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Aminoglycosides phosphate modifying enzyme and detection method for detecting antibiotics

An aminoglycoside and antibiotic technology, applied in the field of aminoglycoside phosphate modification enzymes, can solve the problems of interfering with the binding of antibiotics and ribosomes, and the loss of efficacy of antibiotics, achieving high-throughput detection and analysis, fast detection speed, and convenient operation. Effect

Inactive Publication Date: 2012-03-28
SOUTHEAST UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aminoglycoside phosphate modification enzyme (APH(3')-III) is an inactivating enzyme produced by bacteria, which covalently modifies certain groups of antibiotics, interferes with the combination of antibiotics and ribosomes, and thus makes antibiotics lose their efficacy

Method used

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  • Aminoglycosides phosphate modifying enzyme and detection method for detecting antibiotics
  • Aminoglycosides phosphate modifying enzyme and detection method for detecting antibiotics
  • Aminoglycosides phosphate modifying enzyme and detection method for detecting antibiotics

Examples

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Embodiment 1

[0031] Embodiment 1: the establishment of the standard curve of antibiotic concentration and fluorescent probe intensity: use the light source of 494nm as excitation light source, irradiate to quartz cuvette sample cell, then place receiver in vertical 90 degree direction, detect the fluorescence of emission, and Record data collection.

[0032] Sample preparation: Dissolve known kanamycin A in neutral TrisHCl buffer (pH 7.4, 100mMNaCl) at room temperature, and make standard products with different concentrations (0.1μM, 0.5μM, 1μM, 5μM , 10 μM, 20 μM, 50 μM, 100 μM, 200 μM, 500 μM, 1 mM). Blank experiment: Aminoglycoside phosphate modification enzyme (APH(3')-III), ATP, MgCl 2 Dissolve in neutral TrisHCl buffer (pH 7.4, 100mM NaCl). enzyme, ATP, MgCl 2 Make up the mixed solution, the final concentration of enzyme is 300 μM, the final concentration of ATP is 1 mM, MgCl 2 The final concentration of is 1.5mM, and it is put into the sample cell as a substrate to observe the e...

Embodiment 2

[0043] Embodiment 2: detection of residual neomycin B in milk:

[0044] The problem of antibiotic residues in milk has become one of the hidden dangers of industry development and food safety. At the source of the dairy industry, antibiotics are used frequently, especially for the treatment of bovine mastitis, and are often used repeatedly in large doses. In dairy cow feed, antibiotic additives are also contained. Therefore, the problem of antibiotic residues in milk is very serious. This protocol can be applied to the rapid detection of aminoglycoside antibiotics in milk, taking neomycin B as an example. Weigh 10g of milk, dissolve it in 10ml TrisHCl buffer (pH 7.4, 100mM NaCl), filter or high-speed centrifuge the sample to be tested, some biological macromolecules in the solution, such as protein, will precipitate, and the molecular weight of neomycin B is relatively small , remained in the supernatant solution. Add MgCl to aminoglycoside phosphate modifying enzyme (APH(...

Embodiment 3

[0045] Example 3: Detection of residual tobramycin in honey: Honey is rich in nutrition and is an important product for exporting foreign exchange in my country. However, with the abuse of antibiotics, the United States, Japan and the European Union have strengthened the inspection of antibiotics in honey, which has caused my country's honey export industry to face a severe test. This protocol can be applied to the rapid detection of aminoglycoside antibiotics in honey, taking tobramycin as an example. Weigh a certain volume of honey, add 5ml of water, and mix quickly on a liquid mixer for 1min to completely dissolve the sample. Add 15ml of ethyl acetate, oscillate on the shaker for 10min, centrifuge at 3000r / min for 10min, absorb 12ml of ethyl acetate from the upper layer and transfer it to the evaporation tube of the automatic concentrator, concentrate and evaporate to dryness. Add 5ml of TrisHCl buffer (pH 7.4, 100mM NaCl) to dissolve the residue. Add MgCl to aminoglycosi...

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Abstract

The invention discloses aminoglycosides phosphate modifying enzyme for detecting antibiotics. Fluorophore containing or modifying thiol is labeled on the thiol of 147th-170th peptides of the aminoglycosides phosphate modifying enzyme to serve as a fluorescence probe. The invention also discloses a kit and a detection method for detecting antibiotics, wherein the kit is prepared by aminoglycosides phosphate modifying enzyme for detecting antibiotics. Compared with currently common chromatography method, mass spectrography, enzyme-linked immunosorbent assay and other methods, the detection method has the characteristics of high detection speed, convenience in operation, high sensitivity and the like, and thus is an attractive antibiotic detection method.

Description

technical field [0001] The invention relates to an aminoglycoside phosphate modification enzyme for detecting antibiotics and a method for detecting aminoglycoside antibiotics. Background technique [0002] Since the discovery of penicillin in 1940, thousands of antibiotics have been developed for the clinical treatment of infectious diseases, saving countless lives. However, with the widespread and extensive use of antibiotics, many bacteria have developed resistance to their opponents, and many drugs are no longer as effective as before, and even produce toxic side effects. On August 11, 2010, the world-renowned medical journal "The Lancet" reported that Enterobacteriaceae bacteria with a special gene NDM-1 (New Delhi No. 1 metalloenzyme) are resistant to most commonly used antibiotics[ Kumarasamy K K, et al., The Lancet Infectious Diseases, 2010, 9, 597-602, French G L, International Journal of Antimicrobial Agents, 2010, 36, S3-S7]. The report immediately attracted wid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12Q1/25G01N21/64
Inventor 武灵芝孙峰吴宏文刘全俊陆祖宏
Owner SOUTHEAST UNIV
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