Use of biomarkers to discriminate between acute and chronic phases of brucellosis

Abstract

The application relates to the technical field of markers, in particular to application of biomarkers for identifying acute and chronic stages of brucellosis. A series of biomarkers related to acute and chronic stages of brucellosis are screened and found, and the biomarkers are significantly different between patients with acute and chronic brucellosis. The biomarkers can be used to realize acute and chronic staging of brucellosis, and have high sensitivity and specificity and good diagnostic efficiency. The application provides a new molecular diagnostic tool for acute and chronic staging of brucellosis, solves the problems of low specificity of existing serological detection and incapability of accurately judging clinical stages, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomarker technology, and in particular to the application of biomarkers for differentiating between the acute and chronic phases of brucellosis. Background Technology

[0002] Brucellosis is an important zoonotic systemic immune response disease caused by Brucella infection. It presents with complex and diverse clinical manifestations, has a prolonged course, and seriously endangers human health and livestock development. Based on the time characteristics of disease progression, brucellosis is clinically classified into acute phase (duration ≤ 3 months), subacute phase (duration 3-12 months), and chronic phase (duration > 12 months). Treatment regimens differ between the acute and chronic phases. Acute phase brucellosis is generally treated primarily with antibiotics, using effective and adequate doses of antibiotics for at least 6 weeks. Chronic phase brucellosis typically involves antibiotic treatment, symptomatic and supportive care, and integrated traditional Chinese and Western medicine treatments. The aim is to improve the body's immunity, reduce damage to various organs, alleviate symptoms, and improve quality of life. The treatment course for chronic phase brucellosis is longer than that for acute phase brucellosis. However, excessive or inappropriate use of antibiotics not only increases the burden on patients and the risk of side effects but also significantly accelerates the development of Brucella resistance, posing a serious threat to public health. Therefore, accurately differentiating the clinical stage of patients helps optimize antibiotic use and achieve precision medicine.

[0003] Currently, the diagnosis of brucellosis mainly relies on serological tests (such as SAT, RBPT, and ELISA) and pathogen isolation and culture. However, existing serological testing methods suffer from insufficient specificity, easily resulting in false positives or false negatives; more importantly, these methods often cannot effectively distinguish between the acute and chronic phases of the disease. During diagnosis, physicians often rely on patient descriptions and clinical symptoms to determine the duration and progression of the disease, and this subjective assessment makes it difficult for physicians to accurately determine the patient's clinical stage. These problems affect the standardized treatment and prognosis of brucellosis patients.

[0004] In recent years, with the rapid development of high-throughput bioinformatics, omics technologies such as transcriptomics have been widely applied to the screening of disease biomarkers, the study of pathogenesis, and the analysis of signaling pathways, providing new insights for disease diagnosis and classification. However, for the clinical staging of brucellosis, a specific disease, no staging diagnostic biomarkers with high sensitivity and specificity have yet been discovered. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention has discovered and verified a series of biomarkers related to the acute and chronic stages of brucellosis. Based on this, the following technical solutions are proposed.

[0006] First, the present invention provides the application of reagents for detecting the content of biomarkers in the preparation of products for differentiating between the acute and chronic phases of brucellosis; the biomarkers are at least one of UBE2L6, WARS, CLEC6A, FBXO6, MTHFD2, PSME2, ​​TYMP, AIM2, C2, CD274, GBP1, GBP2, and STAT1.

[0007] In addition, the present invention provides the application of reagents for detecting the expression level of biomarker proteins in the preparation of products for differentiating between the acute and chronic phases of brucellosis; the biomarker is at least one of UBE2L6, WARS, CLEC6A, FBXO6, MTHFD2, PSME2, ​​TYMP, AIM2, C2, CD274, GBP1, GBP2, and STAT1.

[0008] Furthermore, the present invention provides the application of reagents for detecting the expression level of biomarker genes in the preparation of products for differentiating between the acute and chronic phases of brucellosis; wherein the biomarker is at least one of UBE2L6, WARS, CLEC6A, FBXO6, MTHFD2, PSME2, ​​TYMP, AIM2, C2, CD274, GBP1, GBP2, and STAT1.

