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Kit for detecting mycobacterium tuberculosis pncA gene mutation

A Mycobacterium tuberculosis and kit technology, applied in the direction of microorganism-based methods, microorganism measurement/testing, microorganisms, etc., can solve the problems of public reports of undetected mutation sites

Active 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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  • Kit for detecting mycobacterium tuberculosis pncA gene mutation
  • Kit for detecting mycobacterium tuberculosis pncA gene mutation
  • Kit for detecting mycobacterium tuberculosis pncA gene mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1: collecting detection samples

[0048] 16 strains of pyrazinamide-resistant tuberculosis strains were collected, and the clinical patients were all pulmonary tuberculosis patients. According to the test results of drug-susceptible pyrazinamide (PZA content 1 μg / ml), a total of 16 strains of pyrazinamide-resistant tuberculosis strains were obtained. The mean age of patients resistant to pyrazinamide was 40 years. Male patients accounted for 68.75% (11 / 16), aged between 15-69 years old, with an average age of 40.3 years; female patients accounted for 31.25% (5 / 16), aged between 21-49 years old, with an average age of 39.4 age.

Embodiment 2

[0049] Example 2: Extraction of Genomic DNA

[0050] 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);

[0051] 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;

[0052] 3. Add 225 μl buffer A to the cell pellet, shake until the cell is completely suspended;

[0053] 4. Add 10 μl proteinase K solution to the tube and mix by inverting;

[0054] 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.

[0055] 6. Add 250μl buffer B, shake for 5s and ...

Embodiment 3

[0062] Embodiment 3: PCR amplification, electrophoresis result

[0063] Using the extracted whole genome DNA of Mycobacterium tuberculosis as a template, PCR amplification was carried out, and the amplified gene was pncA gene, and the primers used were 5'-TGTCGCTCACTACATCACC-3' and 5'-TCGTAGGTCATAGCGTAGG-3'.

[0064] 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.

[0065] The PCR reaction conditions are: 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 5 minutes. The length of the amplified product is 867bp, 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...

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Abstract

The invention discloses a kit for detecting mycobacterium tuberculosis pncA gene mutation. The kit comprises reagents for detecting mutation sites, namely -12T>C, -11A>G, -7T>C, c.3G>A, c.233G>A, c.408InsA, c.538-561del and +1-+18del, of mycobacterium tuberculosis pncA genes. The kit can be used for detecting the mutation sites of the pncA genes of a patient simply, conveniently, quickly and accurately and can be accordingly used for diagnosis and treatment of the pyrazinamide-resistant tuberculous patient.

Description

technical field [0001] The invention relates to a kit for detecting seven mutations in the mutation hotspots of the pncA gene mutation of the mycobacterium tuberculosis drug-resistant gene pyrazinamide, and belongs to the technical field of detection of tuberculosis drug-resistant gene mutations. Background technique [0002] Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis. Since Koch discovered Mycobacterium tuberculosis, tuberculosis has been a threat to human health, and vaccines, drugs and diagnostic techniques for treating tuberculosis have been the goal of human beings' constant search for solutions. In recent years, due to the abuse of antibiotics, environmental pollution, and AIDS, tuberculosis has made a comeback, and its morbidity and mortality have risen significantly. It has become one of the three major killers of infectious diseases along with malaria and AIDS. To make matters worse, TB bacilli have become more resistant to ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/32
CPCC12Q1/689C12Q2600/106C12Q2600/156
Inventor 张阿梅夏雪山李道群宋玉竹
Owner KUNMING UNIV OF SCI & TECH
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