Application of LLGL2 gene as a biomarker in differential diagnosis of tuberculosis

Abstract

The application can diagnose the infection of a tuberculosis patient by detecting the expression amount of the LLGL2 gene, and the detection method is simple, fast, and has high detection quality and accuracy, and the sensitivity and specificity are all greater than 80%. When the expression amount of the biomarker LLGL2 gene is greater than 1.476, the subject is judged as a latent tuberculosis infection patient; when the expression amount of the biomarker LLGL2 gene is less than or equal to 1.476, the subject is judged as not suspected to be a latent tuberculosis infection patient, and needs further diagnosis. When the expression amount of the biomarker LLGL2 gene is less than 0.8653, the subject is judged as an active tuberculosis patient; when the expression amount of the biomarker LLGL2 gene is greater than or equal to 0.8653, the subject is judged as not suspected to be an active tuberculosis patient, and needs further diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection technology, and in particular to the application of LLGL2 gene in differential diagnosis of tuberculosis. BACKGROUND

[0002] Tuberculosis (TB) is one of the most deadly human chronic infectious diseases caused by Mycobacterium tuberculosis (Mtb) worldwide, and is also one of the major challenges of global public health, and is also the "number one killer" of human immunodeficiency virus (HIV) infected persons, and is also the main cause of death related to antibiotic resistance, and the mortality rate is very high in HIV carriers infected with multi-drug resistant Mycobacterium tuberculosis, and this mixed infection is also the cause of the prevalence of multi-drug resistant tuberculosis. According to the estimate of the World Health Organization, there were more than 1060,000 new tuberculosis patients worldwide in 2021, and the number of deaths was more than 160,000, and only 36% of the patients were treated, and the prevalence of drug-resistant strains and the combination of HIV further aggravated the global TB burden. Tuberculosis is an infectious disease with treatment as the main prevention and control means, so early diagnosis, early treatment and improvement of cure rate are the key links of tuberculosis control in China.

[0003] The most prominent problem in the field of tuberculosis diagnosis in China is the low rate of etiological diagnosis, and the positive rate of tuberculosis etiology in China in 2020 reached 47%, which is still lower than the global average of 57%. In recent years, although some advanced molecular biology detection techniques based on nucleic acid amplification have emerged, the detection limit (LOD) has been reduced, but due to the high cost of equipment and price, it is difficult to popularize and use in clinical practice. The technology of metagenomic sequencing has been well applied in clinical practice, but the main advantage of this technology is the screening of pathogens that have failed to obtain diagnostic clues, and for specimens with very low bacterial load, the detection results are not ideal, and the price is very expensive, the operation is complex, and it cannot be directly carried out in hospitals. Screening of specific molecular markers in body fluid samples is an examination technique independent of the presence of bacteria, and is also the main research direction to solve the diagnostic problem of patients who are not truly bacteria-free. In recent years, some studies have tried to screen specific protein markers, mRNA, metabolites from blood, and study specific components of exosomes in body fluid samples, and have found many relatively specific molecular markers, which can help improve the diagnosis rate of TB.

[0004] LLGL2 is one of the family members of human LGL, located on chromosome 17 of human genome, which encodes LLGL2 protein, a tyrosine protein kinase, involved in cell signaling and regulating cell proliferation, differentiation and metastasis, and is a scaffold protein for regulating the establishment of epithelial cell apical-basal polarity. At present, there is little research on the gene in clinic, and its specific function is not clear. Studies have found that LLGL2 is involved in the occurrence and development of various tumors such as pancreatic cancer, gastric cancer, lung adenocarcinoma, colon cancer and breast cancer, and all show reduced expression. LLGL2 gene has been reported in breast cancer before, and LLGL2 plays a role of tumor growth promoter in breast cancer. Studies have shown that it promotes cell proliferation under nutritional stress, but there is no research report on the relationship between LLGL2 and tuberculosis. + LLGL2 plays a role of tumor growth promoter in breast cancer. Studies have shown that it promotes cell proliferation under nutritional stress, but there is no research report on the relationship between LLGL2 and tuberculosis. SUMMARY

[0005] In a first aspect, the present application provides the use of LLGL2 gene as a biomarker in the preparation of a product for differential diagnosis of tuberculosis infection, wherein the use is achieved by the expression level of LLGL2 gene in the body of the subject.

