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Kit and method for detecting mycobacterium tuberculosis infection and application

A technology of Mycobacterium tuberculosis and reagents, which is applied in the field of biomedical testing, can solve problems such as specificity affecting diagnosis, and achieve the effects of quality assurance, improving reaction efficiency and improving positive detection rate.

Active Publication Date: 2011-04-06
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, some RD region antigens with strong immunogenicity still have cross-reactivity with BCG and non-tuberculous mycobacteria, which affects the specificity of diagnosis

Method used

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  • Kit and method for detecting mycobacterium tuberculosis infection and application
  • Kit and method for detecting mycobacterium tuberculosis infection and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Analysis and screening of Mycobacterium tuberculosis-specific T cell epitopes

[0043] Through the analysis of bioinformatics combined with the detection of T cell immune response in vitro to analyze and screen the T cell antigen epitope of the protein, the specific process is as follows:

[0044] 1. Predict the T cell epitopes of the RD2 region protein Rv1985c and RD11 region protein Rv3425 through bioinformatics technology and corresponding databases such as ProPred, EpiPredict, etc., and analyze the recognition relationship between them and different HLA types.

[0045] 2. Combining with the results of bioinformatics analysis, design polypeptides covering the amino acid sequences of Rv1985c and Rv3425 proteins by means of Overlap (overlapping sequences). Each polypeptide contains 22-25 amino acids, and there are 9 or 10 amino acid repeating sequences between two adjacent polypeptides. Peptides were synthesized by a solid-phase synthesizer.

[0046] 3. Co...

Embodiment 2

[0048] Example 2: Detection of IFN-γ by ELISA method through whole blood IFN-γ release assay

[0049] The whole blood IFN-γ release test was used to test in sputum smear or culture-positive tuberculosis patients and BCG-vaccinated healthy people. The polypeptides of SEQ ID No1-10 are synthesized by a solid-phase synthesizer according to the experimental conditions of the synthesizer instructions, and analyzed and purified by HPLC. The purified peptide dry powder was dissolved in DMSO, prepared as a 10mg / ml storage solution, and stored at -80°C. Before use, dilute with RPMI1640 culture medium to a concentration of 10ug / ml.

[0050] test subject:

[0051] Tuberculosis-infected patients and healthy people were recruited from patients and their family members of Chongqing Pulmonary Hospital and students of Fudan University in Shanghai. Tuberculosis infection includes patients with active tuberculosis and latent tuberculosis infection. Blood samples drawn from subjects were ant...

Embodiment 3

[0074] Embodiment 3: ELISPOT method detects IFN-γ secretion

[0075] 1. Collect blood from tuberculosis infected patients and healthy controls, about 2-5ml each

[0076] 2. Separate peripheral blood mononuclear cells (PBMC) with Ficoll lymphocyte separation medium, and resuspend PBMC in RPMI1640+10% FBS for later use

[0077] 3. Pre-coat overnight with 10ug / ml IFN-γmAB on a 96-well PVDF plate (Millipore);

[0078] 4. Wash twice with medium RPMI 1640, and block with RPMI 1640+10% FBS at 4°C for 1 hour;

[0079] 5. Add 100ul to each well with a concentration of 2.5×10 5 PBMC cells / ml. A single polypeptide or polypeptide mixture was added to the experimental wells, with a final concentration of 10ug / ml for each polypeptide; PHA with a final concentration of 5ug / ml was added to the positive control wells; 100ul AIV-M medium was added to the negative control wells. Each protein in the experimental group is a duplicate well, that is, repeated 2 times;

[0080] 6. Incubate in an...

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Abstract

The invention belongs to the field of biomedicine examination, and particularly relates to a kit and a method for detecting mycobacterium tuberculosis infection and application. The invention discloses a novel mycobacterium tuberculosis detection reagent by screening specific T cell epitope of mycobacterium tuberculosis, wherein the reagent contains polypeptide or analog thereof represented by SEQ ID No.1-10. The method detects cell factors released from T cells by using single or more SEQ ID No.1-10 polypeptides to contact the T cells of mycobacterium tuberculosis infected individuals. The method can effectively detect active tuberculosis or latent tuberculosis infection, and is free from disturbance of Bacilli Calmette Guerin (BCG) inoculation vaccines. The invention also discloses a diagnostic kit and other application based on the polypeptide and the method. Compared with the gamma interferon release experiments in the prior art, the method can obviously improve the detection rate without reducing the specificity and has high clinical application value.

Description

technical field [0001] The invention belongs to the field of biomedical testing, and relates to a microbial molecular biological detection method, in particular to a reagent, method and application for detecting mycobacterium tuberculosis infection. Background technique [0002] Tuberculosis is one of the major public health problems facing the world today. Its pathogen is Mycobacterium tuberculosis, which belongs to the genus Mycobacterium. In recent years, due to the emergence of drug-resistant tuberculosis, human immunodeficiency virus (HIV) combined with Mycobacterium tuberculosis infection, and large-scale population movement, the epidemic situation of tuberculosis has become more serious. According to survey reports, about 9 million people worldwide develop active tuberculosis every year, and 2 to 3 million people die of tuberculosis. my country is one of the 22 countries with a high burden of tuberculosis in the world, with 4.5 million tuberculosis patients, includin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/569
Inventor 张文宏王森陈嘉臻王菲菲邵凌云金嘉琳翁心华
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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