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Kit of Mycobacterium tuberculosis and detection method thereof

A technology for Mycobacterium tuberculosis and kits, applied in biochemical equipment and methods, microbial determination/inspection, analytical materials, etc., can solve the lack of detection methods, limitations of specificity and sensitivity, and long detection time, etc. problems, to achieve the effects of high detection sensitivity and precision, high sensitivity and specificity, and simple in vitro synthesis

Inactive Publication Date: 2011-06-01
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the traditional sputum smear method is used to detect Mycobacterium tuberculosis in the patient's sputum, and it takes 6-8 weeks to culture Mycobacterium tuberculosis in the sputum, which is too long for detection
In the past, PPD (tuberculin pure protein derivative) was also commonly used in the detection and epidemiological investigation of tuberculosis, but its specificity and sensitivity have certain limitations
Another example is that traditional serological antibody detection cannot detect early pathogen antigens, and cannot detect window period patients and patients with latent infection.
Clinically, due to the lack of effective detection methods for patients suspected of being infected with tuberculosis, anti-tuberculosis treatment is often used first, and then testing is carried out based on the effect of treatment. misdiagnosis or delayed treatment

Method used

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  • Kit of Mycobacterium tuberculosis and detection method thereof
  • Kit of Mycobacterium tuberculosis and detection method thereof
  • Kit of Mycobacterium tuberculosis and detection method thereof

Examples

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Effect test

Embodiment 1

[0033] A reagent kit for mycobacterium tuberculosis, comprising a 96-well ELISA plate, biotin-labeled nucleic acid aptamer NK2, horseradish peroxidase-labeled streptavidin and a chromogenic substrate. The preparation process is as follows:

[0034] 1. Synthesize the NK2 sequence and send the NK2 sequence to the company for synthesis. The 5' end of the biotin-labeled NK2 synthesized by the company is: bio-NK2. The sequence of the biotin-labeled nucleic acid adapter NK2 is shown in SEQ ID NO.1 the nucleotide sequence. And diluted with sterile double distilled water to the concentration of 100nM, 200nM, 400nM, stored in -20 ℃ refrigerator.

[0035] 2. Mycobacterium tuberculosis standard strain H37Rv (derived from ATCC 93009, Biochemical and Biophysical Research Communications 357 (2007) 743-748), BCG (derived from Biochemical and Biophysical Research Communications 357 (2007) 743-748), bird branch Bacillus M2 (M.avium, derived from ATCC25291, Clin Chem Lab Med.2009.47(4):405-11...

Embodiment 2

[0042] A method for detecting Mycobacterium tuberculosis with a kit for Mycobacterium tuberculosis, the specific process of kit detection:

[0043] 1. Take the sputum of different tuberculosis patients, the sputum of non-tuberculosis patients and normal people, and use this kit to test the samples of tuberculosis patients and other samples. The process is as follows:

[0044] In a 96-well ELISA plate, 100 μl of diluted sputum (1:10 diluted with normal saline) sample was added to each well, and a negative control well without sputum was set at the same time, incubated at 37° C. for 2 hours. Discard the liquid in the wells, add 300 μl PBST to each well, wash 4 times, then add 400 nM concentration of biotin-labeled aptamers, 100 μl per well, and incubate for 1 hour at 37°C. Discard the liquid in the wells, Add 300 μl PBST to the well, wash 4 times, then add horseradish peroxidase-labeled streptavidin, and incubate at 37°C for half an hour. Discard the liquid in the wells, add 30...

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Abstract

The invention discloses a kit of Mycobacterium tuberculosis and a detection method thereof. The kit contains an enzyme-linked immunosorbent assay (ELISA) plate, a biotin-labeled single-stranded DNA adaptor, horseradish peroxidase-labeled streptavidin and a chromogenic substrate. In the detection method provided by the invention, the biotin-labeled single-stranded DNA adaptor of Mycobacterium tuberculosis H37Rv is used as a probe and a method which is similar to the indirect enzyme-linked immunosorbent assay (ELISA) method is adopted to detect Mycobacterium tuberculosis; and Mycobacterium tuberculosis or sputum to be detected or a body fluid sample, and a control sample are added in the ELISA plate for incubation, Mycobacterium tuberculosis combined with the plate is trapped by the biotin-labeled single-stranded DNA adaptor and then washed, the horseradish peroxidase-labeled streptavidin is added, the substrate is added for developing, and the optical density (OD) value is detected by a microplate reader after developing when the absorption wavelengh is 450nm. When the kit provided by the invention detects Mycobacterium tuberculosis or the Mycobacterium tuberculosis or somatic antigen in sputum, blood and body fluid, the detection speed is high, the detection is convenient and safe, the detection time can be saved, the efficiency is high, the cost is low, the detection sensitivity and accuracy are high, and the application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of infectious disease detection, and in particular relates to a kit for Mycobacterium tuberculosis. At the same time, the invention also relates to a kit for Mycobacterium tuberculosis to detect Mycobacterium tuberculosis. Mycobacterium, and Mycobacterium tuberculosis or bacterial antigens in sputum, blood and body fluids of suspected patients are applicable to all hospitals, including infectious disease and tuberculosis hospitals, for the detection of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a chronic consumptive infectious disease caused by Mycobacterium tuberculosis and zoonotic, which poses a serious threat to human health. According to the statistics of the World Health Organization (WHO), nearly one-third of the people in the world are currently infected with Mycobacterium tuberculosis, and there are 20 million active tuberculosis patients, 3 million people die of tuber...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/52G01N21/31C12Q1/68C12Q1/04
Inventor 章晓联李莹滢
Owner WUHAN UNIV
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