[0006] Further, the detection sample of the expression level of the LLGL2 gene is selected from the peripheral blood sample of the subject.

[0007] Further, the differential diagnosis of tuberculosis infection is to determine whether the subject is a latent infection patient or an active tuberculosis infection patient by the expression level of the LLGL2 gene.

[0008] Further, when the expression level of the biomarker LLGL2 gene is >1.476, the subject is determined to be a latent tuberculosis infection patient; when the expression level of the biomarker LLGL2 gene is ≤1.476, it is determined whether the subject is a latent tuberculosis infection patient or not, and further diagnosis is needed.

[0009] Further, when the expression level of the biomarker LLGL2 gene is <0.8653, the subject is determined to be an active tuberculosis patient; when the expression level of the biomarker LLGL2 gene is ≥0.8653, it is determined whether the subject is an active tuberculosis patient or not, and further diagnosis is needed.

[0010] Further, tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection.

[0011] Further, the Mycobacterium tuberculosis infection includes primary infection, secondary infection and extrapulmonary infection.

[0012] Further, the tuberculosis includes but is not limited to drug-resistant tuberculosis, non-drug-resistant tuberculosis, pulmonary tuberculosis and extrapulmonary tuberculosis.

[0013] Further, the drug-resistant tuberculosis includes, but is not limited to, single drug-resistant tuberculosis, multi-drug resistant tuberculosis, multi-drug resistant tuberculosis, extensively drug-resistant tuberculosis.

[0014] Further, the pulmonary tuberculosis includes primary tuberculosis, secondary tuberculosis, blood type disseminated tuberculosis, trachea-bronchus tuberculosis, tuberculous pleurisy, and bacterin-negative pulmonary tuberculosis.

[0015] Further, the extrapulmonary tuberculosis includes, but is not limited to, intestinal tuberculosis, renal tuberculosis, bone and joint tuberculosis, etc.

[0016] In a second aspect, the present application provides the use of the biomarker in the first aspect in the preparation of a kit for differential diagnosis of tuberculosis, wherein the biomarker is LLGL2, and the use is achieved by the expression level of the LLGL2 gene in the body of a subject.

[0017] In a third aspect, the present application provides a detection kit for differential diagnosis of tuberculosis infection, wherein the kit contains a detection reagent for the expression level of the biomarker LLGL2 gene in the first aspect, an instruction manual, etc.

[0018] Further, when the expression level of the biomarker LLGL2 gene is > 1.476, the subject is determined to be a latent tuberculosis infection patient; when the expression level of the biomarker LLGL2 gene is ≤ 1.476, the subject is determined to be uncertain whether to be a latent tuberculosis infection patient, and needs further diagnosis.

[0019] Further, when the expression level of the biomarker LLGL2 gene is < 0.8653, the subject is determined to be an active tuberculosis patient; when the expression level of the biomarker LLGL2 gene is ≥ 0.8653, the subject is determined to be uncertain whether to be an active tuberculosis patient, and needs further diagnosis.

[0020] In a fourth aspect, the present application provides a diagnosis system for identifying latent tuberculosis infection patients and active tuberculosis infection patients; the system comprises a data acquisition module and a differential diagnosis module; the data acquisition module is used to acquire the biomarker gene expression data of the patient to be tested in the first aspect; the differential diagnosis module is used to take the biomarker gene expression data as an input variable, and provide a diagnosis result after a set data analysis and comparison process.

[0021] Further, when the expression level of the biomarker LLGL2 gene is > 1.476, the subject is determined to be a latent tuberculosis infection patient; when the expression level of the biomarker LLGL2 gene is ≤ 1.476, the subject is determined to be uncertain whether to be a latent tuberculosis infection patient, and needs further diagnosis.

[0022] Further, when the expression amount of the biomarker LLGL2 gene is <0.8653, the subject is determined as an active tuberculosis patient; when the expression amount of the biomarker LLGL2 gene is ≥0.8653, the subject is determined as uncertain whether to be an active tuberculosis patient, and needs further diagnosis.

