CN111521819B - Use of mycobacterium tuberculosis proteins in the preparation of a product for diagnosing tuberculosis latency infected persons and/or active tuberculosis - Google Patents

Abstract

The invention provides the use of the mycobacterium tuberculosis protein in preparing products for diagnosing tuberculosis latent infection and active pulmonary tuberculosis. The method provided by the invention can detect the serum antibody response levels of three different mycobacterium tuberculosis proteins at the same time, so as to better differentiate and diagnose latent tuberculosis infection patients and active tuberculosis patients, with higher sensitivity and better specificity.

Description

Mycobacterium tuberculosis proteins in the preparation of diagnostic latent tuberculosis infection and/or active Uses in tuberculosis products

This application is a divisional application of an invention patent application with an application date of December 30, 2016 and an application number of 2016112595642.

technical field

The invention relates to the field of biomedicine, in particular to the use of mycobacterium tuberculosis protein in preparing products for diagnosing tuberculosis latent infection and/or active pulmonary tuberculosis.

Background technique

Tuberculosis caused by Mycobacterium tuberculosis is still one of the diseases that seriously threaten human health. According to the latest report of the World Health Organization, there were 10.4 million new cases of tuberculosis in the world in 2015, and about 1.8 million people died of tuberculosis. The number of people is 918,000, the death toll is 38,000, and the total number of cases ranks fourth in the world. Whether in the world or in China, there is still a long way to go in the prevention and control of tuberculosis, especially in some underdeveloped countries with poor sanitation, the infection rate of tuberculosis is still high.

After the human body is infected with Mycobacterium tuberculosis, only about 10% of people develop active tuberculosis, while 90% of people do not get sick, but the tuberculosis bacteria in the body are not completely eliminated, but dormant in the human body, called tuberculosis Latent infection, these people do not have any clinical symptoms, and most people do not get sick in their lifetime, and only 5%--10% people can cause the generation of tuberculosis when body immunity declines. At present, about 1/3 of the people in the world are infected with tuberculosis. It can be seen that there are a large number of latent infections. Therefore, it is necessary to monitor the latent infections and give timely drug treatment when the infection status changes, so as to achieve early detection and early detection. Treatment, which can not only prevent the deterioration of the disease, reduce the treatment time and side effects caused by drug treatment, but also reduce the spread of tuberculosis. Therefore, the detection and identification of the two states after tuberculosis infection has a very important effect on the prevention and treatment of tuberculosis.

At present, there are many detection methods for tuberculosis infection, such as tuberculin skin test (TST), interferon-gamma release assay (IGRA), traditional sputum smear of acid-fast bacteria, rapid culture of mycobacteria, and tuberculosis-specific nucleic acid fragments. Amplification (quantitative and qualitative), etc. TST is a skin allergy and has been used for a long time. It is mainly used for outpatient screening. Although the test is simple and easy, when detecting tuberculosis infection, BCG vaccination will produce the same results, and natural infection cannot be identified, and the specificity is not strong. In addition, latent tuberculosis infection cannot be distinguished from active tuberculosis. The gamma-interferon release assay (IGRA) is mainly used to detect cellular immune responses. Although it can rule out the impact of BCG vaccination, it cannot distinguish latent infection from active tuberculosis patients, let alone distinguish treated patients from current infection. However, traditional sputum smears of acid-fast bacteria and rapid culture of mycobacteria are mainly used to detect active tuberculosis patients, which are meaningless for the judgment of latent infection status. In addition, for active tuberculosis patients with atypical clinical symptoms, bacteria may not be found scientific evidence. Therefore, it is of great value to find a method that can accurately identify latent infection and active tuberculosis.

Based on the differences in host protective immune responses after tuberculosis infection, finding serum markers to distinguish latent infection from active tuberculosis has a good application prospect. The serological diagnosis method of antigen-antibody reaction has attracted much attention because of its simplicity, rapidity, and easy access to samples. Now some specific antigens of Mycobacterium tuberculosis have been used for serological diagnosis of tuberculosis infection, such as Secreted protein antigens: antigen 85 complex antigens, namely Ag85A, Ag85B and Ag85C, etc.; lipoprotein antigens: 16kDa, 27kDa, 38kDa antigens, etc.; glycolipid antigens: lipoarabinomannan antigens, tuberculosis carbohydrate lipids Antigens, etc.; however, in actual use, these antigens have low positive detection rates and cross-immunity with other mycobacteria. However, it still cannot be used for the diagnosis of latent Mycobacterium tuberculosis infection.

