Application of LBP as detection marker in spinal tuberculosis serum diagnosis

Abstract

The invention discloses an application of LBP as a detection marker in spinal tuberculosis serum diagnosis. A detection and verification method of the LBP as an early diagnostic marker in spinal tuberculosis peripheral serum comprises the following steps: S1, carrying out liquid chromatography-tandem mass spectrometry detection to obtain protein with expression difference in the spinal tuberculosis peripheral serum; s2, performing experimental verification on the selected differential protein by using ELISA (enzyme-linked immunosorbent assay); s3, clinical correlation analysis is conducted in LBP spinal tuberculosis diagnosis, and the application value of the marker is evaluated, the difference of protein expression level expression in peripheral serum of a spinal tuberculosis patient is screened through proteomics, the marker serves as a spinal tuberculosis diagnostic marker, the accuracy is high when the marker is used for early diagnosis of spinal tuberculosis, and the application value of the marker is evaluated. The LBP can be used for screening and diagnosis of the early diagnosis of spinal tuberculosis, and the complication and risk rate of spinal tuberculosis are reduced, so that the LBP can be prepared into a detection kit for the early diagnosis of spinal tuberculosis, and has a wide prospect in the application of the early diagnosis of spinal tuberculosis.

Description

Technical Field

[0001] The invention belongs to the field of medical molecular biotechnology, and specifically relates to the application of LBP as a detection marker in serum diagnosis of spinal tuberculosis. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that seriously endangers human health. China is a country with a high burden of tuberculosis. According to the 2020 World Health Organization report, the incidence of tuberculosis in China ranks third in the world, second only to India and Indonesia. Pulmonary tuberculosis often causes secondary extrapulmonary tuberculosis, which can occur in various organs throughout the body, such as intestinal tuberculosis, bladder tuberculosis, bone tuberculosis, tuberculous meningitis, etc. Spinal tuberculosis is the most common bone and joint tuberculosis, accounting for about 4% of tuberculosis, 15% of extrapulmonary tuberculosis, and 50% of bone and joint tuberculosis. STB has an insidious onset, slow progression, and no obvious symptoms and signs. It often causes low back pain, limited mobility and other symptoms when it occurs. When STB is severe, it can lead to damage to the spine and nerves, resulting in symptoms such as limb numbness and paralysis, which brings serious economic pressure and burden to families and society. The pathogenesis of STB is complex and affected by many factors, which makes diagnosis and treatment very difficult. Therefore, it is of great significance to find molecular markers for early diagnosis of STB.

[0003] In recent years, proteomics, an emerging basic discipline, has emerged and developed rapidly in the medical field, providing new ideas and methods for the pathogenic mechanism of disease occurrence and development, screening of diagnostic markers, and drug treatment and evaluation in clinical practice. Proteomics mainly uses isotope labeling relative and absolute quantitative technology and liquid chromatography-tandem mass spectrometry detection in proteomics to identify the correlation between differentially expressed proteins in tissues or blood and diseases, and then explore their role as markers in disease diagnosis, treatment and efficacy evaluation. The application of proteomics to explore the differences in peripheral blood of spinal tuberculosis can provide new research directions and ideas for the diagnosis, prevention and treatment, and prognosis of spinal tuberculosis.

[0004] The study found that LBP protein was significantly differentially expressed in the peripheral blood of patients with spinal tuberculosis. It is closely related to the occurrence of spinal tuberculosis and plays an important role in the diagnosis of spinal tuberculosis. It has the potential to be an early molecular diagnostic marker for spinal tuberculosis. However, the shortcomings of this study are that the sample size is small and the research is not in-depth and comprehensive enough, and there may be false positives, low specificity and sensitivity, etc. Therefore, it is necessary to further expand the sample verification and evaluation of the specificity and accuracy of the diagnostic marker. Summary of the invention

[0005] The purpose of the present invention is to use STB patients as research objects, adopt liquid chromatography-tandem mass spectrometry detection technology in proteomics combined with ELISA kit detection and statistical correlation analysis to screen out differentially expressed proteins in the peripheral serum of spinal tuberculosis as diagnostic markers, provide new diagnostic markers for the early diagnosis of clinical spinal tuberculosis, and solve the problems mentioned in the background technology.