[0009] In some implementations, the product includes: a chip, a test strip, a drug, a formulation, a reagent, a kit, or a high-throughput screening platform.

[0010] In some embodiments, the reagent is a reagent used to detect the biomarker in a subject or a sample from a subject.

[0011] In the specific implementation process, the reagents include, but are not limited to, reagents used for detecting the biomarkers by RT-PCR, real-time quantitative PCR, immunoassay (e.g., ELISA, RIA, multiplex immunoassay, immunofluorescence assay, protein blotting, or dot blot assay), flow cytometry, in situ hybridization, or microarray technology.

[0012] Preferably, the sample is a blood sample.

[0013] Preferably, the levels of biomarkers in the acute phase of brucellosis are significantly higher than those in the chronic phase of brucellosis.

[0014] Preferably, the biomarker content in the acute phase of brucellosis is more than 1.5 times that in the chronic phase of brucellosis (e.g., more than 2 times, more than 2.5 times, more than 3 times, more than 3.5 times, more than 4 times, more than 4.5 times, more than 5 times, more than 5.5 times, more than 6 times, more than 6.5 times, more than 7 times, more than 7.5 times, more than 8 times).

[0015] In some implementations, the presence or absence of the biomarker in a subject or a sample from the subject can be used to determine whether the subject is in the acute or chronic phase of brucellosis.

[0016] Furthermore, the present invention provides a kit for detecting acute and chronic stages of brucellosis, comprising: RNA extraction reagent, reverse transcription reagent, qPCR reagent and primers for detecting biomarkers; wherein the biomarkers are at least one selected from UBE2L6, WARS, CLEC6A, FBXO6, MTHFD2, PSME2, ​​TYMP, AIM2, C2, CD274, GBP1, GBP2, and STAT1.

[0017] Preferably, the primers for detecting the biomarkers are shown in SEQ ID No. 1 to SEQ ID No. 26.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] This invention has identified a series of biomarkers associated with the acute and chronic staging of brucellosis, which show significant differences between patients with acute and chronic brucellosis. These biomarkers can be used for acute and chronic staging of brucellosis with high sensitivity and specificity, demonstrating good diagnostic efficacy. This invention provides a new molecular diagnostic tool for the acute and chronic staging of brucellosis, solving the problems of low specificity and inaccurate clinical staging of existing serological tests, and has broad application prospects. Attached Figure Description

[0020] Figure 1 It is a differential gene volcano map.

[0021] Figure 2 This is a heatmap showing the correlation between gene co-expression modules and phenotypes in the acute and chronic phases of brucellosis, healthy individuals, age, and sex.

[0022] Figure 3 This is a Venn diagram showing the differential gene screening results between the core genes identified by WGCNA and the clinical stages of brucellosis.

[0023] Figure 4 yes AIM2 , C2 , CD274 Statistical chart of expression levels.

[0024] Figure 5 yes CLEC6A , GBP1 , GBP2 Statistical chart of expression levels.

[0025] Figure 6 yes MT2A , FBXO6 , MTHFD2 Statistical chart of expression levels.

[0026] Figure 7 yes UBE2L6 , PSME2 , WARS Statistical chart of expression levels.

[0027] Figure 8 yes TYMP, STAT1 Statistical chart of expression levels.

[0028] Figure 9 It is a ROC curve. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] Example 1: Screening of Biomarkers

[0031] This embodiment collects peripheral blood mononuclear cell (PBMC) samples from patients in the acute and chronic phases of brucellosis for transcriptomic RNA sequencing, and performs comprehensive analysis in conjunction with clinical test results. Validation is conducted using an external cohort to identify reliable diagnostic biomarkers. The specific steps are as follows:

[0032] Peripheral blood was collected from 63 subjects (24 in the acute phase, 34 in the chronic phase, and 5 healthy controls). The experiment in this invention was approved by the Ethics Review Committee of the Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. All subjects participated voluntarily, were verbally informed of the purpose of the experiment, and signed informed consent forms. Demographic data of the enrolled subjects were collected, including gender, age, ethnicity, and occupation. Clinical data included the time of first symptom onset, time of diagnosis, sample collection time, laboratory test results, clinical symptoms, complications, and treatment regimen.

