Mycobacterium tuberculosis embb mutant gene and application thereof
A technology of Mycobacterium tuberculosis and mutant genes, which is applied in the fields of genetic engineering, plant gene improvement, application, etc., can solve the problems of no public reports of detected mutation sites, and achieve the effect of an accurate and simple method
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Embodiment 1
[0042] Embodiment 1: collecting detection samples
[0043] Eighteen strains of ethambutol-resistant tuberculosis were collected, and the clinical patients were all pulmonary tuberculosis patients. According to the test results of drug susceptibility to ethambutol (EMB 5 μg / 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 1.5 ml 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 every 5-10 minutes Invert and mix several times, centrifuge at 12 000 rpm (~13 400×g) for 2 min, 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 min, and mix by inverting several times during the process;
[0050] 6. Add 250 μl buffer B, shake for 5 ...
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 445 bp, and then it is detected by agarose gel electrophoresis. The DL2000 model DNA marker is used as the control of the PCR amplification product, and the agarose gel with a gel concentration of 1.5% is prepared. ...
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