A method for isolating and culturing Helicobacter pylori strains

Abstract

The invention belongs to the field of biomedicine, and relates to a method for separating and cultivating bacteria, in particular to a method for isolated culture of a strain of helicobacter pylori. In a sample transfer process of the method, a sample suspected of containing helicobacter pylori is stored in a transport liquid; and the transport liquid includes a brucella broth medium, an adsorbent, a reducing agent, and a helicobacter pylori lipopolysaccharide extract. The helicobacter pylori lipopolysaccharide extract has an inhibitory effect on sundry fungi and has a protective effect on helicobacter pylori thalluses. The method can prolong the transfer time for sample transfer to a bacterial culture research unit and maintain the activity of the helicobacter pylori, so that the difficulty of isolated culture of wild helicobacter pylori strains is reduced, and the positive rate of bacterial culture is improved. The method for culture of the helicobacter pylori can be applied to studies such as bacterial typing, pathogenic mechanism research, construction of animal models, and determination of pathogenic factors.

Description

technical field

[0001] The invention belongs to the field of biomedicine and relates to a method for isolating and cultivating bacteria, in particular to a method for isolating and cultivating Helicobacter pylori strains. Background technique

[0002] Helicobacter pylori (H.pylori) is a spiral Gram-negative microaerophilic bacterium that infects half of the world's population and is the main pathogen that causes gastritis, peptic ulcer and gastric cancer. The discovery of Helicobacter pylori It has deepened human understanding of the relationship between chronic infection, inflammation and cancer, and has been listed as a class I carcinogen for human gastric cancer by the World Health Organization. The isolated and cultured clinical strains of Helicobacter pylori can be used in basic research such as bacterial typing, research on pathogenic mechanism, construction of animal models and determination of pathogenic factors. However, Helicobacter pylori has very strict requirem...

Examples

Embodiment 1

[0043] Embodiment 1: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0044] (1) Materials and instruments

[0045] Microaerophilic culture tank and microaerophilic gas bag, ultra-clean bench (Mitsubishi, Japan); trigas incubator, disposable inoculation loop, Columbia agar (OXOID, UK); Brooke's broth, Gram stain solution (Anhui Chaohu Hongci Medical Company, China), urease test paper (Guangdong Zhuhai Kedi Technology Company, China); fetal bovine serum, cysteine, soluble starch, activated carbon, cyclodextrin, phosphate (PBS) buffer liquid, defibrinated sheep whole blood (Youkang Biotechnology Co., Ltd., China); deoxyribonuclease DNase Ⅰ, ribonuclease RNaseA, tris (hydroxymethyl) aminomethane hydrochloride Tris-HCL (TAKARA); the standard strain NCTC11639 was obtained from Gift from China Helicobacter pylori Strain Management and Preservation Center.

[0046] (1.1) Preparation of Helicobacter pylori lipopolysaccharide extra...

Embodiment 2

[0062] Embodiment 2: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0063] The present embodiment Helicobacter pylori isolation culture material, instrument and process are basically the same as embodiment 1, the difference is:

[0064] (1) Different from (1.2) transport liquid preparation in embodiment 1, the method that present embodiment adopts is:

[0065] To prepare the hpLPS solution, dissolve 15 g of hpLPS in 100 ml of sterile distilled water, and use a 0.22 μm filter membrane to filter and sterilize. Weigh 281g of Brinell broth powder, heat and stir to dissolve in distilled water, add distilled water to the final volume of 100ml to obtain Brinell broth medium, then add 0.5g of activated carbon particles, dissolve and mix well, and then autoclave at 121°C . After cooling, 8 ml of aseptically treated hpLPS solution and 0.1 ml of aseptically treated 0.5 g / ml cysteine ​​solution were added. Then aseptically dispense...

