Determining method capable of quantitatively determining activity of bacterial secretory hemolysin

A determination method and quantitative determination technology, which are applied in the preparation of test samples, measuring devices, analytical materials, etc., can solve the problem that there is no reported quantitative detection method for hemolysin.

Inactive Publication Date: 2017-08-11
新疆畜牧科学院
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  • Abstract
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Problems solved by technology

[0006] At present, there are many methods for the qualitative determination of bacterial hemolysin, the simplest such as 5% sheep blood culture dish, observe the hemolysis after 24-48 hours of culture, PCR method to detect each hemolysin gene carried by bacteria, etc., there is no report of hemolysin Quantitative detection method, especially the detection of the total amount of bacterial exocrine hemolysin

Method used

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  • Determining method capable of quantitatively determining activity of bacterial secretory hemolysin
  • Determining method capable of quantitatively determining activity of bacterial secretory hemolysin
  • Determining method capable of quantitatively determining activity of bacterial secretory hemolysin

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Experimental program
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Embodiment 1

[0033] Embodiment 1, a kind of assay method that can quantitatively measure bacterial secretion hemolysin activity, this assay method comprises the following steps:

[0034] 1. Reagents and preparation

[0035] (1) Preparation of blood cell preservation solution: glucose 2.05g; sodium citrate 0.8g; sodium chloride 0.42g; distill 100mL, mix and dissolve the above ingredients, adjust the pH to 6.1 with citric acid, and sterilize at 115°C for 15 minutes , stored at 4°C for later use;

[0036](2) Buffered saline: stock solution preparation: 2.85 g of disodium hydrogen phosphate dodecahydrate; 0.27 g of potassium dihydrogen phosphate; 17.00 g of sodium chloride; add distilled water to 100 ml, store at 4°C for later use; buffer solution preparation: 5 Add 95 ml of distilled water to 1 ml of stock solution, add 0.1 ml of 10% magnesium sulfate, and use it within 12 hours of preparation on the same day;

[0037] (3) Preparation and storage of 1% sheep red blood cells:

[0038] Asept...

Embodiment 2

[0054] Embodiment 2, have identical step one with embodiment 1, and step two adopts microporous method to measure operation

[0055] (1) Dilute hemolysin: 50 μL of hemolysin to be tested, add 5 mL of buffer solution, and the dilution is 1:100;

[0056] (2) Preparation of standard hemolysis wells: add 800 μL of 1% sheep red blood cells to 1200 μL of distilled water, mix well to form full hemolysis wells, take 250 μL and add to full hemolysis wells; take 600 μL of full hemolysis wells and add 600 μL of buffer to form 50% hemolysis wells , take 250 μL and add to 50% hemolysis well;

[0057] (3) As shown in the table below, add the diluted hemolysin to be tested, buffer and red blood cells into the reaction wells of the “V” bottom microwell plate in turn, mix well, and place in a 37°C water bath for 30 minutes; Hemolysis pairs of wells; make more than three duplicate wells for each well;

[0058] Determination of Bacteriolysin VH50 by Microporous Method

[0059]

[0060] (4)...

Embodiment 3

[0061] Example 3, Quantitative Determination of Staphylococcus aureus Secreted Hemolysin Activity

[0062] Staphylococcus aureus can secrete α, β, γ, δ, ε and other hemolysins, and the amount of hemolysin secreted by different strains and the activity of hemolysin protein are different.

[0063] 1. Material method

[0064] 1.1 Strains: Xinjiang field isolates XJ69, XJNA10, XJ92, XJ50 and XJCP-1

[0065] 1.2 Cultivation: 50% glycerol was used to preserve the bacterial strain, and 50 μL was directly added to 4 mL of Columbia liquid medium, and cultured at 37° C. with shaking at 170 rpm for 20 h.

[0066] 2. Quantitative Determination of Hemolysin

[0067] Carry out according to the test tube method operation step of embodiment 1.

[0068] 3. Results

[0069] Determination of the activity of hemolysin secreted by Staphylococcus aureus

[0070]

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Abstract

The invention belongs to the technical field of design of biological experimental methods, and particularly relates to a determining method capable of quantitatively determining activity of bacterial secretory hemolysin. The determining method comprises the following steps: first, reagent and preparation: (1) preparation of hemocyte storing liquid; (2) preparation of buffering saline and storing liquid, and preparation of buffering liquid; (3) preparation and storage of 1% sheep red blood cells; and (4) preparation and storage of hemolysin to be measured; and second, determination and operation by a test tube method: (1) diluting of the hemolysin; (2) preparation of a hemolysis standard tube; (3) hemolysis contrast; and (4) result determination; or second, determination and operation by a micro-pore method: (1) diluting of the hemolysin; (2) preparation of a hemolysis standard hole; (3) emolysis contrast; and (4) result determination. By the determining method, bacterial hemolysin can be determined well quantitatively.

Description

technical field [0001] The invention belongs to the technical field of design of biological experiment methods, and in particular relates to a method for quantitatively measuring the activity of bacterial secreted hemolysin. Background technique [0002] Hemolysin (hemolysin / haemolysin), also known as cytolysin, is a toxin secreted by bacteria that can dissolve cells. Hemolysin belongs to pore-forming toxin, also known as membrane disrupting type. In 1981, Fussle et al. first proposed the definition of perforating toxins, which are bacterial protein toxins that form pores across the cell membrane. According to the structure of hemolysin, the way it binds to cells, the mechanism of pore formation, and the response of target cells, many hemolysins are classified into repeat toxin family (Repeats in toxin family, RTX), thiol-active cholesterol-binding cytolytic Family of thiol-activated, cholesterol-binding cytolysin. There are also individual hemolysins that cannot be class...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N21/31
CPCG01N1/28G01N21/3103
Inventor 王登峰李建军吴建勇杨学云刘志强蒋晓梅
Owner 新疆畜牧科学院
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