Therefore, it is extremely important to find a new method and marker that can accurately diagnose latent tuberculosis infection and active tuberculosis patients.

Contents of the invention

The technical problem to be solved by the present invention is aimed at the low positive detection rate of products for differential diagnosis of latent tuberculosis infection and/or active pulmonary tuberculosis in the prior art, and the problem that latent Mycobacterium tuberculosis infection cannot be differentially diagnosed. Use of a Mycobacterium tuberculosis protein in preparing products for diagnosing tuberculosis latent infection and/or active pulmonary tuberculosis.

In order to solve the above technical problems, the technical solution provided by the present invention on the one hand is:

Use of the product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in a sample in the preparation of products for identifying, diagnosing and/or screening tuberculosis latent infection and/or active pulmonary tuberculosis.

In the technical solution of the present invention, any product that detects the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in the sample can be used for the preparation of identification, diagnosis and/or screening for tuberculosis latent infection and/or active pulmonary tuberculosis The product.

Preferably, the above-mentioned product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in the sample contains Rv0284 protein, Rv2002 protein and/or Rv2421c protein.

More preferably, the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are engineered to have the same A protein with the same function; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 are modified with the protein shown in SEQ ID NO.2 A protein with the same function; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified and shown in SEQ ID NO.3 protein A protein with the same function.

The above-mentioned term "modification" can use any protein modification method in the prior art, including but not limited to, substitution and/or addition and/or deletion of amino acid residues, so as to form derivatives of the original protein.

Preferably, the detection can be any suitable detection method in the prior art, but as a preference, the detection method can be enzyme-linked immunosorbent assay, agglutination test, precipitation test, E rosette test, small phagocytosis test, immune Fluorescence experiments, colloidal gold immunochromatography experiments, spot gold immunofiltration experiments and/or electrochemiluminescence experiments. More preferably, in one embodiment of the present invention, the detection method is enzyme-linked immunosorbent assay.

Preferably, the sample can be any suitable sample obtained from the object to be detected. But preferably, the sample includes serum, plasma, saliva, urine, ascites and/or cerebrospinal fluid. More preferably, in one embodiment of the present invention, the sample is serum.

Another aspect of the present invention is to provide a marker composition for identifying, diagnosing and/or screening tuberculosis latent infection and/or active pulmonary tuberculosis, said marker composition consisting of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody composition;

Wherein, the Rv0284 is shown in SEQ ID NO.1, or one or several 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 Protein; the Rv2002 is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 have been modified to have the same function as the protein shown in SEQ ID NO.2 Protein; the Rv2421c is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified to have the same function as the protein shown in SEQ ID NO.3 protein.

Another aspect of the present invention is to provide a use of the above marker composition in the preparation of products for identification, diagnosis and/or screening of tuberculosis latent infection and/or active tuberculosis.

Another aspect of the present invention is to provide a kit for identifying, diagnosing and/or screening latent tuberculosis infection and/or active tuberculosis, said kit comprising Rv0284 protein, Rv2002 protein and/or Rv2421c protein.

Preferably, the Rv0284 protein is shown in SEQ ID NO.1, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.1 are modified to have the same protein as the protein shown in SEQ ID NO.1 Functional protein; the Rv2002 protein is shown in SEQ ID NO.2, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.2 have been transformed with the protein shown in SEQ ID NO.2 A protein with the same function; the Rv2421c protein is shown in SEQ ID NO.3, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO.3 are modified and the protein shown in SEQ ID NO.3 proteins with the same function.

The Rv0284 protein, Rv2002 protein and/or Rv2421c protein contained in the above kit can be in any form, including but not limited to direct protein samples, and the form of proteins immobilized on some carrier, such as a detection chip.

In addition to the Rv0284 protein, Rv2002 protein and/or Rv2421c protein, the kit at least includes kit instructions.

Judgment criteria for the results recorded in the instructions: using the combined detection of three proteins, if at least two of the anti-Rv0284 antibodies, anti-Rv2002 antibodies and/or anti-Rv2421c antibodies are positive in the test results of the sample to be tested, then It is judged that the sample to be tested is positive for active tuberculosis, otherwise it is tuberculosis latent infection.

