Method for rapidly screening and evaluating antibiotic bioactivator

A technology for biologically active substances and active substances is applied in the field of rapid screening and evaluation of antibacterial drugs. Effect

Inactive Publication Date: 2011-09-28
HAINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Disc agar diffusion method (K-B test) and test tube broth dilution method as conventional antibacterial drug screening methods are difficult to meet the needs of large-scale screening of new antibacterial drugs
In addition, because the test tub

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  • Method for rapidly screening and evaluating antibiotic bioactivator
  • Method for rapidly screening and evaluating antibiotic bioactivator
  • Method for rapidly screening and evaluating antibiotic bioactivator

Examples

Experimental program
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Effect test

Embodiment 1

[0039]Embodiment 1: A kind of method for rapid screening evaluation antibacterial drug

[0040] According to the preliminary test, ceftazidime was selected as the positive control antibiotic of the experimental bacteria, and the MIC value of ceftazidime to the experimental bacteria was determined to be 12.5 mg / L by the test tube broth dilution method.

[0041] Take a sterile 96-well culture plate, add 100 μL of bacterial suspension and 5 μL of ceftazidime sample solution of different dilutions (200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, 0.380625 mg / L) into each well, Bacterial suspension and sterile water were added to model control wells, and MH broth medium and sterile water were added to blank control wells. After incubating at 37°C for 18 hours, add 50 μL of resazurin solution, and react for 2 hours. Use a microplate reader to measure the absorbance of each well in the 96-well plate at the maximum absorption wavelength of resazurin (λmax=600), and measure the MI...

Embodiment 2

[0045] Example 2: Rapid Screening and Evaluation of Antibacterial Activity of 24 Marine Bioactive Substances

[0046] Accurately weigh 5g of the freeze-dried biological sample, put it into a 34mL extraction pool, add a cellulose filter to the bottom of the extraction pool in advance, and use petroleum ether, ethyl acetate, ethanol (volume fraction 95%), methanol, and distilled water as extraction Solvent, set ASE extraction parameters (extraction pressure is 10MPa, extraction temperature is 120°C, extraction time is 10min, cycle 2 times), the organic solvent extract is concentrated to dryness at 45°C under reduced pressure, and the water extract is vacuum freeze-dried to obtain 24 a biologically active substance. Accurately weigh a certain amount of biologically active substances and dissolve and disperse with a small amount of 20% dimethyl sulfoxide (DMSO), centrifuge to get the supernatant, and use 20% DMSO solution to dilute the sample in multiples for use.

[0047] Accord...

Embodiment 3

[0053] Embodiment 3: the antibacterial activity comparison of 6 kinds of plant bioactive substances

[0054] Accurately weigh 5g each of the root, stem, and leaf dry products of the plant sample, and put them into a 34mL extraction pool respectively. A cellulose filter is installed at the bottom of the extraction pool in advance, and distilled water is used as the extraction solvent, and the ASE extraction parameters are set (extraction pressure is 10MPa , the extraction temperature is 120° C., the extraction time is 10 min, and the cycle is 2 times), the water extract is vacuum freeze-dried, and the bioactive substances of the roots, stems, and leaves of Rauwolfia are obtained respectively. Accurately weigh an appropriate amount of the biologically active substance, dissolve it in a small amount of distilled water, and perform a multiple dilution for use.

[0055] According to the preliminary test, ceftazidime was selected as the positive control antibiotic of the experimenta...

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Abstract

The invention discloses a method for rapidly screening and evaluating an antibiotic bioactivator, in particular a test method for screening the antibiotic bioactivator by combining an accelerated solvent extraction apparatus and a microplate resazurin assay. The method comprises the following steps of: preparing a bioactivator by the accelerated solvent extraction apparatus; adding the bioactivator and known antibiotic into a microplate according to concentration gradient, and adding experimental bacterial suspension into various holes; placing the microplate under suitable conditions, incubating, adding resazurin solution, and mixing uniformly; determining light absorption values of the holes of the microplate at 600nm by using a microplate reader; evaluating a minimum inhibitory concentration (MIC) value of the bioactivator to be screened according to light absorption values in known antibiotic holes and light absorption values in bioactivator holes at different concentrations; and further evaluating the antibiotic activity of the bioactivator. The method is rapid, simple and convenient, and the antibiotic activity of the bioactivator can be objectively and accurately evaluated, so that the high-throughput screening of the antibiotic bioactivator becomes possible.

Description

technical field [0001] The invention relates to a method for rapid screening and evaluation of antibacterial drugs, in particular to a test method for rapid screening and evaluation of antibacterial bioactive substances through rapid solvent extraction technology combined with microporous plate resazurin chromogenic method. Used in the field of biomedicine. Background technique [0002] Antibacterial drugs are an indispensable class of drugs in modern medicine, and are widely used in clinical practice. However, there are many problems in the application of this class of drugs at present, such as toxic and side effects, allergic reactions, bacterial drug resistance, etc., especially the rapid rise of bacterial drug resistance, making serious infections caused by these bacteria There is no effective drug available, which poses a great threat to human health. Due to the specificity of bioactive substances, bacteria are less resistant to bioactive substances. Therefore, the r...

Claims

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Application Information

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IPC IPC(8): C12Q1/04
Inventor 刘平怀刘洋洋时杰杨勋郝宗娣陈德力汪春牛许琼情
Owner HAINAN UNIVERSITY
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