[0023] Beneficial effects:

[0024] The application differentiates and diagnoses the infection of tuberculosis patients by detecting the expression amount of LLGL2 gene, and the method is simple and fast, and the sensitivity and specificity are both >80%, and the detection quality is high and accurate. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 LLGL2 expression level in blood of active tuberculosis and tuberculosis latent infection in data set GSE62525;

[0026] Figure 2 ROC analysis of relative expression level of LLGL2 in blood of active tuberculosis and tuberculosis latent infection in data set GSE62525;

[0027] Figure 3 LLGL2 expression level in PBMCs of active tuberculosis patients, tuberculosis latent infection and healthy controls;

[0028] Figure 4 ROC analysis of relative expression level of LLGL2 in PBMCs of active tuberculosis patients and tuberculosis latent infection;

[0029] Figure 5 ROC analysis of relative expression level of LLGL2 in PBMCs of active tuberculosis patients and healthy controls;

[0030] Figure 6 ROC analysis of relative expression level of LLGL2 in PBMCs of tuberculosis latent infection and healthy controls. DETAILED DESCRIPTION

[0031] In order to better understand the present application, but not limited to the present application. The experimental methods in the following examples are all conventional methods, unless otherwise specified. The experimental materials used in the following examples are all purchased from conventional biochemical reagent companies, unless otherwise specified. The quantitative tests in the following examples are all set with three repeated experiments, and the average value is taken as the result.

[0032] Example 1: LLGL2 expression level detection and correlation analysis with tuberculosis

[0033] 1. Data set acquisition

[0034] Gene expression profile GSE62525 from GEO database was used as an independent validation dataset.

[0035] 2. Analysis of LLGL2 expression level difference between active tuberculosis patients and tuberculosis latent infection

[0036] Background correction, inter-array normalization and group difference analysis were performed using the "limma" package of R software; data visualization was performed using the "ggpubr" package, and the results are shown in Figure 1 LLGL2 expression level in the latent tuberculosis infection group was significantly higher than that in the active tuberculosis group.

[0037] 3. ROC analysis of LLGL2 expression level in active tuberculosis patients and tuberculosis latent infection

[0038] ROC curve analysis was performed using the "pROC" package of R software to identify the diagnostic value of LLGL2 as a latent tuberculosis infection biomarker, and the results are shown in Figure 2 The area under the ROC curve was 0.811 (95% CI: 0.597-0.974), indicating that LLGL2 expression level had diagnostic value in distinguishing or identifying tuberculosis latent infection and active tuberculosis.

[0039] Example 2: LLGL2 gene expression level for differential diagnosis of tuberculosis

[0040] Experimental materials and methods

[0041] 1. Obtaining of peripheral blood samples

[0042] Peripheral blood samples of subjects: 33 active tuberculosis patients, 26 tuberculosis latent infection and 33 healthy subjects.

[0043] Healthy controls: aged 18-65 years; no clinical symptoms of tuberculosis; negative gamma interferon release test; no lung lesions found by chest X-ray or CT; no other serious medical history.

[0044] Tuberculosis latent infection: no clinical symptoms of tuberculosis; positive gamma interferon release test, no history of tuberculosis treatment.

[0045] Active tuberculosis patients: aged 18 years or older; have clinical symptoms of tuberculosis; lung lesions found by chest X-ray or CT.

[0046] Method for obtaining peripheral blood samples: 4-5 ml of peripheral blood was drawn from each subject and placed in an EDTA anticoagulant blood collection tube, and the anticoagulant was mixed evenly with the peripheral blood by turning it upside down 5-6 times.

[0047] 2. Isolation of peripheral blood mononuclear cells (PBMCs)

[0048] First dilute the anticoagulant peripheral blood 1:1 with 1xPBS, then use human peripheral blood mononuclear cell separation solution for density gradient centrifugation (Ficoll, Solabio), collect the cells in the middle mononuclear cell layer, and then wash twice with 1xPBS medium to obtain PBMCs.