Embodiment 3

[0070] Embodiment 3: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0071] The present embodiment Helicobacter pylori isolation culture material, instrument and process are basically the same as embodiment 1, the difference is:

[0072] (1) Different from (1.2) transport liquid preparation in embodiment 1, the method that present embodiment adopts is:

[0073] To prepare the hpLPS solution, dissolve 15 g of hpLPS in 100 ml of sterile distilled water, and use a 0.22 μm filter membrane to filter and sterilize. Weigh 281g of Brooke’s broth powder, heat and stir to dissolve in distilled water, add distilled water to the final volume of 100ml to obtain Brooke’s broth medium, then add 1g of cyclodextrin, dissolve and mix well, and then autoclave at 121°C . After cooling, 3 ml of aseptically treated hpLPS solution and 0.1 ml of aseptically treated 0.5 g / ml cysteine ​​solution were added. Then aseptically dispense the Brooke's ...

technical field

[0001] The invention belongs to the field of biomedicine and relates to a method for isolating and cultivating bacteria, in particular to a method for isolating and cultivating Helicobacter pylori strains. Background technique

[0002] Helicobacter pylori (H.pylori) is a spiral Gram-negative microaerophilic bacterium that infects half of the world's population and is the main pathogen that causes gastritis, peptic ulcer and gastric cancer. The discovery of Helicobacter pylori It has deepened human understanding of the relationship between chronic infection, inflammation and cancer, and has been listed as a class I carcinogen for human gastric cancer by the World Health Organization. The isolated and cultured clinical strains of Helicobacter pylori can be used in basic research such as bacterial typing, research on pathogenic mechanism, construction of animal models and determination of pathogenic factors. However, Helicobacter pylori has very strict requirem...

Examples

Embodiment 1

[0043] Embodiment 1: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0044] (1) Materials and instruments

[0045] Microaerophilic culture tank and microaerophilic gas bag, ultra-clean bench (Mitsubishi, Japan); trigas incubator, disposable inoculation loop, Columbia agar (OXOID, UK); Brooke's broth, Gram stain solution (Anhui Chaohu Hongci Medical Company, China), urease test paper (Guangdong Zhuhai Kedi Technology Company, China); fetal bovine serum, cysteine, soluble starch, activated carbon, cyclodextrin, phosphate (PBS) buffer liquid, defibrinated sheep whole blood (Youkang Biotechnology Co., Ltd., China); deoxyribonuclease DNase Ⅰ, ribonuclease RNaseA, tris (hydroxymethyl) aminomethane hydrochloride Tris-HCL (TAKARA); the standard strain NCTC11639 was obtained from Gift from China Helicobacter pylori Strain Management and Preservation Center.

[0046] (1.1) Preparation of Helicobacter pylori lipopolysaccharide extra...

Embodiment 2

[0062] Embodiment 2: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0063] The present embodiment Helicobacter pylori isolation culture material, instrument and process are basically the same as embodiment 1, the difference is:

[0064] (1) Different from (1.2) transport liquid preparation in embodiment 1, the method that present embodiment adopts is:

[0065] To prepare the hpLPS solution, dissolve 15 g of hpLPS in 100 ml of sterile distilled water, and use a 0.22 μm filter membrane to filter and sterilize. Weigh 281g of Brinell broth powder, heat and stir to dissolve in distilled water, add distilled water to the final volume of 100ml to obtain Brinell broth medium, then add 0.5g of activated carbon particles, dissolve and mix well, and then autoclave at 121°C . After cooling, 8 ml of aseptically treated hpLPS solution and 0.1 ml of aseptically treated 0.5 g / ml cysteine ​​solution were added. Then aseptically dispense...

Embodiment 3

[0070] Embodiment 3: Adopt the method of the present invention to the isolation culture of wild-type Helicobacter pylori

[0071] The present embodiment Helicobacter pylori isolation culture material, instrument and process are basically the same as embodiment 1, the difference is:

[0072] (1) Different from (1.2) transport liquid preparation in embodiment 1, the method that present embodiment adopts is:

[0073] To prepare the hpLPS solution, dissolve 15 g of hpLPS in 100 ml of sterile distilled water, and use a 0.22 μm filter membrane to filter and sterilize. Weigh 281g of Brooke’s broth powder, heat and stir to dissolve in distilled water, add distilled water to the final volume of 100ml to obtain Brooke’s broth medium, then add 1g of cyclodextrin, dissolve and mix well, and then autoclave at 121°C . After cooling, 3 ml of aseptically treated hpLPS solution and 0.1 ml of aseptically treated 0.5 g / ml cysteine ​​solution were added. Then aseptically dispense the Brooke's ...

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