Another aspect of the present invention is to provide a use of the above kit in the preparation of products for identifying, diagnosing and/or screening tuberculosis latent infection and/or active pulmonary tuberculosis.

The beneficial effects of the present invention are:

The invention can simultaneously detect the serum antibody response levels of three different Mycobacterium tuberculosis proteins, thereby better distinguishing and diagnosing tuberculosis latent infection patients and active tuberculosis patients, with higher sensitivity and better specificity.

Description of drawings

Fig. 1 is the statistic graph of anti-Rv0284 antibody, anti-Rv2002 antibody and anti-Rv2421c antibody expression level in ELISA detection tuberculosis latent infection patient (n=93) and active tuberculosis patient (n=92) serum;

Fig. 2 is the ROC curve diagram of joint detection of antibodies corresponding to three antigens;

sequence description

SEQ ID NO.1 is the amino acid sequence of the Rv0284 protein in the embodiment of the present invention;

SEQ ID NO.2 is the amino acid sequence of the Rv2002 protein in the embodiment of the present invention;

SEQ ID NO.3 is the amino acid sequence of the Rv2421c protein in the embodiment of the present invention.

Detailed ways

The invention discloses the use of a product for detecting the level of anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody in a sample in the preparation of products for identifying, diagnosing and/or screening tuberculosis latent infection and/or active pulmonary tuberculosis. Those skilled in the art can refer to the content of this article to appropriately improve the process parameters to achieve. It needs to be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention, and relevant personnel can obviously make changes without departing from the content, spirit and scope of the present invention. Changes or appropriate changes and combinations are made to the content described herein to realize and apply the technology of the present invention.

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art.

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below in conjunction with specific examples.

Example 1: Collection and processing of samples

The sample detected by the present invention is serum, and the collection method is to collect the blood of the subject on an empty stomach, use an ordinary biochemical test tube without anticoagulant, let it stand at room temperature for natural coagulation, centrifuge at 3000 rpm for 10 minutes at room temperature, and collect the serum fraction. Packed in cryopreservation tubes and stored in a -80°C low-temperature refrigerator 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 those who have no clinical complaint symptoms, TST test and T-SPOT test are positive, and chest X-ray films are negative; The active tuberculosis patient group refers to the confirmed tuberculosis patients with positive smear or culture, who have not started or just started systemic chemotherapy; both groups exclude diabetes, HIV infection and other autoimmune diseases.

The samples of the latent infection group used in the present invention come from the epidemiological investigation project of the Epidemiology Research Office of the Beijing Institute of Tuberculosis and Chest Tumors, and the samples of the active tuberculosis patient group come from inpatients in each ward of the Department of Tuberculosis, Beijing Chest Hospital Affiliated to Capital Medical University. This project has been approved by the Research Ethics Committee of Beijing Chest Hospital Affiliated to Capital Medical University, and all subjects gave informed consent.

Embodiment 2: Utilize ELISA experiment to the detection of sample

1. Establish various buffers, diluents, reaction solutions and stop solutions required for ELSIA experiments:

(1) Coating buffer (pH9.6, 0.05mol/L carbonate buffer):

Na ₂ CO ₃ 1.59g, NaHCO ₃ 2.93g, add double distilled water to 1000ml, adjust pH to 9.6;

(2) PBS solution with pH7.4:

NaCl 8.0g, KCl 0.2g, KH ₂ PO ₄ 0.2g, Na ₂ HPO ₄ 12H ₂ O 2.9g,

Add double distilled water to 1000ml, adjust pH to 7.4;

(3) PBST washing solution with pH7.4:

Add 0.5ml Tween-20 to 1000ml PBS solution, adjust the pH to 7.4;

(4) Blocking solution (pBS solution containing skimmed milk powder at pH 7.4):

Add 5g skimmed milk powder to 100ml PBS solution;

(5) Substrate buffer: 0.2M NaH ₂ PO ₄ (28.4g/L) 25.7ml,

0.1M citric acid (19.2g/L) 24.3ml, add 50ml of distilled water.

(6) TMB (tetramethylbenzidine) use solution: TMB (10mg/5ml absolute ethanol) 0.5ml substrate

Buffer (pH5.5) 10ml 0.75% H ₂ O ₂ 32μl;

(7) Stop solution (2M H ₂ SO ₄ ): 21.32ml H ₂ SO ₄ , 178.68ml H ₂ O.

