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In-vitro abortive phthisic test method

A technology for active tuberculosis and in vitro detection, which is applied in the field of detection, can solve the problems of poor repeatability of molecular diagnostic methods, false positive test results, and long time consumption, and achieve the effects of wide coverage and practicability, accurate judgment results, and shortened detection time

Inactive Publication Date: 2017-03-29
GUANGZHOU DEAOU MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the patient is found, the patient does not take any preventive measures. In the process of close contact with family members, colleagues, classmates, etc., the contacts are prone to be infected by tuberculosis.
At present, the main treatment for tuberculosis is mainly drug treatment, but such treatment cannot completely prevent the spread of tuberculosis
Bacillus Calmette-Guerin (BCG) vaccination is currently the most effective way to prevent tuberculosis. China's planned immunization program stipulates that newborns must be vaccinated with BCG, but its protection efficiency has not reached 100%. Therefore, the primary measure to prevent tuberculosis It is to detect new patients hidden in the crowd as early as possible
[0003] Among the methods commonly used in clinical diagnosis of active tuberculosis at present, imaging methods have poor specificity, the diagnostic process is closely related to the doctor’s experience, and the diagnostic results are highly subjective; sputum culture method is the gold standard for the diagnosis of tuberculosis, but its sensitivity is low , and it takes a long time (2-8 weeks), which is not conducive to the early diagnosis of tuberculosis; the sensitivity of acid-fast staining method is only 20-30%; Certain requirements; tuberculin test (TST) is susceptible to BCG vaccination, resulting in false positive test results
In recent years, T cell-based γ-interferon release test has appeared, but this method can only judge whether the patient has been infected (over) Mycobacterium tuberculosis, and cannot judge whether it is active tuberculosis
Among the above detection methods, although sputum culture, acid-fast staining and molecular diagnostic methods have good specificity, they only target bacillus-positive tuberculosis (about 30%) in tuberculosis patients and cannot diagnose bacillus-negative tuberculosis. A direct method for the detection of active tuberculosis

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Separation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation

[0025] Use fresh heparin lithium or heparin sodium blood collection tubes to aseptically extract 4-5ml of peripheral venous blood from the person to be tested, and invert the blood to mix the anticoagulant and blood. Prepare two 15ml centrifuge tubes, and add physiological saline for injection and lymphocyte separation solution equal to the volume of blood collection respectively. After mixing the fresh heparin anticoagulated blood with normal saline, slowly add it to the separation liquid at a uniform speed. When adding, keep the separation liquid and blood layered, and then centrifuge at 22°C and 1800g for 20 minutes. After centrifugation, PBMCs cells can be seen in the form of clouds layer. Aspirate the PBMCs cell layer into a new 15ml centrifuge tube, make up to 12ml with RPMI-1640 culture medium, and centrifuge at 600g for 10min at 22°C. Pour off the supe...

Embodiment 2

[0026] Example 2 Enzyme-linked immunosorbent assay (ELISA) detects the level of IFN-γ and IL-2 secreted by PBMC after the induction of clinically diagnosed active tuberculosis patients and healthy people

[0027] 1) Select the heparin sodium anticoagulated peripheral venous blood samples of 10 patients clinically diagnosed as active tuberculosis and 10 healthy people, separate PBMCs according to the density stratification method in Example 1, and use serum-free medium after cell counting Dilute PBMCs to 2.5 x 10 6 cells / ml concentration, the cells were seeded into 96-well plates, and 2.5×10 5 Then add specific stimulating antigen (fusion protein CFP-10-ESAT-6-Rv1985c, concentration 2 μg / ml) diluted with serum-free medium, 37°C, 5% CO 2 After incubation for 18 hours under the same conditions, the cell culture fluid was taken to detect IFN-γ and IL-2 cytokines.

[0028] 2) Use the ELISA plate pre-coated with anti-human IFN-γ monoclonal antibody, add the cell culture supernatan...

Embodiment 3

[0031] Example 3 Enzyme-linked immunospot method (ELISPOT) detects the level of IFN-γ and IL-2 secreted by PBMC after the induction of clinically diagnosed active tuberculosis patients and healthy people

[0032]1) Select the heparin sodium anticoagulated peripheral venous blood samples of 10 patients with active tuberculosis and 10 healthy people, separate PBMCs according to the density stratification method in Example 1, and use serum-free medium to dilute it to 2.5×10 6 A / ml concentration for use.

[0033] 2) Select the PVDF membrane detection plate (pre-coated with anti-human IFN-γ monoclonal antibody and anti-human IL-2 monoclonal antibody respectively, and store at 4°C) as the reaction plate, add 2.5×10 5 For each PBMC prepared in the previous step, three detection wells were set for each cytokine in each sample, which were blank control (adding serum-free medium), experimental well (adding specific stimulating antigen for stimulation, the concentration was 2 μg / ml) and...

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Abstract

The invention discloses an in-vitro abortive phthisic test method. The method is based on the in-vitro phthisic test application of a reagent for the quantitative test of phthisic correlation factor IFN-gamma and IL-2. Compared with the prior art of abortive phthisic test method, the method is more accurate, and has a higher specificity in the judgment results of abortive phthisic. Compared with the current golden standard sputum culture, the test time is greatly shortened, the shortened time provides the basis for clinicians to have a timely diagnosis of the patients and give drug serving instructions. The in-vitro abortive phthisic test method bears extremely positive significance for controlling the phthisic. The reagent is used for testing all types of phthisic including tuberculosis, scrofula, enterophthisis, bone tuberculosis, nephrophthisis and the like with wide coverage and even wider application scope.

Description

technical field [0001] The invention belongs to the detection field, and in particular relates to a method for in vitro detection of active tuberculosis. Background technique [0002] Tuberculosis is a common chronic infectious disease caused by Mycobacterium tuberculosis, which can invade many organs, with pulmonary tuberculosis infection being the most common. At present, nearly 1 / 3 of the world's people have been infected with Mycobacterium tuberculosis. Statistics show that in 2013, 1.5 million people died of tuberculosis and 9 million new cases. Tuberculosis has become one of the main diseases that cause adult deaths from infectious diseases all over the world. . China is one of the 22 countries with a high burden of tuberculosis in the world, and the number of active tuberculosis patients ranks second in the world. The transmission of tuberculosis mainly occurs before the patient is discovered and treated. One tuberculosis patient can infect as many as 10-15 people t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/5695G01N2800/12
Inventor 黄曦胡鹏男李妍
Owner GUANGZHOU DEAOU MEDICAL TECH CO LTD
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