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A kit and method for double staining of Mycobacterium tuberculosis and its antigen

A technology of mycobacterium tuberculosis and staining method, which is applied in the field of double staining kits for mycobacterium tuberculosis and its antigen, can solve the problems of low sensitivity, MTB time-consuming and labor-intensive, etc. The effect of clinical extension value

Active Publication Date: 2017-08-25
BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used MTB detection method is the Ziehl-Neelsen staining method, but there are disadvantages such as low sensitivity and time-consuming and labor-intensive search for MTB.

Method used

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  • A kit and method for double staining of Mycobacterium tuberculosis and its antigen
  • A kit and method for double staining of Mycobacterium tuberculosis and its antigen
  • A kit and method for double staining of Mycobacterium tuberculosis and its antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, staining method of tuberculosis tissue specimen

[0050] 1. Reagent configuration:

[0051] 0.01mol / L, the sodium citrate buffer solution that pH value is 6.0: 2.41g sodium citrate (Na 3 C 6 h 5 o 7 2H 2 O) and 0.38g citric acid (C 6 h 8 o 7 ·H 2 O) Dissolve in 1 L of distilled water.

[0052] Concentration of 0.01M, pH value of 7.4 PBS formulation: 2.9g Na 2 HPO 4 12H 2 O, 0.3g NaH 2 PO 4 2H 2 O and 9gNaCl were dissolved in 1L of distilled water to adjust the pH value to 7.4.

[0053] Polyclonal antibody against MTB antigen: New Zealand white rabbits were immunized with MTB antigen (amino acid sequence is sequence 1), rabbit serum was collected, and polyclonal antibody against MTB antigen was obtained by purification with proteinG.

[0054] DAB staining solution: first prepare 20×DAB dyeing mother solution: pour 0.1g of diaminobenzidine (3,3'-diaminobenzidine, DAB) into 10ml of distilled water to obtain a mixed solution, add 3-5 drops of 10M...

Embodiment 2

[0105] Embodiment 2, staining method of tuberculosis tissue specimen

[0106] In order to explore the clinical application value of the kit prepared in Example 1, 212 clinically confirmed tuberculosis tissue samples (provided by Beijing Chest Hospital Affiliated to Capital Medical University, and informed by the patient) and 63 cases of control disease tissue samples (clinically confirmed) were collected. The non-tuberculosis tissue samples were provided by Beijing Chest Hospital Affiliated to Capital Medical University, and the patients were informed).

[0107] Above-mentioned sample is tested according to dyeing method 1 shown in embodiment 1;

[0108] At the same time, traditional lindensl staining was used as the control.

[0109] Traditional method of linseed and Nissl staining:

[0110] 1) Prepare 4 μm thick paraffin tissue sections, routine xylene dewaxing, gradient alcohol hydration.

[0111] 2) Add phenol fuchsin staining solution dropwise, cover the tissue to be t...

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Abstract

The invention discloses a kit and a method for staining Mycobacterium tuberculosis and its expressed protein. The present invention provides a method for staining Mycobacterium tuberculosis and its expressed protein, comprising the following steps: 1) sequentially incubating a sample containing Mycobacterium tuberculosis with a primary antibody and a secondary antibody to obtain an antibody binding product; 2) ) The antibody-binding product is sequentially stained with DAB staining solution, phenolic fuchsin staining solution and hematoxylin staining solution to achieve staining of Mycobacterium tuberculosis and its expressed protein. The experiments of the present invention prove that the present invention has developed a new kit for pathological diagnosis of tuberculosis, which can simultaneously detect Mycobacterium tuberculosis cells and their expressed proteins in a tissue specimen slice, Thereby improving the sensitivity of pathological diagnosis of tuberculosis. The kit is easy to use, suitable for routine detection in pathology department, and has good clinical promotion value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for double staining of mycobacterium tuberculosis and its antigen and a method thereof. Background technique [0002] Tuberculosis is an infectious disease that seriously threatens human health. One-third of the world's population is infected with Mycobacterium tuberculosis (MTB), and 2 million people die from tuberculosis every year. The death rate is only the lowest among infectious diseases. Ranked second to AIDS. The current gold standard for TB diagnosis is sputum bacteriology. However, the positive rate of sputum smears is low, and it takes one month for sputum culture to produce results. Therefore, the most commonly used sputum bacteriological detection methods cannot meet the needs of clinical diagnosis. In addition, in recent years, new technologies such as ELISPOT based on immunology and PCR based on nucleic acid detection have also been applied to the diagnosis of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N1/30G01N33/569
Inventor 车南颖张海青
Owner BEIJING CHEST HOSPITAL CAPITAL MEDICAL UNIV
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