[0006] In order to solve the above problems, the present invention provides a technical solution:

[0007] The application of LBP as a detection marker in the serum diagnosis of spinal tuberculosis, including the detection and verification method of LBP as an early diagnostic marker in the peripheral serum of spinal tuberculosis;

[0008] The steps of the detection and verification method are as follows:

[0009] S1. Differentially expressed proteins in peripheral serum of spinal tuberculosis were obtained by liquid chromatography-tandem mass spectrometry;

[0010] S2. Experimental verification of selected differentially expressed proteins using ELISA;

[0011] S3. Conduct clinical correlation analysis on the diagnosis of LBP spinal tuberculosis and evaluate its application value as a marker.

[0012] Preferably, the method for detecting differential proteins in step S1 is:

[0013] Liquid chromatography-tandem mass spectrometry was used to detect proteins in the peripheral serum of the normal control group and the experimental group of spinal tuberculosis patients, and the differential proteins in the peripheral serum of the two groups were determined.

[0014] Preferably, the implementation steps of S1 are as follows:

[0015] S11. Determine the experimental group and the control group;

[0016] S12, collect and preserve peripheral serum from the experimental group and the control group;

[0017] S13, proteomic analysis of serum;

[0018] S14. Screening for early diagnostic markers of spinal tuberculosis;

[0019] S15. Identify the control group and the experimental group by early diagnostic markers of spinal tuberculosis;

[0020] S16. Conduct large sample validation of the screened early markers;

[0021] S17. Clinical correlation analysis was used to evaluate the value of markers in the diagnosis of spinal tuberculosis.

[0022] Preferably, the experimental group in S11 is peripheral serum of spinal tuberculosis confirmed by clinical symptoms, laboratory tests, imaging examinations, pathological diagnosis, etc. before treatment;

[0023] The control group was normal peripheral blood serum from subjects who underwent physical examination at the same period.

[0024] Preferably, the verification method of step S2 is:

[0025] S21. Peripheral sera from 30 patients with spinal tuberculosis were collected as the experimental group and 30 normal subjects as the control group;

[0026] S22. Use ELISA to detect the stability and specificity of differential proteins in peripheral serum of spinal tuberculosis.

[0027] Preferably, the correlation analysis method in step S3 is:

[0028] S31. Collect relevant clinical data of patients with spinal tuberculosis;

[0029] S32. Conduct correlation analysis based on laboratory, pathological and imaging examinations.

[0030] The beneficial effects of the present invention are: by screening the differences in protein expression levels in the peripheral serum of patients with spinal tuberculosis through proteomics, it has high accuracy as a diagnostic marker for spinal tuberculosis and is used for the early diagnosis of spinal tuberculosis, and can be used for the screening and diagnosis of early diagnosis of spinal tuberculosis, thereby reducing the complications and risk rates of spinal tuberculosis. Therefore, LBP can be prepared as a detection kit for the early diagnosis of spinal tuberculosis, and has broad prospects in the application of early diagnosis of spinal tuberculosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] For ease of explanation, the present invention is described in detail with reference to the following specific implementations and the accompanying drawings.

[0032] Figure 1 This is the heat map of differential protein clustering analysis between peripheral serum of patients with spinal tuberculosis and healthy subjects

[0033] Figure 2 It is the volcano distribution map of differential proteins in peripheral serum of patients with spinal tuberculosis;

[0034] Figure 3 This is the analysis diagram of the differential protein LBP results verified by ELISA;

[0035] Figure 4 This is the ROC curve result diagram of LBP in STB examination;

[0036] Figure 5This is the correlation analysis chart of CRP and ESR in the LBP test of spinal tuberculosis;

[0037] Figure 6 This is the ROC curve analysis diagram for the combined diagnosis of LBP and spinal tuberculosis. DETAILED DESCRIPTION

[0038] like Figure 1-6 As shown, this specific implementation adopts the following technical solutions:

