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DNA methylation marker, diagnosis model, methylation probe and kit for detecting tuberculosis

A methylation marker and diagnostic model technology, applied in the direction of recombinant DNA technology, DNA/RNA fragment, microorganism determination/inspection, etc., can solve the problem of inability to monitor, limit the application of genomic biomarkers, and RNA instability hinders feasibility and other problems, to achieve the effect of strong diagnostic ability

Pending Publication Date: 2022-03-29
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite their strong DNA stability, the inability to monitor the progression from latent to active phase limits the utility of genomic biomarkers
Transcriptional biomarkers can reflect the interaction between M. tuberculosis and the host, but RNA instability hinders its feasibility in clinical application

Method used

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  • DNA methylation marker, diagnosis model, methylation probe and kit for detecting tuberculosis

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Embodiment 1

[0020] The present invention provides a DNA methylation marker for detecting tuberculosis, including one or more of the following three methylated regions: chr3: 195635643-195636243, chr6: 29691631-29692475 and chr11: 65315205-65315625 . Preferably, the methylated region 1: chr3: 195635643-195636243 is derived from the target gene TNK2-AS1, the methylated region 2: chr6: 29691631-29692475 is derived from the target gene HLA-F, and the methylated region 3: chr11: 65315205 -65315625 is derived from the target gene LTBP3. Preferably, the methylated region 1: chr3: 195635643-195636243 has the gene sequence shown in SEQ ID NO.1. Methylation region 2: chr6: 29691631-29692475 has the gene sequence shown in SEQ ID NO.2; methylation region 3: chr11: 65315205-65315625 has the gene sequence shown in SEQ ID NO.3.

[0021] the information is as follows:

[0022]

Embodiment 2

[0024] Embodiment 2 of the present invention provides a diagnostic model for detecting tuberculosis, according to the 3 tuberculosis-related differentially methylated regions (DMRs) involved in embodiment 1, constructing a 3-DMR diagnostic classifier based on the above-mentioned DMRs by Logistic regression .

[0025] 2.1 Collect and retrieve data set samples:

[0026] The NCBI Gene Expression Omnibus database and the European Bioinformatics Institute ArrayExpress were searched for tuberculosis-associated DNA methylation array datasets up to October 3, 2020.

[0027] 2.2 Model building process:

[0028] Using all tuberculosis-related DNA methylation chip datasets in the GEO database, Minfi was used for data processing of the original DNA methylation files, and sva was used to correct for batch effects between different datasets. LIMMA is used to identify different methylation probes with |fold change (FC)|>1.5 (p<0.05). After using DMRcate to find differentially methylated re...

Embodiment 3

[0031] This embodiment provides a process of detecting and verifying three tuberculosis-related methylated regions using a 3DMRs diagnostic classifier. The validation data set samples are clinical TB patients and healthy control (HC) specimens collected through inclusion and exclusion criteria. Specific steps are as follows:

[0032] 3.1 Collect buffy coat after centrifugation of EDTA anticoagulated blood

[0033] DNA extraction:

[0034] 1. Add 100 microliters of Qiagen protease to the bottom of a 15ml centrifuge tube.

[0035] 2. Add 1ml of whole blood.

[0036] Note: When the volume of whole blood is less than 1ml, make up 1ml with PBS.

[0037] 3. Add 1.2ml of AL buffer solution, mix thoroughly, and shake on the shaker for at least one minute to make the solution a homogeneous solution.

[0038] 4. Incubate in a 70°C water bath for 10 minutes.

[0039] 5. Add 1ml of absolute ethanol and shake fully.

[0040] 6. Transfer the liquid to the adsorption column, centrifuge ...

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Abstract

The invention provides a DNA (deoxyribonucleic acid) methylation marker, a diagnosis model, a methylation probe and a kit for detecting tuberculosis, and relates to the technical field of medical detection, the DNA methylation marker comprises one or more of the following three methylation regions: chr3: 195635643-195636243, chr6: 29691631-29692475 and chr11: 65315205-65315625. By detecting one or more of the three methylation regions, the kit has a strong diagnosis capability on tuberculosis, and meanwhile, DNA methylation is used as a biomarker to make up the limitation of instability of a sample and reflect the interaction between mycobacterium tuberculosis and a host. The invention further provides a 3DMRs diagnosis classifier constructed on the basis of the three DNA methylation regions through Logistic regression and elastic network regression methods, and the 3DMRs diagnosis classifier has high sensitivity, specificity and area under curve (AUC) and has strong diagnosis capacity for tuberculosis.

Description

technical field [0001] The invention relates to the technical field of medical detection and diagnosis, in particular to a DNA methylation marker, a diagnostic model, a methylation probe and a kit for detecting tuberculosis. Background technique [0002] Tuberculosis is a preventable and curable disease whose pathogenesis involves the host, Mycobacterium tuberculosis, the environment, and the dynamic interactions between these factors. Although genomic biomarkers have strong DNA stability, the application of genomic biomarkers is limited by the inability to monitor the process from latent to active phase. Transcriptional biomarkers can reflect the interaction between M. tuberculosis and the host, but the instability of RNA hinders its feasibility in clinical application. Existing tuberculosis detection methods include pathogenic detection such as Xpert MTB / RIF, sputum smear, culture, etc., as well as IFN-γ release assay (IGRA) and tuberculin test based on the detection abil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/154
Inventor 吕梦媛应斌武周健
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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