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Method for rapidly determining drug tolerance of strain

A rapid determination and drug resistance technology, applied in the field of rapid determination of bacterial resistance, rapid determination of bacterial susceptibility to antibiotics, and can solve problems such as inability to use

Inactive Publication Date: 2013-04-24
CHINESE ACAD OF INSPECTION & QUARANTINE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the drug resistance genes of several bacteria have been studied more, and the detection method of drug resistance genes has been developed, but for most bacteria, this method cannot be used yet.

Method used

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  • Method for rapidly determining drug tolerance of strain
  • Method for rapidly determining drug tolerance of strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0024] According to one embodiment of the rapid determination method of bacterial strain drug resistance of the present invention, the determination method comprises the steps:

[0025] A. Prepare thiazolyl blue with PBS to make a 5±0.5% stock solution, sterilize it, and store it in a refrigerator at about 4°C. Add the thiazole blue stock solution to the brain heart infusion culture solution to make the final concentration reach 0.1±0.05%, sterilize it, and store it in a refrigerator at about 4°C for later use;

[0026] B. Prepare a bacterial suspension of 1-10CFU / ml;

[0027] C. prepare antibiotic solution;

[0028] D. Add 178 ± 20 μl of brain heart infusion containing thiazolium blue to each well of the culture plate, and add 20 ± 5 μl of bacterial suspension, and then add 2 ± 1 μl of antibiotics, serially dilute the antibiotic solution, and the final concentration of the antibiotic solution is a series of concentrations, such as 0.1, 1, 10 and 100 μg / ml series concentrati...

Embodiment 1

[0034] Take Staphylococcus aureus as an example of a Gram-positive bacterium. The thiazolyl blue was prepared into a 5% stock solution with PBS, sterilized, and stored in a refrigerator at 4°C. The thiazole blue stock solution was added to the brain heart infusion culture solution to make the final concentration reach 0.1%, sterilized, and stored in a 4°C refrigerator for later use. Add 178 μl of brain heart infusion containing thiazolium blue to each well of a 96-well culture plate. Staphylococcus aureus (strain IQCC22004) bacterial suspension was prepared so that its concentration reached the order of 10 CFU / ml, and 20 μl of bacterial suspension was added to the microwells. Among them, the three lines of A, B, and C were repeated three times, A1, B1, and C1 were blank controls, and 2 μl of sterile water was added; A2, B2, and C2 were added with 2 μl of amoxicillin solution with a concentration of 100 mg / ml, so that The final concentration is 1mg / ml; A3, B3, C3~A11, B11, C1...

Embodiment 2

[0037] Take Enterobacter sakazakii as an example of a Gram-positive bacterium. Add 178 μl of brain heart infusion containing thiazolium blue to each well of a 96-well culture plate. Prepare a bacterial suspension of Enterobacter sakazakii (strain IQCC 10424) so ​​that its concentration reaches the order of 10 CFU / ml, and add 20 μl of the bacterial suspension to the microwells. Repeat the test with two wells. A1 and A2 are negative controls, add 20 μl of sterile water instead of bacterial suspension; A3 and A4 are blank controls, add 2 μl of sterile water instead of antibiotics. A5, A6, A7, A8 are 0.1 μg / ml, 0.05 μg / ml vancomycin, and so on, A9, A10~H11, H12 are oxacillin, cefazolin, cefpodoxime, cephalothin, penicillin G , cefoxitin, amoxicillin, ampicillin, imipenem, pefloxacin, gentamicin, carbenicillin, netilmicin, cefotaxime, cefnicillin, nitrofurantoin, compound Sumophen, ciprofloxacin, ceftazidime, norfloxacin, tetracycline, tobramycin solution. All the other steps a...

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Abstract

The invention discloses a method for rapidly determining drug tolerance of a strain. The method comprises the following steps: A, preparing nutrient fluid containing methyl thiazolyl tetrazolium; B, preparing bacterial suspension; C, preparing antibiotic solution; D, adding the nutrient fluid containing the methyl thiazolyl tetrazolium, the antibiotic solution and the bacterial suspension into holes of a culture plate, and performing serial dilution on the antibiotic solution; and E, scanning and determining the diluent in real time, and determining the drug tolerance of the strain to antibiotic according to the scanning result. Compared with the prior art, the method has the advantages of simply, rapidly, economically and high-efficiently determining the drug tolerance of the strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for quickly measuring the drug resistance of bacterial strains, in particular to a method for rapidly measuring the sensitivity of bacterial strains to antibiotics by using the growth curve of bacterial strains obtained by scanning. Background technique [0002] At present, the World Health Organization believes that with the abuse of antibiotics, all major infectious disease pathogens will gradually develop resistance to drugs. The rapid emergence, rapid spread, and high rate of drug resistance of drug-resistant bacteria have attracted worldwide attention. The drug resistance of bacterial strains not only weakens the efficacy of drugs, but also makes them lose their efficacy, leading to increased patient mortality. The research on the drug resistance of bacterial strains has become a worldwide hotspot. Quick and easy determination of drug resistance of bacterial strains is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02
Inventor 陈颖袁飞徐宝梁赵贵明杨海荣赵勇胜张菲菲
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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