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Application of deoxyribozyme probe in escherichia coli drug-resistant phenotype high-throughput sensing

A DNAzyme probe, Escherichia coli technology, applied in the field of analysis and detection, can solve problems such as inability to achieve phenotype analysis, and achieve the effects of preventing the formation of multidrug-resistant bacteria, accurate sensing results, and preventing the abuse of antibiotics

Pending Publication Date: 2022-02-18
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, most of the high-throughput and rapid detection methods for drug-resistant bacteria are gene analysis methods, which can only complete genotype analysis and cannot achieve phenotypic analysis.
However, drug resistance genes do not directly indicate the presence of drug-resistant bacteria in the sample, and the bacterial resistance phenotypes are diverse and variable. In order to obtain more detailed drug resistance information, a new type of high-throughput drug-resistant bacteria phenotype analysis should be developed method is important

Method used

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  • Application of deoxyribozyme probe in escherichia coli drug-resistant phenotype high-throughput sensing
  • Application of deoxyribozyme probe in escherichia coli drug-resistant phenotype high-throughput sensing
  • Application of deoxyribozyme probe in escherichia coli drug-resistant phenotype high-throughput sensing

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Rapid detection of tetracycline-resistant Escherichia coli using RFD-EC recognition molecules

[0040] Rapid detection of tetracycline-resistant Escherichia coli using RFD-EC recognition molecules, the principle is as follows figure 2 As shown, the specific operation is as follows:

[0041] a) Cultivate engineering bacteria of tetracycline-resistant Escherichia coli, add 10 μL of tetracycline-resistant bacteria to 5 mL of SOB medium, and carry out overnight culture, the temperature of the shaking table is 37 ° C, and the shaking speed is 150 rpm;

[0042] b) After culturing overnight, use a microplate reader to measure the bacterial concentration of Escherichia coli, and when OD600=0.5, the bacteria are taken out from the shaker, and the bacterial concentration is about 10 8 CFU / mL;

[0043] c) Dilute the tetracycline-resistant Escherichia coli with sterile water so that the bacterial concentration is 10 5 CFU / mL; by calculation in a 10.5 μL bacterial samp...

Embodiment 2

[0049] Example 2 Rapid determination of kanamycin-resistant Escherichia coli using RFD-EC recognition molecules, and bacterial counting

[0050] Using RFD-EC recognition molecules to quickly detect kanamycin-resistant Escherichia coli, the specific operation is as follows:

[0051] a) Cultivate engineering bacteria of kanamycin-resistant Escherichia coli, add 10 μL of kanamycin-resistant bacteria to 5 mL of SOB medium, and carry out overnight culture, the temperature of the shaking table is 37 ° C, and the shaking speed is 150 rpm;

[0052] b) After culturing overnight, use a microplate reader to measure the bacterial concentration of Escherichia coli, and when OD600=0.5, the bacteria are taken out from the shaker, and the bacterial concentration is about 10 8 CFU / mL;

[0053] c) Dilute kanamycin-resistant Escherichia coli with sterile water so that the bacterial concentration is 10 4 ~10 6 CFU / mL, by calculation in a 10.5 μL bacterial sample, the number of bacteria is 100,...

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Abstract

The invention discloses application of a deoxyribozyme probe in escherichia coli drug-resistant phenotype high-throughput sensing, and belongs to the field of analysis and detection. The deoxyribozyme probe comprises deoxynucleotide chains as shown in SEQ ID NO.1 and SEQ ID NO.2, the SEQ ID NO.2 serves as a substrate chain, the SEQ ID NO.2 contains an RNA base A, and T bases on the two sides of the base are connected with a fluorophore F and a fluorescence quenching group Q respectively. After a specific target protein is recognized, the SEQ ID NO.1 cuts the SEQ ID NO.2 chain into a short chain, and then fluorescence is generated. The detection principle is combined with a droplet microfluidic system with high-throughput detection capability, so that rapid and high-throughput detection of escherichia coli can be realized, drug-resistant escherichia coli and non-drug-resistant escherichia coli can be effectively distinguished, bacterial drug-resistant phenotype analysis is performed, accurate determination is realized, and the deoxyribozyme probe can be widely applied to diagnosis of drug-resistant types of escherichia coli in actual samples, and has great significance in the aspects of realizing pollution control, further preventing abuse of antibiotics, preventing formation of multidrug-resistant bacteria and the like.

Description

technical field [0001] The invention belongs to the field of analysis and detection, and in particular relates to the application of a deoxyribozyme probe in high-throughput sensing of Escherichia coli drug-resistant phenotype. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Drug-resistant bacteria have become a major threat to human health and ecosystem security. Rapid, high-throughput detection of bacterial drug-resistant phenotypes is the key to effective control of drug-resistant bacteria. At present, drug-resistant bacteria and resistance genes have been widely detected in my country's soil, rivers, waste treatment plants, medical pharmaceutical factories and breedin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816C12Q1/10C12N15/11
CPCC12Q1/6816C12Q2521/345C12Q2563/107Y02A50/30
Inventor 刘猛迟艳琛石蒙常洋洋
Owner DALIAN UNIV OF TECH
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