[0039] The application of LBP as a detection marker in the serum diagnosis of spinal tuberculosis, including the detection and verification method of LBP as an early diagnostic marker in the peripheral serum of spinal tuberculosis;

[0040] The steps of the detection and verification method are as follows:

[0041] S1. Differentially expressed proteins in peripheral serum of spinal tuberculosis were obtained by liquid chromatography-tandem mass spectrometry;

[0042] S2. Experimental verification of selected differentially expressed proteins using ELISA;

[0043] S3. Analyze the clinical relevance of LBP in the diagnosis of spinal tuberculosis and evaluate its application value as a marker;

[0044] Among them, LBP is used as a diagnostic marker in spinal tuberculosis;

[0045] Furthermore, in the application of LBP as a diagnostic marker for spinal tuberculosis, the peripheral serum of spinal tuberculosis is collected and stored in a routine clinical test tube;

[0046] Furthermore, in the application of LBP as a diagnostic marker for spinal tuberculosis, LBP is derived from the peripheral serum of patients with spinal tuberculosis;

[0047] Furthermore, in the application of LBP as a diagnostic marker for spinal tuberculosis, LBP in peripheral serum is detected by conventional methods;

[0048] Furthermore, in the application of LBP as a diagnostic marker for spinal tuberculosis, the above-mentioned detection method is liquid chromatography-tandem mass spectrometry detection.

[0049] In the application of LBP as a diagnostic marker for spinal tuberculosis, the above detection method and conditions are:

[0050] The samples were separated by EasynLC at a nano-flow rate, loaded onto a C18 reverse-phase analytical column, and separated in buffer A (0.1% formic acid) with a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL / min;

[0051] Select the corresponding liquid phase gradient:

[0052] i. 1 h gradient: 6% buffer B for 3 min, 6-28% buffer B for 42 min, 28-38% buffer B for 5 min, 38-100% buffer B for 5 min, 100% buffer B for 5 min;

[0053] ii. 1.5 h gradient: 6% buffer B for 5 min, 6-28% buffer B for 63 min, 28-38% buffer B for 10 min, 38-100% buffer B for 7 min, hold at 100% buffer B for 5 min;

[0054] After chromatographic separation, the samples were analyzed by mass spectrometry using a QExactiveplus mass spectrometer;

[0055] The analysis duration was 60 / 90 min, the detection mode was positive ion, the parent ion scan range was 350-1800 m / z, the primary mass spectrometry resolution was 70000, the AGC standard was 3e6, the primary maximum IT was 50 ms, the secondary mass spectrometry resolution was 17500, the AGC standard was 2e5, the minimum AGC target was 2e3, the secondary maximum IT was 45 ms, the collection charge was between 2-6, the ion dynamic exclusion time was 30 s, and the collision energy was 30 eV;

[0056] The application of LBP in the preparation of drugs for treating spinal tuberculosis and the evaluation of therapeutic efficacy is a protein detection kit for diagnosing spinal tuberculosis. The detection kit is the LBP protein standard of the present invention, and the standard is an LBP chemical monomer or mixture.

[0057] Furthermore, the protein detection kit comprises a sample buffer and a color developing solution.

[0058] Furthermore, the protein detection kit comprises a standard diluent and a concentrated washing solution.

[0059] Furthermore, the LBP detection kit plays a role in the early diagnosis of spinal tuberculosis and the prognosis evaluation of drug efficacy.

[0060] Example:

[0061] Application of products that screen differentially expressed proteins LBP in the peripheral serum of patients with spinal tuberculosis by proteomics as diagnostic markers for spinal tuberculosis, such as Figure 1 The following is a cluster heat map of differentially expressed proteins in proteomic analysis of spinal tuberculosis patients and healthy subjects. Figure 2 The figure shows the differences in the presence of differential proteins in the peripheral blood of patients with spinal tuberculosis, where Diff: difference exists; NoDiff: no difference; Mark: marker symbol; Figure 3The following is the test result of the designed ELISA kit on the peripheral serum of healthy people and patients with spinal tuberculosis, where Normal: healthy people; STB: patients with spinal tuberculosis; Figure 4 The figure shows the results of peripheral serum test of LBP protein in patients with spinal tuberculosis and ROC analysis of spinal tuberculosis diagnosis. The results show that AUC = 0.880 (P < 0.01), indicating that LBP has important value in diagnosing spinal tuberculosis. Figure 5 As shown in the figure, the correlation analysis of LBP based on the evaluation index of spinal tuberculosis inflammation found that the values ​​of CRP and ESR in the LBP blood routine were linearly correlated, indicating that LBP is sensitive to abnormalities in the peripheral blood of spinal tuberculosis and has important value for the diagnosis of spinal tuberculosis; Figure 6 The results of ROC analysis of LBP measurement values ​​and spinal tuberculosis diagnosis show that LBP has important value in the diagnosis of spinal tuberculosis and can be used as an early diagnostic marker for spinal tuberculosis in clinical practice;

[0062] Study subjects:

[0063] Three peripheral sera from patients with spinal tuberculosis were selected as the experimental group, and three peripheral sera from healthy subjects were selected as the control group. In addition, 30 peripheral blood samples from patients with confirmed spinal tuberculosis and laboratory test and diagnosis-related data from 100 patients were collected.

[0064] Proteomics experiments:

[0065] Proteomics was performed to determine the differential proteins in peripheral serum as follows:

[0066] 2 ml of peripheral blood from the experimental group and the control group was taken and placed in an anticoagulant tube. The blood was centrifuged within 2 h. According to the operation method in the corresponding protocol of Agilent, high-abundance proteins were removed to obtain low-abundance solutions. The solution was concentrated by ultrafiltration using a 5KD ultrafiltration tube, and then SDT lysis buffer was added. The solution was placed in a boiling water bath for 10 min and centrifuged using a 14000 g centrifuge for 15 min. The supernatant was taken and then protein BCA quantification was performed. The samples were packaged and stored at -80 °C.

[0067] Peripheral plasma proteins were enzymatically hydrolyzed, and the products after enzymatic hydrolysis were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS). The samples were separated by EasynLC at a nanoliter flow rate, and the samples were loaded onto a C18 reverse phase analytical column and separated in buffer A (0.1% formic acid) using a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 300 nL / min;

[0068] Select the corresponding liquid phase gradient:

[0069] i. 1 h gradient: 6% buffer B for 3 min, 6-28% buffer B for 42 min, 28-38% buffer B for 5 min, 38-100% buffer B for 5 min, 100% buffer B for 5 min;

[0070] ii. 1.5 h gradient: 6% buffer B for 5 min, 6-28% buffer B for 63 min, 28-38% buffer B for 10 min, 38-100% buffer B for 7 min, hold at 100% buffer B for 5 min;

[0071] After chromatographic separation, the samples were analyzed by mass spectrometry using a QExactiveplus mass spectrometer. The analysis time was 60 / 90 min, the detection mode was positive ion, the parent ion scanning range was 350-1800 m / z, the primary mass spectrometry resolution was 70000, the AGC standard was 3e6, the primary maximum IT was 50 ms, the secondary mass spectrometry resolution was 17500, the AGC standard was 2e5, the minimum AGC target was 2e3, the secondary maximum IT was 45 ms, the collection charge was between 2 and 6, the ion dynamic exclusion time was 30 s, and the collision energy was 30 eV;

[0072] TMT quantitative proteomics analysis was performed using a high-resolution mass spectrometer QExactiveplus. ProteomeDiscoverer2.2 software was used to screen protein data based on a standard of 0.01 relative deviation between peptide and protein levels to obtain highly reliable protein qualitative results.

[0073] ELISA experimental verification:

[0074] An LBP protein antibody ELISA detection kit was designed to detect the expression level of LBP protein in the peripheral blood serum of spinal tuberculosis patients and healthy people. The operation method was to set standard and sample wells, add different concentrations of standard wells to the standard wells; add the sample to be tested to the sample wells, then add the diluent, and gently shake to mix; add enzyme-labeled reagent to each well, seal the plate with a sealing film for a period of time, add the washing solution, and then add the color developing solution to react for 15 minutes, pour out the sealing solution and add the stop solution, measure the relevant absorbance, and finally calculate the differential protein concentration. The results are shown as follows Figure 3 As shown;

[0075] Correlation analysis of clinical counting data was performed using curve regression and Spesrman correlation analysis.

