Method for rapidly evaluating in-vitro antagonistic helicobacter pylori activity

A Helicobacter pylori, antagonistic technology, applied in the field of microbial detection, can solve the problems of large number of plates, flow away, small inhibition zone, etc., and achieve the effects of simplified operation steps, intuitive observation results, and practical detection methods

Pending Publication Date: 2021-03-26
SHANGHAI ACAD OF AGRI SCI
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  • Claims
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Problems solved by technology

At present, the methods used to antagonize Helicobacter pylori in vitro mainly include disk method, viable count method, punching diffusion method and Oxford cup agar diffusion method; but these methods have certain shortcomings. The amount of sample that can be loaded on the drug-sensitive paper is small, and the active substance is easily adsorbed on the paper and difficult to diffuse into the culture medium, resulting in a very small inhibition zone around the paper, which is not easy to determine; the viable count method takes a long time, The number of plates required is large, and the difference in the number of colonies in each repeated experiment is relatively large, which is not conducive to judgment; the disadvantage of the punching diffusion method is that the sample solution flows away from the hole, and the expected antibacterial activity cannot be detected; Oxford cup agar diffusion The method adopts the principle of double-layer plates, that is, the lower layer is the basic medium, and the upper layer of indicator culture medium needs to be mixed with a certain amount of Helicobacter pylori bacteria liquid. After the upper layer of medium is solidified, a sterile Oxford cup is placed, and the sample solution is poured in. Determination; when preparing the upper medium, the thickness of the upper medium, the uniformity of Helicobacter pylori dispersion and the survival rate of the bacteria should be controlled, so the experimental conditions of this method are strict, and it is not suitable for the rapid evaluation of antibacterial activity
At present, there is a lack of a rapid method for evaluating the activity of antagonizing Helicobacter pylori in vitro

Method used

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  • Method for rapidly evaluating in-vitro antagonistic helicobacter pylori activity
  • Method for rapidly evaluating in-vitro antagonistic helicobacter pylori activity
  • Method for rapidly evaluating in-vitro antagonistic helicobacter pylori activity

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preparation example Construction

[0024] In the present invention, a sheep blood agar medium plate is taken, a bacterial suspension of Helicobacter pylori is coated on the sheep blood agar medium plate, an Oxford cup is placed, the sample solution to be tested is added into the Oxford cup, cultured for 3 to 5 days, and measured The diameter of the zone of inhibition. In the present invention, the culture conditions are preferably 37° C., 10% carbon dioxide, and 5% oxygen. In the present invention, the raw materials for the preparation of the sheep blood agar medium include the following components in parts by weight: 8-10 parts of brain heart extract powder, 20-25 parts of Columbia agar, 700-850 parts of water and defibrated sheep blood 30-40 servings. In the present invention, the preparation method of the sheep blood agar medium includes: mixing brain heart extract powder, Columbia agar and water, and adding defibrated sheep blood after autoclaving and cooling to 45-50°C. In the present invention, the thic...

Embodiment 1

[0030] 1. Cultivation and passage of Helicobacter pylori: Take the cultured sheep blood agar medium plate with the Helicobacter pylori ATCC43504 strain, add 800 μL of brain heart infusion broth (BHI) washing solution under sterile conditions, and the bacteria liquid Transfer to a 2mL sterile sample tube, take 100 μL of the bacterial solution onto the sheep blood agar medium, shake well, and incubate at 37°C with 10% carbon dioxide and 5% oxygen for later use.

[0031] The components of sheep blood agar medium are: brain heart extract powder 9.6g, Columbia agar 23.4g, distilled water 780mL, after autoclaving and cooling down to 46°C, add 33-40mL of defibrated sheep blood, pour plate.

[0032] The components of BHI lotion are: peptone 10.0g / L, dehydrated calf brain extract powder 12.5g / L, dehydrated beef heart extract powder 5.0g / L, sodium chloride 5.0g / L, glucose 2.0g / L, phosphoric acid Sodium hydrogen disodium 2.5g / L, water balance, pH 7.4.

[0033] 2. Prepare the sample soluti...

Embodiment 2

[0040] 1. Cultivation and passage of Helicobacter pylori: Take the cultured sheep blood agar medium plate with Helicobacter pylori Sydney SS1 strain, add 800 μL of brain heart infusion broth (BHI) washing solution under sterile conditions, and the bacteria The solution was transferred to a 2mL sterile sample tube, and 100 μL of the bacterial solution was placed on the sheep blood agar medium, and after shaking, it was cultured at 37°C with 10% carbon dioxide and 5% oxygen for later use. The components of the sheep blood agar medium and the components of the BHI washing solution are the same as in Example 1.

[0041] 2. The configuration of the sample solution is the same as in Example 1.

[0042] 3. Determination of the diameter of the inhibition zone by the Oxford cup method: take a sheep blood agar medium plate with a diameter of 9 cm and a thickness of about 5 mm, and add about 100 μL to each plate to a concentration of 1×10 8 The bacterial suspension of Helicobacter pylor...

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Abstract

The invention relates to a method for rapidly evaluating in-vitro antagonistic helicobacter pylori activity, and belongs to the technical field of microbiological detection. The method comprises the following steps of taking a sheep blood agar culture medium plate, coating the sheep blood agar culture medium plate with a bacterial suspension of helicobacter pylori, placing an oxford cup, adding ato-be-detected sample solution into the oxford cup, culturing for 3-5d, and measuring the diameter of an inhibition zone; and preparing to-be-detected sample solutions with different concentrations, respectively adding the to-be-detected sample solutions into 96-well plates, adding the sheep blood agar culture medium into each well while the goat blood agar culture medium is hot, mixing, adding the bacterial suspension of the helicobacter pylori into each well after the sheep blood agar culture medium is solidified, culturing for 3-5d, and determining the minimum inhibition concentration of the to-be-detected sample solutions on the helicobacter pylori. The method has the characteristics of simplicity, high efficiency, short judgment time, visual observation result and the like, meets therequirements of a helicobacter pylori in-vitro antagonism experiment method, and is suitable for multi-sample detection.

Description

technical field [0001] The invention relates to the technical field of microorganism detection, in particular to a method for rapidly evaluating the activity of antagonizing Helicobacter pylori in vitro. Background technique [0002] Helicobacter pylori (H. pylori) was successfully isolated from gastric mucosa in 1983 by Warren and Marshall. It has been confirmed that H.pylori is closely related to chronic gastritis, peptic ulcer, gastric mucosa-associated lymphoid tissue (MALT) lymphoma and gastric cancer. In 1994, the World Cancer Research Agency listed it as a class I carcinogen. Currently, triple or quadruple antibiotic therapy is commonly used in clinical treatment of H. pylori infection. Due to the disadvantages of this therapy, such as large side effects and easy drug resistance, the search for antibiotic alternative therapy is currently a hot spot in international and domestic research. In the process of seeking new anti-H.pylori drugs, in vitro antagonism is the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/18C12R1/01
CPCC12Q1/18G01N2333/195
Inventor 张忠杨焱吴迪王雨阳李文陈万超张劲松刘艳芳
Owner SHANGHAI ACAD OF AGRI SCI
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