阅读说明：本技术 一种基于结核分枝杆菌pfge法分型中的dna提取方法 (DNA extraction method based on mycobacterium tuberculosis PFGE method typing ) 是由 吴利先 王国富 聂恒 *** 郝贤斌 李萍 李丛哲 于 2020-06-04 设计创作，主要内容包括：本发明公开了一种基于结核分枝杆菌PFGE法分型中的DNA提取方法,包括将培养的结核分枝杆菌在磨菌管中制成菌悬液,菌悬液进行灭活、离心、沸水加热处理后,再加入溶菌酶,置于plug模具中,凝固成胶栓备用；对胶栓进行多次细胞溶解及水浴震荡后,获得结核分枝杆菌DNA,采用本方法能够方便去除M.tb细胞壁,获得M.tb的DNA；并且能够降低成本,推广性、实用性较强。(The invention discloses a DNA extraction method based on mycobacterium tuberculosis PFGE method typing, which comprises the steps of preparing a bacterial suspension from cultured mycobacterium tuberculosis in a bacterium grinding tube, inactivating the bacterial suspension, centrifuging, heating with boiling water, adding lysozyme, placing in a plug mould, and solidifying into a plug for later use; the cell lysis and water bath oscillation are carried out on the glue suppository for many times to obtain the DNA of the mycobacterium tuberculosis, and the method can be adopted to conveniently remove the cell wall of M.tb and obtain the DNA of the M.tb; and can reduce cost, and popularization nature, practicality are stronger.)

1. A DNA extraction method based on Mycobacterium tuberculosis PFGE method typing is characterized by comprising the following steps:

(1) preparing pure mycobacterium tuberculosis cultured on the inclined plane into a bacterial suspension in a bacterial grinding tube, and inactivating the bacterial suspension at 65-90 ℃ for 30 min;

(2) taking 1ml of the bacterial suspension into an EP tube, placing the tube in a centrifuge for 5min at 12000/rpm, removing supernatant, and suspending the precipitate in 500 mu L of TEN buffer;

(3) placing the bacterial suspension in the step (2) in a centrifuge to centrifuge at 4500/rpm for 10min, removing supernatant, suspending the precipitate in 200 μ L EC buffer of 100-;

(4) cooling the solution in the step (3), adding lysozyme to enable the final concentration of the lysozyme to be 25mg/ml, adding 2% low-melting-point agarose, uniformly mixing, and placing the mixture in a 56 ℃ water bath for later use;

(5) placing 80 μ L of the mixture in a plug mold, and solidifying into a suppository at 4 deg.C;

(6) placing the glue bolt containing M.tb genomic DNA into a centrifuge tube, adding 10ml of cell lysis buffer I, and shaking in water bath at 37 ℃ for 24 h;

(7) after the cell lysis buffer I is removed, 9800 mu L of cell lysis buffer II is added, 200 mu L of protease K solution is added, and the mixture is shaken in water bath at 50 ℃ for 24 h;

(8) after removing the cell lysis buffer II, adding 10ml TE buffer to wash for 3 times, each time for 30min, and after the 3 rd time washing, transferring the glue plug to a sterile test tube to obtain the mycobacterium tuberculosis DNA.

2. The method as claimed in claim 1, wherein the concentration of the bacterial suspension in the step (1) is 10 or more¹⁰cfu/ml, inactivation temperature 85 ℃.

3. The method according to claim 1, wherein the TEN buffer in step (2) is 300 μ L.

4. The method according to claim 1, wherein the EC buffer in step (3) is 150 μ L.

5. The method according to claim 1, wherein the heating time in boiling water in step (3) is 15 min.

6. The method according to claim 1, wherein the low melting point agarose of an equal volume to the mixed solution is added in the step (6).

7. The method according to claim 1, wherein proteinase K in step (7) is 20 mg/ml.

8. The DNA extraction method according to claims 1 to 7, wherein the DNA extraction method is applied to typing by the PFGE method of Mycobacterium tuberculosis.

Technical Field

The invention relates to the technical field of biological extraction, in particular to a DNA extraction method based on mycobacterium tuberculosis PFGE method typing.

Background

Tuberculosis (Tuberculosis) is a major infectious disease that causes death of adults 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 been an important identification method in molecular epidemiological studies of infectious diseases. The use of PFGE in Mycobacterium tuberculosis (m.tb) genotyping aids in the epidemiological investigation of tuberculosis and in distinguishing between new infections and inciting infections of old lesions. Previous investigator studies found that PFGE had the same resolution in genotyping m.tb clinical isolates of tuberculosis patients as the genotyping gold standard insert sequence 6110 restriction fragment length polymorphism analysis (IS6110-RFLP) of m.tb. However, in the process of mycobacterium tuberculosis PFGE genotyping research, due to the particularity of the cell structure of M.tb, the cell wall of the M.tb is difficult to remove, so that the application of PFGE in M.tb genotyping is controversial among researchers, and the controversial reason is the relevant report of PFGE genotyping in recent M.tb genotyping research reports. In view of the above difficulties, later researchers continuously improve experimental techniques and try to find a method capable of solving the problem that the M.tb cell wall is difficult to remove. Some researchers have added antibiotics such as ampicillin and cycloserine to the medium during the log phase of m.tb cultures; some researchers have attempted to remove the cell wall of m.tb using an eluent of cytolytic enzymes and chemolysis.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a DNA extraction method based on the typing of Mycobacterium tuberculosis by a PFGE method.

