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Fluorescently-labeled aminoglycoside adenosine modification enzyme and method for detecting antibiotics with same

An aminoglycoside and glycoside adenosine technology, which is applied in the field of fluorescently labeled aminoglycoside adenosine modification enzymes and the detection of antibiotics, can solve the problems of interfering with the binding of antibiotics and ribosomes, and the loss of efficacy of antibiotics, and achieves enzymatic catalysis. Fast response, expensive reagents and consumables, and the effect of curbing application prospects

Inactive Publication Date: 2013-05-01
NANJING UNIV OF POSTS & TELECOMM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aminoglycoside adenosine-modifying enzyme (ANT(4')) 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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  • Fluorescently-labeled aminoglycoside adenosine modification enzyme and method for detecting antibiotics with same
  • Fluorescently-labeled aminoglycoside adenosine modification enzyme and method for detecting antibiotics with same
  • Fluorescently-labeled aminoglycoside adenosine modification enzyme and method for detecting antibiotics with same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The establishment of the standard curve of antibiotic concentration and fluorescent probe intensity: use a 494nm light source as the excitation light source, irradiate the sample cell of the quartz cuvette, then place the receiver in a vertical 90-degree direction, detect the emitted fluorescence, and record the data collection .

[0035] Sample preparation: Dissolve the known kanamycin A in neutral HEPES buffer (pH 7.5, 100mM KCl) at room temperature, and make successively different concentrations of standards (0.1μM, 0.5μM, 1μM, 5μM, 10μM, 20μM, 50μM, 100μM, 200μM, 500μM, 1mM). Blank experiment: First, on the free sulfhydryl Cys198 of the aminoglycoside adenosine modifying enzyme, fluorescein-5-maleimide (Fluorescein-5-maleimide) containing or modifying the sulfhydryl group is labeled, and its structure is as follows figure 2 shown. Then the labeled aminoglycoside adenosine modifying enzyme (ANT(4')), ATP, MgCl 2 Dissolve in neutral HEPES buffer (pH 7.5, 100mM KCl)...

Embodiment 2

[0046] Example 2: Detection of residual neomycin B in milk:

[0047] 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. Take 5ml of milk (such as Yili) on the market, add a small amount of neomycin to make the concentration 10μM, and mix well. Then centrifuge on a high-speed centrifuge with a rotating speed of 21000 rpm for 2.5 hours. After centrifugation, the milk is separated, and large proteins are precipitated. Neomycin B has a relatively small molecular weight and remains in the supern...

Embodiment 3

[0048] Example 3: Detection of residual kanamycin A in milk:

[0049] This protocol can be applied to the rapid detection of aminoglycoside antibiotics in milk, taking kanamycin A as an example. Take 5ml of milk (such as Yili) on the market, add a small amount of kanamycin to make the concentration 6μM, and mix well. Then centrifuge on a high-speed centrifuge with a rotating speed of 21000 rpm for 2.5 hours. After centrifugation, the milk is separated, and large proteins are precipitated. Kanamycin A has a relatively small molecular weight and remains in the supernatant aqueous solution. Carefully transfer the aqueous supernatant to a new centrifuge tube for fluorescence testing. Then the reaction system was prepared, and the fluorescently labeled aminoglycoside adenosine modifying enzyme, ATP, and metal ions were configured in neutral HEPES buffer (pH 7.5, 100mM KCl) according to a certain concentration, wherein the modifying enzyme was 10 μM, and the ATP concentration wa...

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Abstract

The invention discloses a fluorescently-labeled aminoglycoside adenosine modification enzyme and a method for detecting antibiotics with the same. A fluorophore containing or modifying a sulfydryl is labeled on the free sulfydryl Cys198 of the fluorescently-labeled aminoglycoside adenosine modification enzyme, and is fluorescein-5-maleimide. The invention further discloses the method for detecting antibiotics with the fluorescently-labeled aminoglycoside adenosine modification enzyme. As the fluorescently-labeled aminoglycoside adenosine modification enzyme prepared according to the invention is simple to reagent preparation, low in cost, and fast and sensitive in enzyme catalytic reaction, residual antibiotic medicines in foods and environments can be detected conveniently, fast and efficiently without the limitation of laboratory operation.

Description

technical field [0001] The invention relates to a fluorescently labeled aminoglycoside adenosine-modifying enzyme and a method for detecting antibiotics, which are mainly used for the detection and quantitative analysis of antibiotic drug residues in the fields of food safety, medical hygiene, environmental monitoring and the like. 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 Lan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12Q1/48G01N21/64
Inventor 武灵芝胡栋
Owner NANJING UNIV OF POSTS & TELECOMM
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