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Method for detecting ampicillin based on aptamer

An ampicillin, nucleic acid aptamer technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complex analysis process, low specificity and sensitivity, long time-consuming analysis and detection, etc. Fluorescence signal, improved sensitivity, low detection cost

Inactive Publication Date: 2017-08-18
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, both methods have their own inevitable shortcomings and limitations.
For example, the analysis and detection of microbial analysis takes a long time, and the specificity and sensitivity are relatively low; for high-performance liquid chromatography, the entire analysis process is relatively complicated and cumbersome, and it also requires expensive sample pretreatment, so this method is not suitable for being used. extensive promotion

Method used

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  • Method for detecting ampicillin based on aptamer
  • Method for detecting ampicillin based on aptamer
  • Method for detecting ampicillin based on aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] a. Mix 1 μL of different concentrations of Hairpin (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Replacement template (1 μL, 10 μM), phi29 DNA polymerase (1 μL, 10×) , dNTPs (1 μL), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the substance to be tested (ampicillin) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 30 minutes;

[0037] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it for 10 minutes to inactivate the enzyme in the system, then add Circular template (1 μL, 10×), T4 DNA into the EP tube Ligase (1 μL, 10×), incubated at 16°C for 12 h;

[0038] c. After heat preservation treatment at 16°C, put it in a water bath at 65°C for 15 minutes, then add Molecularbeacon (1 μL, 10×) and phi29 DNA polymerase (1 μL, 10×) into the EP tube, and place in a water bath at 37°C Water bath for 30min. Take out the mixed solution from the water bath, and measure the ...

Embodiment 2

[0041] a. Mix 1 μL of different concentrations of Replacement template (final concentrations are 0.4 μM, 0.6 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.4 μM), Hairpin (1 μL, 10 μM), phi29 DNA polymerase (1 μL, 10×) , dNTPs (1 μL), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the substance to be tested (ampicillin) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 30 minutes;

[0042] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it for 10 minutes to inactivate the enzyme in the system, then add Circular template (1 μL, 10×), T4 DNA into the EP tube Ligase (1 μL, 10×), incubated at 16°C for 12 h;

[0043] c. After heat preservation treatment at 16°C, put it in a water bath at 65°C for 15 minutes, then add Molecularbeacon (1 μL, 10×) and phi29 DNA polymerase (1 μL, 10×) into the EP tube, and place in a water bath at 37°C Water bath for 30min. Take out the mixed solution from the water bath, and measure the ...

Embodiment 3

[0046] a. Add Hairpin (1μL, 10μM), Replacement template (1μL, 10μM), phi29 DNA polymerase (1μL, 10×), dNTP (1μL), Nb.BbvCI (1μL, 10×), NEB buffer (5μL, 10×) and the substance to be tested (ampicillin) were added to the EP tube, shaken for 30s, and placed in a water bath at 37°C for 30 minutes;

[0047] b. Take the reacted solution out of the water bath, put it into a water bath at 85°C and heat it for 10 minutes to inactivate the enzyme in the system, and then add 1 μL of Circular template of different concentrations to the EP tube (the final concentrations are respectively 0.4μM, 0.6μM, 0.8μM, 1.0μM, 1.2μM, 1.4μM), T4 DNA ligase (1μL, 10×), incubate at 16°C for 12 h;

[0048] c. After heat preservation treatment at 16°C, put it in a water bath at 65°C for 15 minutes, then add Molecularbeacon (1 μL, 10×) and phi29 DNA polymerase (1 μL, 10×) into the EP tube, and place in a water bath at 37°C Water bath for 30min. Take out the mixed solution from the water bath, and measure ...

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Abstract

The invention provides a method for detecting ampicillin based on an aptamer. According to the invention, signal amplification is realized by applying a target object circulation technology, a strand displacement reaction technology and a rolling circle replication amplifying technology. The method provided by the invention is low in price, high in analysis and detection speed, high in sensitivity and strong in specificity.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a fluorescence detection method for detecting ampicillin. Background technique [0002] Ampicillin is a broad-spectrum semi-synthetic penicillin with similar antibacterial properties to penicillin. Ampicillin has been widely used now, especially in medicine and the treatment of bacterial infections. It can effectively kill a variety of bacteria, such as: Haemophilus influenzae, Escherichia coli, Salmonella, Shigella, etc. However, the widespread use of ampicillin will also have a serious impact on people's healthy life. For example, the abuse of ampicillin in animal husbandry leads to drug residues in food and agricultural products and diseases caused by these residues, such as: allergic reactions, respiratory Difficulty and seizures etc. Therefore, for people's health, it is very important to determine the residual amount of ampicillin in food and agricultural products...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q2521/101C12Q2521/301C12Q2521/501C12Q2531/119C12Q2531/125C12Q2563/107
Inventor 李慧徐成功王玉张雪刘素冷雪琪裴倩倩崔雪君韩聪刘学娇秦艺菲涂玉琴
Owner UNIV OF JINAN
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