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MTB (mycobacterium tuberculosis) rpoB mutant gene and application thereof

A technology of Mycobacterium tuberculosis and mutant genes, which can be applied in the fields of genetic engineering, plant genetic improvement, application, etc., and can solve the problems of no public reports of detected mutation sites.

Pending Publication Date: 2016-02-17
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Through literature search, there is no public report identical with the mutation site detected by the present invention

Method used

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  • MTB (mycobacterium tuberculosis) rpoB mutant gene and application thereof
  • MTB (mycobacterium tuberculosis) rpoB mutant gene and application thereof
  • MTB (mycobacterium tuberculosis) rpoB mutant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: collecting detection samples

[0044] A total of 33 rifampicin-resistant tuberculosis strains were collected, and all clinical patients were pulmonary tuberculosis patients. According to the test results of drug-sensitivity rifampicin (RIF1.0μg / ml), a total of 33 isorifampicin-resistant tuberculosis strains were obtained. The average age of rifampicin-resistant patients was 41.3 years. Male patients accounted for 63.64% (21 / 33), aged between 20-80 years old, with an average age of 46.2 years; female patients accounted for 36.36% (12 / 33), aged between 14-55 years old, with an average age of 32.7 years .

Embodiment 2

[0045] Example 2: Extraction of Genomic DNA

[0046] 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);

[0047] 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;

[0048] 3. Add 225 μl buffer A to the cell pellet, shake until the cell is completely suspended;

[0049] 4. Add 10 μl proteinase K solution to the tube and mix by inverting;

[0050] 5. Add 25 μl lysis buffer S, invert and mix; place in a water bath at 57°C for 20 minutes, and invert and mix several times during this time.

[0051] 6. Add 250μl buffer B, shake for 5s and ...

Embodiment 3

[0058] Embodiment 3: PCR amplification, electrophoresis result

[0059] Using the extracted whole genome DNA of Mycobacterium tuberculosis as a template, PCR amplification was carried out, and the amplified gene was RpoB gene, and the primers used were 5'-CAAGGAGTTCTTCGGCACC-3' and 5'-CGTCCATGTAGTCCACCTC-3'.

[0060] 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.

[0061] 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 30 seconds), and finally extension at 72°C for 5 minutes. The length of the amplified product is 443bp, and then it is detected by agarose gel electrophoresis. The DL2000 model DNAmarker is selected as the PCR amplification product control, and the agarose gel with a gel concentration of 1.5% is pr...

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Abstract

The invention discloses an MTB (mycobacterium tuberculosis) rpoB mutant gene and an application thereof. Compared with a normal rpoB gene, the mutant gene has a c.1843G>A mutation site; a candidate gene screening method is adopted to detect 33 rifampicin-resistant tuberculosis strains in China, the gene mutation site is found for the first time, and the rpoB gene c.1843G>A mutation site in the rifampicin-resistant tuberculosis strains has certain occurrence frequency, so that the rpoB gene c.1843G>A mutation site can be taken as diagnostic basis of drug-resistance molecular mechanisms of rifampicin-resistant strains clinically.

Description

Technical field [0001] The present invention involves a type of tuberculosis RPOB mutation gene, as well as a kit for detecting the RPOB mutant C.1843G> A of RPOB mutations that cause tuberculosis, which belongs to the field of tuberculosis drug resistance gene mutation detection technology. Background technique [0002] Tuberculosis ( tuberculosis , TB) is a tuberculosis ( Mycobacteriumtuberculosis , MTB) Chronic infectious diseases caused by).It is mainly involved in the lungs, and in addition, it will invade the skin and bones of multiple tissues and organs throughout the body.At present, the number of tuberculosis patients in my country ranks second in the world, and the resistance rate is ranked first in the world.About 2 / 3 of the patients with tuberculosis are in the dangers of Rifampicin (MDR-TB) at least Rifampicin and ISONIAZID (ISONIAZID) at the same time).It is reported that there are as many as 150,000 patients with newly resistant multi -drug tuberculosis each y...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12Q1/68C12Q1/04C12R1/32
Inventor 张阿梅宋玉竹夏雪山李道群
Owner KUNMING UNIV OF SCI & TECH
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