[0033] PBMCs were isolated using Ficoll density gradient centrifugation. RNA was extracted using the TRIZOL reagent method, followed by next-generation sequencing, and the data were analyzed using bioinformatics.

[0034] Transcriptomics sequencing and bioinformatics analysis:

[0035] Transcriptome sequencing was performed using the Illumina HiSeq platform, and clean reads were obtained after Fastp quality control.

[0036] Differential gene expression analysis: DESeq2 was used to screen for differentially expressed genes between groups (|log2FC|≥1, p <0.05), there were 751 DEGs between the acute and chronic phase groups, with 449 upregulated genes and 302 downregulated genes, respectively.

[0037] WGCNA Analysis: Constructing a scale-free network (soft threshold β=4, R 2 >0.8), identifying 18 gene modules. The brown module (ME.brown) was positively correlated with the acute phase ( R =0.807, p <0.01), negatively correlated with the chronic phase ( R =-0.661, p <0.01), and was significantly positively correlated with liver function indicators (ALT, AST and γ-GGT), inflammatory markers (CRP) and brucellosis clinical symptoms (fever and muscle pain), and significantly negatively correlated with spondylitis complications;

[0038] The two analytical methods jointly screened out 103 core differentially expressed genes. Through differential expression analysis and weighted gene co-expression network analysis (WGCNA), 14 core genes were finally obtained: AIM2 , C2 , CD274 , CLEC6A , FBXO6 , GBP1 , GBP2 , MT2A , MTHFD2 , PSME2 , STAT1 , TYMP , UBE2L6 , WARS Among them, the differential gene volcano diagram is as follows: Figure 1 As shown in the heatmap, the correlation between gene co-expression modules and brucellosis phenotypes in acute and chronic phases, healthy individuals, age, and sex is as follows. Figure 2 As shown in the figure, the Venn diagram of differentially expressed genes related to clinical stages of brucellosis, based on the core genes screened by WGCNA, is as follows. Figure 3 As shown.

[0039] Example 2: Validation of the marker

[0040] First, based on the results of Example 1, this example constructs a brucellosis acute and chronic staging detection kit for the clinical diagnosis of acute and chronic brucellosis. The kit consists of RNA extraction reagent, reverse transcription reagent, qPCR reagent, and primers for 14 biomarkers (primers for biomarker detection are shown in SEQ ID No. 1 to SEQ ID No. 28, with β-actin as an internal reference gene). β-actin The primers are shown in SEQ ID No. 29 to SEQ ID No. 30, and the primer information is shown in Table 1.

[0041] Table 1 Primer Information

[0042]

[0043] Furthermore, this embodiment collected PBMC samples from 26 additional clinically diagnosed brucellosis patients at different disease stages (11 in the acute phase and 15 in the chronic phase), different from those in Embodiment 1. The above-mentioned kit was used to analyze the clinical staging diagnostic performance of brucellosis using qRT-PCR. Expression levels were determined by 2... –ΔΔCt The method was used to calculate the result, with β-actin as the internal reference gene.

[0044] The specific detection method for qRT-PCR is as follows:

[0045] 1. Sample preparation

[0046] RNA samples: RNA was extracted from PBMC cells using the TRIZOL method and then reverse transcribed into cDNA (Tiangen KR116) (suitable for qRT-PCR detection of RNA). Ensure sample purity (A260 / A280 ratio between 1.8 and 2.0) and avoid protein or inhibitor contamination.

[0047] 2. Preparation of qPCR reaction system

[0048] Reaction solution composition: qPCR reagent: SuperReal PreMix Plus SYBR qPCR Mix (Tiangen FP205).

[0049] Core components: qPCR premix (containing Taq DNA polymerase, dNTPs, buffer, K) + NH4 + (etc.), forward and reverse primers (final concentration usually 0.6 μM), template DNA or cDNA (1 μL), dye method: add SYBR Green dye (premixed in the reagent), and make up to the final volume with nuclease-free water.

