 1000ml for 2.93g, and adjusting the pH to 9.6;
(2) PBS solution ph 7.4:
NaCl 8.0g，KCl 0.2g，KH2PO40.2g，Na2HPO4·12H2O 2.9g，
adding double distilled water to 1000ml, and adjusting the pH value to 7.4;
(3) PBST wash ph 7.4:
adding Tween-200.5 ml into 1000ml of PBS solution, and adjusting the pH to 7.4;
(4) blocking solution (PBS solution with skim milk powder, ph 7.4):
5g of skimmed milk powder is added into 100ml of PBS solution;
(5) substrate buffer: 0.2M NaH2PO4(28.4g/L)25.7ml，
24.3ml of 0.1M citric acid (19.2g/L) and 50ml of distilled water were added.
(6) TMB (tetramethylbenzidine) use solution 0.5ml of TMB (10mg/5ml of absolute ethanol) substrate
Buffer (pH5.5)10ml 0.75% H2O232μl；
(7) Stop solution (2M H)2SO4)：21.32ml H2SO4，178.68ml H2O。
(8) Secondary antibody: horseradish peroxidase (HRP) labeled goat anti-human IgG;
(9) a 96-hole enzyme label plate; nunc Incorporated (Theromo, Gebenhagen, Denmark)
2. A detection step:
(1) coating: the Rv0284, Rv2002 and Rv2421c antigens were dissolved in coating buffer (5ug/ml) respectively; adding 100ul per well into a 96-well enzyme label plate, and standing overnight at 4 ℃;
(2) washing the plate: PBST cleaning solution 300 ul/hole, 3min, wash 3 times;
(3) and (3) sealing: blocking solution, 300 ul/hole, and incubating for 1h at room temperature;
(4) washing the plate: washing solution 300 ul/hole for 3min for 3 times;
(5) adding serum: incubating the serum to be tested at room temperature for 1h at 100 ul/hole after dilution at a ratio of 1: 400;
(6) washing the plate: washing solution 300 ul/hole for 3min for 5 times;
(7) adding an enzyme-labeled secondary antibody: 100ul of freshly diluted enzyme-labeled antibody (1:30000) per well, and incubating for 1h at room temperature;
(8) washing the plate: washing solution 300 ul/hole for 3min for 5 times;
(9) color development: incubation is carried out for 5-10min at room temperature in a dark place with 100 ul/hole of TMB developing solution prepared freshly;
(10) and (4) terminating: stop solution 2M H2SO450 ul/well;
(11) and (3) determination: the microplate reader was adjusted to 450nm and the OD of each well was measured after zeroing with the first blank PBS control well.
3. Results analysis and criteria for determination:
using SPSS22.0 software to draw an ROC curve, obtaining specificity and sensitivity corresponding to different critical points according to coordinate values, then calculating a Yoden index (the Yoden index is specificity + sensitivity-1), and obtaining the critical point OD values of three antigens respectively, wherein Rv0248 is 0.3475, Rv2002 is 0.3048, and Rv2421c is 0.4173;
the results are shown in figure 1, and show that the three proteins have the function of distinguishing latent tuberculosis infection and active tuberculosis.
And (3) judging standard:
and (3) using the three proteins for joint detection, if at least 2 antibodies in the anti-Rv 0284 antibody, the anti-Rv 2002 antibody and/or the anti-Rv 2421c antibody are positive in the detection result of the sample to be detected, judging that the sample to be detected is positive in active tuberculosis, otherwise, judging that the sample to be detected is latent tuberculosis infection.
Example 3: specificity and sensitivity of method for differential diagnosis of active tuberculosis patients and latent infected patients by applying ELISA
Adopting 92 parts of serum of a patient clinically diagnosed with active tuberculosis and 93 parts of serum of a latent infected patient; the test was carried out by the ELISA method in examples 1 and 2, and the test subject was judged to be positive for active tuberculosis by the judgment criteria in example 2, as shown in FIG. 2, with the result that the assay specificity was 83.87% and the sensitivity was 79.35%. In FIG. 2, the abscissa 1-specificity represents the false positive rate, and the ordinate sensitivity represents the true positive rate.