In order to achieve the purpose, the technical scheme of the invention is as follows: designing a DNA extraction method based on the typing of a mycobacterium tuberculosis PFGE method, wherein the method comprises the following steps:

(1) preparing the cultured mycobacterium tuberculosis into a bacterial suspension in a bacterium grinding tube, and inactivating the bacterial suspension at 65-90 ℃ for 30 min;

(2) taking 1ml of the bacterial suspension into an EP tube, placing the tube in a centrifuge for 5min at 12000/rpm, removing supernatant, and suspending the precipitate in 500 mu L of TEN buffer;

(3) placing the bacterial suspension in the step (2) in a centrifuge to centrifuge at 4500/rpm for 10min, removing supernatant, suspending the precipitate in 200 μ L EC buffer of 100-;

(4) cooling the solution in the step (3), adding lysozyme to enable the final concentration of the lysozyme to be 25mg/ml, adding 2% low-melting-point agarose, uniformly mixing, and placing the mixture in a 56 ℃ water bath for later use;

(5) placing 80 μ L of the mixture in a plug mold, and solidifying into a suppository at 4 deg.C;

(6) placing the glue bolt containing M.tb genomic DNA into a centrifuge tube, adding 10ml of cell lysis buffer I, and shaking in water bath at 37 ℃ for 24 h;

(7) after the cell lysis buffer I is removed, 9800 mu L of cell lysis buffer II is added, 200 mu L of protease K solution is added, and the mixture is shaken in water bath at 50 ℃ for 24 h;

(8) after removing the cell lysis buffer II, adding 10ml TE buffer to wash for 3 times, each time for 30min, and after the 3 rd time washing, transferring the glue plug to a sterile test tube to obtain the mycobacterium tuberculosis DNA.

Preferably, the concentration of the bacterial suspension in the step (1) is more than or equal to 10¹⁰cfu/ml, inactivation temperature 85 ℃.

Preferably, the TEN buffer in the step (2) is 300 mu L.

Preferably, the EC buffer in the step (3) is 150 μ L.

Preferably, the heating time in boiling water in the step (3) is 15 min.

Preferably, the low melting point agarose equal to the volume of the mixed solution is added in the step (6).

Preferably, the proteinase K in the step (7) is 20 mg/ml.

Preferably, the DNA extraction method is applied to typing of Mycobacterium tuberculosis by a PFGE method.

Compared with the prior art, the invention has the beneficial effects;

1. the method removes most cell walls by boiling treatment, and then adds lysozyme to remove the remaining cell walls, thereby overcoming the difficulty of removing M.tb cell walls in PFGE typing;

2. the method reduces the using amount of lysozyme and the experiment cost;

3. in the method, the using concentration of the proteinase K is only 0.4mg/ml, while the concentration of the proteinase K is mostly about 1mg/ml, so that the cost is reduced again;

4. after the DNA extracted by the method is subjected to enzyme digestion by Dra I, the gene fragments are clear through electrophoresis display, the typing effect is obviously improved, and the research on tuberculosis is facilitated;

drawings

FIG. 1 is a fingerprint of a clinical isolate Dra I enzyme digestion PFGE of Mycobacterium tuberculosis experimental group 1;

FIG. 2 is a fingerprint of the clinical isolate Dra I enzyme-digested PFGE of Mycobacterium tuberculosis experimental group 2;

FIG. 3 is a diagram of cluster analysis of typing by the MtbPFGE method;

Detailed Description

The present invention is further described in detail with reference to the following specific examples, but the scope of the present invention is not limited by the specific examples, which are defined by the claims. In addition, any modification or change that can be easily made by a person having ordinary skill in the art without departing from the technical solution of the present invention will fall within the scope of the claims of the present invention.

The technical scheme of the invention is further described in detail by combining the drawings and the detailed implementation mode:

a DNA extraction method based on Mycobacterium tuberculosis PFGE method typing, which comprises the following steps:

(1) preparing the cultured mycobacterium tuberculosis into a bacterial suspension in a bacterium grinding tube, and inactivating the bacterial suspension at 65-90 ℃ for 30 min;

(2) taking 1ml of the bacterial suspension into an EP tube, placing the tube in a centrifuge for 5min at 12000/rpm, removing supernatant, and suspending the precipitate in 500 mu L of TEN buffer;

(3) placing the bacterial suspension in the step (2) in a centrifuge to centrifuge at 4500/rpm for 10min, removing supernatant, suspending the precipitate in 200 μ L EC buffer of 100-;

(4) cooling the solution in the step (3), adding lysozyme to enable the final concentration of the lysozyme to be 25mg/ml, adding 2% low-melting-point agarose, uniformly mixing, and placing the mixture in a 56 ℃ water bath for later use;

(5) placing 80 μ L of the mixture in a plug mold, and solidifying into a suppository at 4 deg.C;

(6) placing the glue bolt containing M.tb genomic DNA into a centrifuge tube, adding 10ml of cell lysis buffer I, and shaking in water bath at 37 ℃ for 24 h;

(7) after the cell lysis buffer I is removed, 9800 mu L of cell lysis buffer II is added, 200 mu L of protease K solution is added, and the mixture is shaken in water bath at 50 ℃ for 24 h;

(8) after removing the cell lysis buffer II, adding 10ml TE buffer to wash for 3 times, each time for 30min, and after the 3 rd time washing, transferring the glue plug to a sterile test tube to obtain the mycobacterium tuberculosis DNA.

Preferably, the concentration of the bacterial suspension in the step (1) is more than or equal to 10¹⁰cfu/ml, inactivation temperature 85 ℃.

Preferably, the TEN buffer in the step (2) is 300 mu L.

Preferably, the EC buffer in the step (3) is 150 μ L.

Preferably, the heating time in boiling water in the step (3) is 15 min.

Preferably, the low melting point agarose equal to the volume of the mixed solution is added in the step (6).

Preferably, the proteinase K in the step (7) is 20 mg/ml.

Preferably, the DNA extraction method is applied to typing of Mycobacterium tuberculosis by a PFGE method.