[0050] Dispensing and control settings:

[0051] Negative control: template-free (replaced with nuclease-free water).

[0052] Positive control: Target sequence at known concentration.

[0053] Internal reference gene (for relative quantification): housekeeping gene (β-actin).

[0054] After the reaction, confirm the amplification curve, melting curve, CT value, standard curve, etc. of Real Time qPCR, and perform qRT-PCR quantitative data analysis.

[0055] 3. qPCR program settings

[0056] Instrument parameters: Select the detection channel (SYBR Green) according to the fluorescent dye.

[0057] Set up the reaction procedure:

[0058] Pre-denaturation: 95℃, 15 minutes (to activate Taq enzyme). Amplification cycle (repeated 40 times): Denaturation: 95℃, 10 seconds. Annealing: 55℃, 30 seconds. Extension: 72℃, 20 seconds.

[0059] Melting curve analysis: The temperature was gradually increased from 60-95℃, and fluorescence changes were detected to confirm product specificity.

[0060] 4. Data Analysis

[0061] Threshold line and Ct value

[0062] Ct value (Threshold Cycle): The number of cycles required for the fluorescence signal to reach a set threshold, which is inversely proportional to the amount of initial template.

[0063] Use 2 -ΔΔct Method calculation, 2 -ΔΔct =2 - [(Target gene CT in experimental group - Internal reference gene CT in experimental group) - Target gene CT in control group - Internal reference gene CT in control group)], and plotted a bar chart of the relative expression levels and trends of the core genes. See the results below. Figures 4-8 (in the picture, This indicates a statistically significant change between groups. 2 -ΔΔct It is expressed as Mean±SD. P<0.001, P<0.01, (P<0.05) and Table 2.

[0064] Table 2. Expression levels of core genes in the acute and chronic phases of brucellosis.

[0065]

[0066] The results showed that, except MT2A In addition, the expression levels of the remaining 13 genes differed significantly between the acute and chronic phases of brucellosis.

[0067] Furthermore, the diagnostic performance of 13 biomarkers that showed significant differences between the acute and chronic phase groups in the validation experiment was analyzed individually for acute and chronic brucellosis staging. The ROC curve results are as follows: Figure 9 As shown, the results indicate that AIM2 The AUC value is 0.818. C2 The AUC value is 0.870. CD274 The AUC value is 0.818. CLEC6A The AUC value was 0.857. GBP1 The AUC value was 0.909. GBP2 The AUC value is 1.000. FBXO6 The AUC value is 0.818. MTHFD2 The AUC value is 0.844. UBE2L6 The AUC value was 0.948. PSME2 The AUC value was 0.779. TYMP The AUC value is 0.818. WARS The AUC value was 0.779. STAT1 The AUC was 0.961 (P<0.05).

[0068] The above results demonstrate that using 13 biomarkers alone for acute and chronic staging of brucellosis has good sensitivity and specificity.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a reagent for detecting the content of a biomarker in the preparation of a product for identifying acute and chronic phases of brucellosis; the biomarker being CLEC6A.

2. Use of a reagent for detecting the expression level of a biomarker protein in the preparation of a product for identifying acute and chronic phases of brucellosis; the biomarker being CLEC6A.

3. Use of a reagent for detecting the expression level of a biomarker gene in the preparation of a product for identifying acute and chronic phases of brucellosis; the biomarker being CLEC6A.

4. The use according to any one of claims 1 to 3, characterized in that, The product comprises a chip, a test paper or a kit.

5. The use according to any one of claims 1 to 3, characterized in that, The product is used for detecting a blood sample.

6. The application according to any one of claims 1 to 3, characterized in that, The content of the biomarker in the acute phase of brucellosis is significantly higher than that in the chronic phase of brucellosis.

7. Use according to claim 6, characterized in that, The content of the biomarker in the acute phase of brucellosis is more than 1.5 times that in the chronic phase of brucellosis.

8. Use according to claim 7, characterized in that, The content of the biomarker in the acute phase of brucellosis is more than 2 times that in the chronic phase of brucellosis.

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