The results prove that the kit can detect the level of the serum antibody response of three different mycobacterium tuberculosis proteins, can better identify and diagnose latent tuberculosis infectors and active tuberculosis patients, and has higher sensitivity and better specificity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Beijing thoracic Hospital affiliated to capital medical university
<120> use of protein of mycobacterium tuberculosis in preparing products for diagnosing latent tuberculosis infection and/or active tuberculosis
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Claims (10)
1. Use of a product for detecting the levels of anti-Rv 0284, anti-Rv 2002 and anti-Rv 2421c antibodies in a sample in the manufacture of a product for the identification, diagnosis and/or screening of active tuberculosis.
2. The use of claim 1, wherein the product for detecting the levels of anti-Rv 0284, anti-Rv 2002 and anti-Rv 2421c antibodies in a sample comprises an Rv0284 protein, an Rv2002 protein and an Rv2421c protein.
3. The use according to claim 2, wherein the Rv0284 protein is a protein shown as SEQ ID No.1, or a protein which has the same function as the protein shown as SEQ ID No.1 after being modified by one or more amino acid residues in the amino acid sequence shown as SEQ ID No. 1; the Rv2002 protein is shown in SEQ ID NO.2, or the protein which has the same function with the protein shown in SEQ ID NO.2 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified; the Rv2421c protein is shown in SEQ ID NO.3, or the protein which has the same function with the protein shown in SEQ ID NO.3 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified.
4. Use according to any one of claims 1 to 3, wherein the detection method comprises an enzyme-linked immunosorbent assay, an agglutination assay, a precipitation assay, an E rosette assay, a phagocytosis assay, an immunofluorescence assay, a colloidal gold immunochromatography assay, a dot immunogold diafiltration assay and/or an electrochemiluminescence assay.
5. The use according to any one of claims 1 to 3, wherein the sample comprises serum, plasma, saliva, urine, pleural effusion and/or cerebrospinal fluid.
6. A marker composition for use in identifying, diagnosing and/or screening active tuberculosis, wherein the marker composition consists of an anti-Rv 0284 antibody, an anti-Rv 2002 antibody and an anti-Rv 242 2421c antibody;
wherein, the Rv0284 is a protein which is shown in SEQ ID NO.1 or has the same function with the protein shown in SEQ ID NO.1 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified; the Rv2002 is a protein shown in SEQ ID NO.2, or one or more amino acid residues in an amino acid sequence shown in SEQ ID NO.2 are modified to have the same function with the protein shown in SEQ ID NO. 2; the Rv2421c is a protein shown in SEQ ID NO.3, or one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified to have the same function with the protein shown in SEQ ID NO. 3.
7. Use of a marker composition according to claim 6 in the manufacture of a product for the identification, diagnosis and/or screening of active tuberculosis.
8. A kit for identifying, diagnosing and/or screening active tuberculosis, wherein the kit comprises Rv0284 protein, Rv2002 protein and Rv2421c protein.
9. The kit according to claim 8, wherein the Rv0284 protein is a protein shown in SEQ ID No.1, or a protein which has the same function as the protein shown in SEQ ID No.1 after being modified by one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 1; the Rv2002 protein is shown in SEQ ID NO.2, or the protein which has the same function with the protein shown in SEQ ID NO.2 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified; the Rv2421c protein is shown in SEQ ID NO.3, or the protein which has the same function with the protein shown in SEQ ID NO.3 after one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified.
10. Use of a kit according to claim 8 or 9 for the manufacture of a product for the identification, diagnosis and/or screening of active tuberculosis.
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| CN201611259564.2A CN108267589B (en) | 2016-12-30 | 2016-12-30 | Application of mycobacterium tuberculosis protein in preparing product for diagnosing latent tuberculosis infected person and/or active tuberculosis |
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| CN108267589A (en) | 2018-07-10 |
| CN111521819B (en) | 2023-06-20 |
| CN108267589B (en) | 2020-10-23 |
| CN111551741B (en) | 2023-06-16 |
| CN111551741A (en) | 2020-08-18 |
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