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Mycobacterium tuberculosis EmbB mutant gene and application thereof

A technology of Mycobacterium tuberculosis and mutant gene, which is applied in the directions of genetic engineering, plant gene improvement, application, etc., can solve the problem of undetected mutation sites in public reports, etc., and achieve the effect of an accurate and simple method

Active Publication Date: 2016-02-10
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Through literature search, there is no public report identical with the mutation site detected by the present invention

Method used

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  • Mycobacterium tuberculosis EmbB mutant gene and application thereof
  • Mycobacterium tuberculosis EmbB mutant gene and application thereof
  • Mycobacterium tuberculosis EmbB mutant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1: collecting detection samples

[0043] Eighteen ethambutol-resistant tuberculosis strains were collected, and the clinical patients were all pulmonary tuberculosis patients. According to the test results of drug susceptibility to ethambutol (EMB5μg / m), a total of 18 ethambutol-resistant tuberculosis strains were obtained. The average age of ethambutol-resistant patients was 33.8 years. Male patients accounted for 72.22% (13 / 18), aged between 15 and 60, with an average age of 33.5; female patients accounted for 27.78% (5 / 18), aged between 14 and 60, with an average age of 34.8 .

Embodiment 2

[0044] Example 2: Extraction of Genomic DNA

[0045] 1. Use a disposable inoculation loop to scrape the cultured colony of Mycobacterium tuberculosis and place it in a 1.5ml EP tube (try not to scrape the culture medium);

[0046] 2. Add 500 μl of cell suspension to the centrifuge tube of the bacterial sediment (first check whether Lysozyme has been added), use a pipette or a vortex shaker to thoroughly suspend the tuberculosis cell pellet, and incubate at 37°C for 30 minutes and invert every 5-10 minutes Mix several times, centrifuge at 12000rpm (~13400×g) for 2min, and try to absorb the supernatant;

[0047] 3. Add 225 μl buffer A to the cell pellet, shake until the cell is completely suspended;

[0048] 4. Add 10 μl proteinase K solution to the tube and mix by inverting;

[0049] 5. Add 25 μl lysis buffer S, mix by inverting; place in a water bath at 57°C for 20 minutes, and mix by inverting several times during the process;

[0050] 6. Add 250μl buffer B, shake for 5s...

Embodiment 3

[0057] Embodiment 3: PCR amplification, electrophoresis result

[0058] Using the extracted whole genome DNA of Mycobacterium tuberculosis as a template, PCR amplification was carried out, and the amplified gene was EmbB gene, and the primers 5'-TGATTGGCTTTGTGTTGTCG-3' and 5'-GAACACCCCGACAATCTGCG-3' were used. PCR amplification system: 25 μL of 2×PCR master mix (containing rTaq enzyme, TAKARA), 1 μM of forward and reverse primers, 50 ng of template DNA, and 21 μL of deionized water.

[0059] The PCR reaction conditions were: denaturation at 94°C for 5 minutes, followed by 35 cycles (denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 1 minute), and final extension at 72°C for 7 minutes. The length of the amplified product is 445bp, and then it is detected by agarose gel electrophoresis. The DL2000 model DNAmarker is used as the control of the PCR amplified product, and the agarose gel with a gel concentration of 1.5% is prepared. min...

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Abstract

The invention discloses a mycobacterium tuberculosis EmbB mutant gene and an application thereof. Compared with the common EmbB gene, the mutant gene has the c.233A>G mutation site. According to the invention, by utilizing the candidate gene screening method, 18 Chinese ethambutol-resisting tuberculosis strains are detected, the gene mutation site is found for the first time, and in the ethambutol-resisting tuberculosis strains, EmbB gene mutation c. 233A>G has certain frequency of occurrence, and therefore, the EmbB gene mutation c. 233A>G can be taken as the diagnostic basis for the drug-resisting molecular mechanism of the ethambutol-resisting strains in clinic.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis EmbB mutation gene and a kit for detecting the EmbB mutation c.233A>G of the ethambutol drug resistance gene of mycobacterium tuberculosis, belonging to the technical field of tuberculosis drug resistance gene mutation detection. Background technique [0002] Human tuberculosis is a highly contagious disease caused by Mycobacterium tuberculosis, which can affect multiple organs and tissues throughout the body. The occurrence of the disease seriously affects the life and work of patients, and brings a heavy economic burden to the family and society. The incidence of tuberculosis in underdeveloped countries is higher than that in developed countries due to the low standard of living and medical care in developing countries. China is a country with a large population and a developing country, so the incidence of tuberculosis is among the highest in the world. Although with the improvement of peopl...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12Q1/68C12Q1/04C12R1/32
Inventor 张阿梅张成林夏雪山李道群
Owner KUNMING UNIV OF SCI & TECH
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