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Method for determining minimal inhibitory concentration of drug

A technology of minimum inhibitory concentration and drug, applied in material excitation analysis, fluorescence/phosphorescence, etc., can solve the problem of long measurement time, and achieve the effect of low cost, good application prospect and market prospect, fast, accurate and simple.

Active Publication Date: 2013-08-07
BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, their common disadvantage is that the measurement time is long, especially when the relevant drug information needs to be given quickly in critical situations, the traditional method is slightly insufficient

Method used

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  • Method for determining minimal inhibitory concentration of drug
  • Method for determining minimal inhibitory concentration of drug
  • Method for determining minimal inhibitory concentration of drug

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The rapid fluorescence method described in embodiment 1 detects the MIC value of ampicillin, levofloxacin, vancomycin to Enterococcus faecalis ATCC 29212

[0033] (1) Antibiotic preparation: refer to the MIC reference value of each antibiotic against Enterococcus provided by CLSI (2010 version), set the drug concentration gradient as 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / ml. Antibacterial drugs of different concentrations after serial dilution were added to sterile 96-well polystyrene plates, 100 μl per well, and three replicate wells were made for each gradient. At the same time, negative control wells (70% ethanol treatment group) and no drug-treated wells were set as growth control (positive control).

[0034] (2) Preparation of bacterial suspension: Dilute the bacterial suspension of 0.5 McFarland turbidimetric standard 10 times with MH broth (Mueller-Hinton Broth, OXOID), and add it to each well containing different concentrations of dru...

Embodiment 2

[0042] (1) Antibiotic preparation: refer to the MIC reference value of each antibiotic against Enterococcus provided by CLSI (2010 version), set the drug concentration gradient as 512, 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125 μg / ml. Antibacterial drugs of different concentrations after serial dilution were added to sterile 96-well polystyrene plates, 100 μl per well, and three replicate wells were made for each gradient. At the same time, negative control wells (70% ethanol treatment group) and no drug-treated wells were set as growth controls (positive control).

[0043] (3) Preparation of bacterial suspension: Dilute the 0.5 McFarland turbidimetric standard bacterial suspension 10 times with MH broth (Mueller-Hinton Broth, OXOID), and add it to each well containing different concentrations of drugs, 100 μl / well, the final concentration is 2.5×10 6 CFU / ml. After sealing, put it in a normal air incubator at 34.5°C and incubate for 4 hours (the effect of vancomyci...

Embodiment 3

[0049] Example 3 The method of the present invention was applied to 90 strains of enterococcus clinically isolated, and compared with the MIC results obtained by the microdilution method performed in parallel, to verify the feasibility of the rapid fluorescence method for detecting MIC.

[0050] 1. Materials and methods

[0051] 1. Strains and Antibiotics

[0052] 1.1 Strains: Enterococcus faecalis standard strain ATCC 29212; 90 clinically isolated Enterococcus strains, 90 of which were identified as Enterococcus faecalis, of which 75 were Enterococcus faecalis and 15 were Enterococcus faecium.

[0053] 1.1 Antibiotics: Standard products of levofloxacin, ampicillin and vancomycin were purchased from China National Institutes for Food and Drug Control. The three drug standard products were made into stock solutions with sterile deionized water, aliquoted and stored at -70°C.

[0054] 2. Method

[0055] 2.1 Determination of MIC

[0056] In this study, the minimum inhibitory ...

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Abstract

The invention discloses a method capable of rapidly, simply, conveniently and quantitatively detecting minimal inhibitory concentration, and the method comprises the following steps of: in accordance with the standard of CLSI (Clinical and Laboratory Standards Institute) (Version 2010), adding a fresh enterococcus suspension of a certain concentration into a sterile 96-well plate containing concentration gradient antibacterials for co-incubation; upon the ending of the 4-hour incubation, adding a fixed amount of fluorescent dyes SYTOX Green and DAPI (4,6-diamino-2-phenyl indole) to all the wells, protecting the wells from light for 15 minutes at room temperature, reading the fluorescent intensity of the two dyes in the wells by use of a fluorescent microplate reader respectively; after relevant background fluorescence is deducted, drawing a corresponding curve between bacterial fluorescence intensity ratio (Pdead / livel) and concentration of drug (CDrug), and determining the minimal concentration of drug corresponding to the moment the Pdead / livel is no longer fluctuated as the MIC (Minimal Inhibitory Concentration) of the antibiotic to the bacterium. The detection method provided by the invention has the characteristics of being simple, convenient, rapid and objective and being capable of performing mathematic statistics and analysis directly on detected data, becomes a new detection method for determining the minimal inhibitory concentration of antibacterials, and has a wide application prospect.

Description

technical field [0001] The invention relates to a method for determining the bacteriostatic concentration, in particular to a method for determining the minimum bacteriostatic concentration of drugs based on fluorescent dyes. Background of the invention [0002] Commonly used methods for determining the minimum inhibitory concentration (MIC) of antibacterial drugs include: broth dilution method, agar dilution method and E-test. The above methods are all based on the NCCLS antimicrobial susceptibility test operating standards, select the drug concentration range including drug resistance, intermediate and sensitive cut-off points (except for special cases), use bacteria in media containing different concentrations of drugs (broth or The difference in the growth state after long-term culture in agar) is used to determine the minimum drug concentration that inhibits bacterial growth. The general culture time is 20-24 hours, and the time to obtain the results is relatively long...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
Inventor 苏建荣孙伟
Owner BEIJING FRIENDSHIP HOSPITAL CAPITAL MEDICAL UNIV
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