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DNA extraction method in pulse field gel electrophoresis (PFGE) method parting of mycobacterium tuberculosis (M. tb)

A technology of Mycobacterium tuberculosis and extraction method, which is applied in the field of DNA extraction based on Mycobacterium tuberculosis PFGE method typing, can solve the problems such as the difficulty of removing the cell wall, and achieve the effect of reducing experimental costs, reducing dosage, and clear gene fragments

Pending Publication Date: 2020-09-29
DALI UNIV
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Problems solved by technology

However, in the process of PFGE typing of Mycobacterium tuberculosis, it is difficult to remove the cell wall due to the particularity of M.tb's own cell structure, so the application of PFGE in M.tb genotyping is controversial among researchers. The main reason why there are few related reports on M.tb genotyping in recent years.

Method used

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  • DNA extraction method in pulse field gel electrophoresis (PFGE) method parting of mycobacterium tuberculosis (M. tb)
  • DNA extraction method in pulse field gel electrophoresis (PFGE) method parting of mycobacterium tuberculosis (M. tb)
  • DNA extraction method in pulse field gel electrophoresis (PFGE) method parting of mycobacterium tuberculosis (M. tb)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Subjects

[0048] Take sputum samples from 27 clinical pulmonary tuberculosis patients;

[0049] 2. Grouping

[0050] Divide 27 oral sputum samples into experimental group 1 and experimental group 2;

[0051] 3. Specimen processing

[0052] Take the mouth sputum specimens of pulmonary tuberculosis patients for acid treatment for 20 minutes, and neutralize with alkali;

[0053] 4. Cultivate

[0054] Inoculate in L-J (Roche) medium by partition marking method, and culture at 37°C;

[0055] 5. Purification

[0056] Observe every day, if there is colony growth, carry out: A. Observe the size, shape, edge, surface, color, etc. of the colony; B. Identification of acid-fast staining and identification medium; For biochemical reactions, if necessary, use the BacT / ALERT 3D 360 automatic culture instrument of the French Mérieux company and the matching BacT / ALERT MP Mycobacterium tuberculosis culture bottle for culture and identification; D. After the identification is c...

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Abstract

The invention discloses a DNA extraction method in pulse field gel electrophoresis (PFGE) method parting of mycobacterium tuberculosis (M. tb). The DNA extraction method comprises the following steps:preparing a bacterial suspension from cultured M. tb in a bacterium grinding tube, performing inactivation, centrifugation and boiling water heating treatment on the bacterial suspension, then, adding a lysozyme, and placing the bacterial suspension in a plug mold to be solidified into a gel plug for later use; and carrying out cytolysis and water-bath oscillation on the gel plug repeatedly to obtain DNA of the M. tb. The method can conveniently remove cell walls of the M. tb and obtain the DNA of the M. tb; and besides, the method can reduce the cost and has higher promotion performance andpracticability.

Description

technical field [0001] The invention relates to the technical field of biological extraction, in particular to a DNA extraction method based on the PFGE typing of Mycobacterium tuberculosis. Background technique [0002] Tuberculosis is a major infectious disease causing adult mortality worldwide. With the development of molecular microbiology, many new methods for identifying microorganisms have been established and applied in recent years, but pulse field gel electrophoresis (PFGE) has always been a method of molecular epidemiology research on infectious diseases. important method of identification. The application of PFGE in the genotyping of Mycobacterium tuberculosis (M.tb) is helpful for the investigation of outbreaks of tuberculosis and the distinction between new infections and priming infections of old lesions. In the previous research, the researchers found that the resolution of PFGE in the genotyping of M.tb clinical isolates from pulmonary tuberculosis patient...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 吴利先王国富聂恒王旭东郝贤斌李萍李丛哲
Owner DALI UNIV
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