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Mycobacterium tuberculosis KatG mutant gene and application thereof

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

Inactive Publication Date: 2016-02-10
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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  • Mycobacterium tuberculosis KatG mutant gene and application thereof
  • Mycobacterium tuberculosis KatG mutant gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: collecting detection samples

[0044] 1. Collected 33 isoniazid-resistant tuberculosis strains, all clinical patients were pulmonary tuberculosis patients. According to the test results of drug-susceptible isoniazid (INH0.1ug / ml), a total of 34 isoniazid-resistant tuberculosis strains were obtained. The average age of isoniazid-resistant patients was 38.6 years. Male patients accounted for 58.82% (20 / 34), aged between 20-69 years old, with an average age of 41.6 years; female patients accounted for 41.18% (14 / 34), aged between 14-67 years old, with an average age of 34.3 years .

Embodiment 2

[0045] Example 2: Extraction of Mycobacterium tuberculosis 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, mix by inverting; place in a water bath at 57°C for 20 minutes, and mix by inverting several times during the process;

[0051] 6. Add 2...

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 the KatG gene, and the primers 5'-CCGCCTTTGCTGCTTTCTC-3' and 5'-GGGGCTGATCTACGTGAAC-3' were used.

[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 1 minute and 30 seconds), and finally extension at 72°C for 7 minutes. The length of the amplified product is 983bp, and then it is detected by agarose gel electrophoresis. The DL2000 model DNAmarker is used as the control of the PCR amplification product, and the agarose gel with a gel concent...

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Abstract

The invention discloses a mycobacterium tuberculosis KatG mutant gene and an application thereof. Compared with the normal KatG gene, the mutant gene has the c.906C>A mutation site. According to the invention, by utilizing the candidate gene screening method, 33 Chinese isoniazide-resisting tuberculosis strains are detected, the gene mutation site is found for the first time, and in the isoniazide-resisting tuberculosis strains, KatG gene mutation c.906C>A has certain frequency of occurrence, and therefore, the KatG gene mutation c.906C>A can be taken as the diagnostic basis for the drug-resisting molecular mechanism of the isoniazide-resisting strains clinically.

Description

technical field [0001] The invention relates to a mycobacterium tuberculosis KatG mutation gene and a kit for detecting the KatG mutation c.906C>A of the isoniazid drug-resistant gene of mycobacterium tuberculosis, belonging to the technical field of detection of tuberculosis drug-resistant gene mutations. Background technique [0002] Mycobacterium tuberculosis causes tuberculosis, a common chronic respiratory disease, which mainly affects the lungs, and also invades multiple tissues and organs throughout the body, such as the skin and bones. Tuberculosis is prevalent in the world, but the number of patients in developing countries is relatively large. With the application of tuberculosis drugs and the improvement of living and medical standards, the incidence of tuberculosis has decreased. In recent years, the incidence and drug resistance rate of tuberculosis caused by antibiotic abuse, environmental pollution and AIDS have increased. According to the report of the Wo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12Q1/68C12Q1/04C12R1/32
Inventor 张阿梅夏雪山宋玉竹李道群
Owner KUNMING UNIV OF SCI & TECH
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