[0076] Linear analysis was performed on the LBP protein content in the patients and the CRP and ESR values, which were related to spinal tuberculosis laboratory examinations. It was found that the LBP measurement value was positively correlated with the changes in CRP and ESR values, which was statistically significant (P≤0.01). Figure 5As shown: It suggests that LBP has a synergistic effect with CRP and ESR in the detection of infection or inflammatory diseases;

[0077] According to the determination of LBP content, ROC analysis was performed to determine the value of LBP in the diagnosis of spinal tuberculosis:

[0078] ROC curve analysis was used to analyze the pathology, bacteriology, acid-fast staining, T-SPOT, and imaging related to the diagnosis of spinal tuberculosis. The results showed that Figure 6 As shown in Figure 2, the AUCs for STB diagnosis were 0.677, 0.707, 0.656, 0.751, and 0.714, respectively (all P < 0.01). The results showed that LBP was of great value in the diagnosis of STB. The results of LBP combined with the above spinal tuberculosis diagnosis were as follows: Figure 4 The AUC shown was 0.880, indicating that LBP has the potential to be used as a marker for peripheral serum diagnosis of STB.

[0079] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The application of LBP as a detection marker in the serological diagnosis of spinal tuberculosis is characterized by: Including the detection and validation methods of LBP as an early diagnostic marker in peripheral serum of spinal tuberculosis; The steps of the detection and verification method are as follows: S1. Differentially expressed proteins in peripheral serum of spinal tuberculosis were obtained by liquid chromatography-tandem mass spectrometry; S2. Experimental verification of selected differentially expressed proteins using ELISA; S3. Conduct clinical correlation analysis on the diagnosis of LBP spinal tuberculosis and evaluate its application value as a marker.

2. The use of LBP as a detectable marker in serological diagnosis of spinal tuberculosis according to claim 1, characterized in that: The method for detecting differential proteins in step S1 is: Liquid chromatography-tandem mass spectrometry was used to detect proteins in the peripheral serum of the normal control group and the experimental group of spinal tuberculosis patients, and the differential proteins in the peripheral serum of the two groups were determined.

3. The use of LBP as a detectable marker in serological diagnosis of spinal tuberculosis according to claim 2, characterized in that: The implementation steps of S1 are as follows: S11. Determine the experimental group and the control group; S12, collect and preserve peripheral serum from the experimental group and the control group; S13, proteomic analysis of serum; S14. Screening for early diagnostic markers of spinal tuberculosis; S15. Identify the control group and the experimental group by early diagnostic markers of spinal tuberculosis; S16. Conduct large sample validation of the screened early markers; S17. Clinical correlation analysis was used to evaluate the value of markers in the diagnosis of spinal tuberculosis.

4. The use of LBP as a detectable marker in serological diagnosis of spinal tuberculosis according to claim 3, characterized in that: The experimental group in S11 was peripheral serum of spinal tuberculosis diagnosed by clinical symptoms, laboratory tests, imaging examinations, pathological diagnosis, etc. before treatment; The control group was normal peripheral blood serum from subjects who underwent physical examination at the same period.

5. The use of LBP as a detectable marker in serological diagnosis of spinal tuberculosis according to claim 1, characterized in that: The verification method of step S2 is: S21. Peripheral sera from 30 patients with spinal tuberculosis were collected as the experimental group and 30 normal subjects as the control group; S22. Use ELISA to detect the stability and specificity of differential proteins in peripheral serum of spinal tuberculosis.

6. The use of LBP as a detectable marker in serological diagnosis of spinal tuberculosis according to claim 1, characterized in that: The correlation analysis method in step S3 is: S31. Collect relevant clinical data of patients with spinal tuberculosis; S32. Conduct correlation analysis based on laboratory, pathological and imaging examinations.

Images