(8) Secondary antibody: goat anti-human IgG labeled with horseradish peroxidase (HRP);

(9) 96-well ELISA plate; Nunc Incorporated (Theromo, Gebenhagen, Denmark)

2. Detection steps:

(1) Coating: Rv0284, Rv2002 and Rv2421c antigens were respectively dissolved in coating buffer (5ug/ml); 100ul/well was added to a 96-well microtiter plate, and overnight at 4°C;

(2) Plate washing: PBST washing solution 300ul/well, 3min, wash 3 times;

(3) Blocking: blocking solution, 300ul/well, incubate at room temperature for 1h;

(4) Plate washing: washing solution 300ul/well, 3min, wash 3 times;

(5) Add serum: 1:400 diluted serum to be tested, 100ul/well, incubate at room temperature for 1h;

(6) Plate washing: wash solution 300ul/well, 3min, wash 5 times;

(7) Add enzyme-labeled secondary antibody: freshly diluted enzyme-labeled antibody (1:30000) 100ul/well, incubate at room temperature for 1h;

(8) Plate washing: wash solution 300ul/well, 3min, wash 5 times;

(9) Color development: freshly prepared TMB color development solution, 100ul/well, incubate at room temperature in the dark for 5-10min;

(10) Termination: stop solution 2M H ₂ SO ₄ , 50ul/well;

(11) Determination: Adjust the microplate reader to 450nm, measure the OD value of each well after zeroing the first blank PBS control well.

3. Result analysis and judgment criteria:

Use SPSS22.0 software to draw the ROC curve, and according to the coordinate values, the specificity and sensitivity corresponding to different critical points can be obtained, and then the Youden index can be calculated (Youden index = specificity + sensitivity - 1), and three antigens can be obtained The critical point OD value of Rv0248 is 0.3475, Rv2002 is 0.3048, Rv2421c is 0.4173;

The results are shown in Figure 1, and the results indicated that all three proteins have the function of distinguishing tuberculosis latent infection from active tuberculosis.

judgement standard:

Using the combined detection of three proteins, if at least two of the anti-Rv0284 antibody, anti-Rv2002 antibody and/or anti-Rv2421c antibody are positive in the test results of the test sample, it is determined that the test sample is positive for active tuberculosis , otherwise latent tuberculosis infection.

Example 3: Specificity and sensitivity of the method for differential diagnosis of active tuberculosis patients and latent infections using ELISA

Adopt clinical diagnosis as 92 parts of serum of patients with active pulmonary tuberculosis, 93 parts of serum of latent infection; Utilize the ELISA method among the embodiment 1 and 2 to detect, judge whether the test subject is active pulmonary tuberculosis positive by the judgment standard of embodiment 2 , the results are shown in Figure 2, the specificity of the results analysis was 83.87%, and the sensitivity was 79.35%. In Figure 2, the abscissa 1-specificity represents the false positive rate, and the ordinate sensitivity represents the true positive rate.

The above results prove that the present invention detects the serum antibody response levels of three different Mycobacterium tuberculosis proteins, can better differentiate and diagnose latent tuberculosis infection patients and active tuberculosis patients, and has higher sensitivity and better specificity.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (4)

1. Use of a product for detecting levels of an anti-Rv 0284 antibody, an anti-Rv 2002 antibody and an anti-Rv 2421c antibody in a sample for the preparation of a product for the identification, diagnosis and/or screening of active tuberculosis;

the sample is serum.

2. A use according to claim 1, wherein the products for detecting levels of anti-Rv 0284, anti-Rv 2002 and anti-Rv 2421c antibodies in a sample comprise Rv0284, rv2002 and Rv2421c proteins.

3. The use according to claim 2, wherein the Rv0284 protein is shown in SEQ ID No. 1; the Rv2002 protein is shown as SEQ ID NO. 2; the Rv2421c protein is shown in SEQ ID NO. 3.

4. The use according to any one of claims 1 to 3, wherein the method of detection comprises an enzyme-linked immunosorbent assay, an agglutination assay, a precipitation assay, an E-garland assay, a small phagocytosis assay, an immunofluorescence assay, a colloidal gold immunochromatography assay, a spot gold immunodiafiltration assay and/or an electrochemiluminescence assay.

Images