[0049] 3. Extraction of RNA

[0050] Use the Super Total RNA Extraction Kit of Promega Company to extract total RNA in PBMCs. The specific steps are as follows:

[0051] (1) Discard the cell culture medium;

[0052] (2) Wash the cells 3 times with sterile 1xPBS;

[0053] (3) Add 300ul of RNA lysis solution, mix well, and transfer to a new nuclease-free ep tube;

[0054] (4) Add 300ul of RNA dilution solution, mix well, and incubate at room temperature for 3-5min;

[0055] (5) Centrifuge at 12000rpm for 5min, and transfer the supernatant to a new nuclease-free ep tube;

[0056] (6) Add 0.5 times the volume of absolute ethanol to the supernatant, mix well;

[0057] (7) Transfer the mixture to a centrifugal column, centrifuge at 12000-14000rpm for 1min, and discard the filtrate;

[0058] (8) Add 600ul of RNA washing solution, centrifuge at 12000-14000rpm for 1min, and discard the filtrate;

[0059] (9) Add 50ul of DNAase I (10xDNAase I buffer: 5ul, DNAase I: 5ul, nuclease-free water: 40ul, mix gently, do not shake), to the center of the adsorption membrane, and incubate at room temperature for 15min;

[0060] (10) Add 600ul of RNA washing solution, centrifuge at 12000-14000rpm for 1min, and discard the filtrate;

[0061] (11) Add 600ul of RNA washing solution, centrifuge at 12000-14000rpm for 1min, and discard the filtrate; empty, centrifuge at 12000-14000rpm for 2min;

[0062] ​(12) Transfer the centrifuge column to the elution tube, add 50-200 μL of nuclease-free water to the center of the centrifuge column membrane, incubate at room temperature for 2 min, centrifuge at 12000-14000 rpm for 1 min, and store the RNA at -80℃.

[0063] 4. Obtaining cDNA

[0064] Using Promega The Reverse Transcription System reverse transcribes the extracted RNA (see Table 1) to obtain cDNA. The specific steps are as follows:

[0065] Will After thawing each component of the reverse transcription system on ice, add the components according to the table below to prepare an RT-Mix (calculated based on a total reaction volume of 20 μL; usually, prepare 1-2 more RT-Mix units than the actual number of samples to prevent volume loss due to liquid adhering to the walls):

[0066] Table 1. Reverse transcription reaction system

[0067]

[0068]

[0069] Distribute the prepared RT-Mix into each reaction tube, add total RNA (1-21g), and add water to bring the volume to 20μL; set the program to perform the reverse transcription reaction, including extension at 42℃ for 15min and reverse transcriptase inactivation at 70℃ for 15min.

[0070] 5. Primer Sequence and Synthesis

[0071] The primer sequences for the internal reference gene β-actin and the target gene LLGL2 are shown in Table 2.

[0072] Table 2. Primer sequences for the internal reference gene β-actin and the target gene LLGL2

[0073]

[0074] 6. Real-time quantitative PCR

[0075] The reaction system is as follows: qPCR 10 μL Green Master Mix, 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), 2 μL cDNA (5-20 ng), and make up to 20 μL with nuclease-free water. The forward and reverse primers are LLGL2-F and LLGL2-R, respectively.

[0076] β-actin-F and β-actin-R were detected using a Bio-Rad CFX96 Real-Time System. Reaction conditions: pre-denaturation: 95℃, 3 min; amplification reaction: 95℃, 5 s, 58℃, 30 s, fluorescence signal detected during extension, 40 cycles. Two... -ΔΔCt Calculate the relative expression levels of genes.

[0077] 7. Statistical Analysis

[0078] Statistical analysis was performed using GraphPad Prism 9.2.

[0079] Experimental results

[0080] 1. Relative expression level of LLGL2 in PBMCs of patients with active tuberculosis and those with latent tuberculosis infection

[0081] The relative expression level of LLGL2 in 33 patients with active tuberculosis and 26 patients with latent tuberculosis infection was detected by RT-PCR. The results are as follows: Figure 3 As shown, the relative expression level of LLGL2 in PBMCs of patients with active tuberculosis was significantly lower than that in patients with latent tuberculosis infection (P<0.001), indicating that the expression level of LLGL2 has important value in distinguishing between patients with active tuberculosis and those with latent tuberculosis infection.

[0082] 2. Relative expression levels of LLGL2 in PBMCs of patients with active tuberculosis and healthy individuals

[0083] The relative expression level of LLGL2 in 33 patients with active tuberculosis and 33 healthy individuals was detected by RT-PCR. The results are as follows: Figure 3 As shown, the relative expression level of LLGL2 in PBMCs of patients with active tuberculosis was significantly lower than that in healthy individuals (P<0.001). This indicates that the expression level of LLGL2 has important value in the diagnosis of active tuberculosis.

[0084] 3. Relative expression level of LLGL2 in PBMCs of latent tuberculosis infected individuals and healthy individuals

[0085] The relative expression levels of LLGL2 in 26 patients with latent tuberculosis infection and 33 healthy individuals were detected by RT-PCR. Results are as follows: Figure 3 As shown, the relative expression level of LLGL2 in PBMCs of latent tuberculosis infected individuals was significantly higher than that in healthy individuals (P = 0.008). This indicates that the expression level of LLGL2 has important value in the diagnosis of latent tuberculosis infected individuals.

[0086] 4. ROC curve analysis of patients with active tuberculosis and those with latent tuberculosis infection

[0087] According to the relative expression amount of LLGL2 in PBMCs of active tuberculosis patients and tuberculosis latent infection, the receiver operating characteristic curve (ROC) was drawn. The results are shown in Figure 4 The area under the ROC curve was 0.976. It showed that the expression amount of LLGL2 had important value in distinguishing active tuberculosis patients from tuberculosis latent infection.

[0088] 5. ROC curve analysis of active tuberculosis patients and healthy controls

[0089] According to the relative expression amount of LLGL2 in PBMCs of active tuberculosis patients and healthy controls, the ROC curve was drawn. The results are shown in Figure 5 The area under the ROC curve was 0.906. It showed that the expression amount of LLGL2 had important value in diagnosing active tuberculosis.

[0090] 6. ROC curve analysis of tuberculosis latent infection and healthy controls

[0091] The relative expression amount of LLGL2 in PBMCs of tuberculosis latent infection and healthy controls, the ROC curve was drawn. The results are shown in Figure 6 The area under the ROC curve was 0.782. It showed that the expression amount of LLGL2 had important value in diagnosing tuberculosis latent infection and healthy.

[0092] 7. Interpretation of results in practical application of LLGL2 gene

[0093] In practical application, active tuberculosis can be diagnosed according to the following judgment criteria: if the relative expression amount of LLGL2 gene in the peripheral blood of the testee is lower than 0.8653 (sensitivity is 90.91%, confidence interval is 76.43%-96.86%; specificity is 84.91%, confidence interval is 72.95%-92.15%), the testee is or suspected to be an active tuberculosis patient; if the relative expression amount of LLGL2 gene in the peripheral blood of the testee is equal to or higher than 0.8653, the testee is not or suspected not to be an active tuberculosis patient; if the relative expression amount of LLGL2 gene in the peripheral blood of the testee is higher than 1.476 (sensitivity is 80.77%, confidence interval is 62.12%-91.49%; specificity is 86.36%, confidence interval is 76.07%-92.66%), the testee is or suspected to be a tuberculosis latent infection; if the relative expression amount of LLGL2 gene in the peripheral blood of the testee is equal to or lower than 1.476, the testee is not or suspected not to be a tuberculosis latent infection

[0094] The application has been described in detail. For those skilled in the art, the application can be implemented in a wider range under the same parameters, concentrations and conditions without departing from the spirit and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. The application of the LLGL2 gene as a biomarker in the preparation of products for the differential diagnosis of tuberculosis, wherein the application is achieved by measuring the expression level of the LLGL2 gene in a subject; the sample for detecting the LLGL2 gene expression level is selected from the peripheral blood sample of the subject; the differential diagnosis of tuberculosis is based on the expression level of the LLGL2 gene to determine whether the subject is a latently infected patient or an active tuberculosis patient; when the expression level of the biomarker LLGL2 gene is >1.476, the subject is determined to be a latent .... When the expression level of the biomarker LLGL2 gene is ≤1.476, it is determined that the subject is uncertain whether they have latent tuberculosis infection and further diagnosis is required; when the expression level of the biomarker LLGL2 gene is <0.8653, the subject is determined to have active tuberculosis; when the expression level of the biomarker LLGL2 gene is ≥0.8653, it is determined that the subject is uncertain whether they have active tuberculosis and further diagnosis is required; the tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis infection, and the Mycobacterium tuberculosis infection includes: Primary infection, secondary infection, extrapulmonary infection.

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