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Fluorescence detection method for trace tetracycline antibiotics

A tetracycline and fluorescence detection technology, which is applied in the direction of fluorescence/phosphorescence, material excitation analysis, etc., can solve the problems of complex operation and poor sensitivity, and achieve the effect of simple operation, high sensitivity and maintaining binding ability

Active Publication Date: 2013-05-22
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention solves the problems of complex operation and poor sensitivity of the existing methods for detecting tetracycline antibiotics, and provides a method for detecting trace tetracycline antibiotics with high sensitivity and high selectivity

Method used

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  • Fluorescence detection method for trace tetracycline antibiotics
  • Fluorescence detection method for trace tetracycline antibiotics
  • Fluorescence detection method for trace tetracycline antibiotics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1. Determination of Oxytetracycline Content in Milk Samples

[0026] (1) Preparation of circular DNA template

[0027] Take 5 μL of phosphorylated padlock probe (final concentration 4 μM) and 10 μL DNA primer (final concentration 5.6 μM), mix well in 50 μL 1×T4 DNA ligase buffer, heat the water bath to 95 °C and keep 5 min, then annealing and cooling to 37 °C for 1 h, then adding T4 DNA ligase (final concentration 3.5 U / μL), 5 μL 0.05% BSA, and 50 μL high-purity water at 16 °C overnight. After the reaction is over, add 1 μL 10×Exonuclease I buffer, 1 μL 10×Exonuclease I buffer, Exonuclease I (final concentration 0.1 U / μL), and Exonuclease III (final concentration 0.1 U / μL) Incubate at 37 °C for 1 h to degrade unreacted single-stranded DNA primers. Heat the water bath to 90 °C and incubate for 10 min to terminate the reaction. Finally, the prepared circular DNA template was separated and purified with nanofiltration membrane (10 K, Pall, USA) to remove macromolecula...

Embodiment 2

[0043] Example 2. Determination of tetracycline antibiotics in lake water

[0044] (1) Preparation of circular DNA template

[0045] Take 5 μL of phosphorylated padlock probe (final concentration 2 μM) and 10 μL DNA primer (final concentration 4 μM) and mix well in 50 μL 1×T4 DNA ligase buffer, heat the water bath to 80 °C and keep 5 min, then annealing and cooling to 37 °C for 0.5 h, then adding T4 DNA ligase (final concentration 2 U / μL), 5 μL 0.05% BSA, and 50 μL high-purity water at 10 °C overnight. After the reaction, add 1 μL 10×Exonuclease I buffer, 1 μL 10×Exonuclease I buffer, Exonuclease I (final concentration 0.05 U / μL), and Exonuclease III (final concentration 0.05 U / μL) at 37 °C. Incubate for 0.5 h to degrade unreacted single-stranded DNA primers. Heat the water bath to 80 °C and incubate for 5 min to terminate the reaction. Finally, the prepared circular DNA template was separated and purified with nanofiltration membrane (10 K, Pall, USA) to remove macromolecular en...

Embodiment 3

[0061] Example 3 Determination of Oxytetracycline Content in Lake Water Sample

[0062] (1) Preparation of circular DNA template

[0063] Take 5 μL of phosphorylated padlock probe (final concentration 6 μM) and 10 μL DNA primer (final concentration 8 μM) and mix well in 50 μL 1×T4 DNA ligase buffer. Heat in a water bath to 95 °C and keep After 15 min, annealing and cooling to 37 °C for 2 h, then adding T4 DNA ligase (final concentration 6 U / μL), 5 μL 0.05% BSA, and 50 μL high-purity water at 25 °C overnight. After the reaction, add 1 μL 10×Exonuclease I buffer, 1 μL 10×Exonuclease I buffer, Exonuclease I (final concentration 0.2 U / μL), and Exonuclease III (final concentration 0.2 U / μL). Incubate at 37 °C for 2 h to degrade unreacted single-stranded DNA primers. Heat the water bath to 95 °C and incubate for 15 minutes to terminate the reaction. Finally, the prepared circular DNA template was separated and purified with nanofiltration membrane (10 K, Pall, USA) to remove macromolec...

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Abstract

The invention relates to a fluorescence detection method for trace tetracycline antibiotics. According to the method, by utilizing the combined action of tetracycline antibiotics molecules and the specificity of an aptamer thereof, an amplification action of a rolling ring amplification signal and high selection action of a graphene molecular beacon as well as combining a fluorescence spectrophotometer, the fluorescence signal of the graphene molecular beacon is obtained, so that quantitative detection of trace tetracycline antibiotics can be realized. According to the method, a plurality of defects of the prior art such as complexity in detection are overcome, the sensitivity and selectivity are improved, and a new method is provided for detection of other small molecule substances or pollutants.

Description

Technical field [0001] The invention belongs to the technical field of environmental monitoring, and relates to a fluorescence detection method of trace tetracycline antibiotics. Background technique [0002] Tetracycline antibiotics are currently one of the most widely used broad-spectrum antibiotics in clinical practice, such as tetracycline, oxytetracycline, and doxycycline. In addition, because tetracycline antibiotics can promote animal growth and improve feed utilization, about 70% of antibiotics are used as feed additives for animal husbandry and aquaculture. Through various metabolic pathways and migration and transformation pathways, tetracyclic antibiotics have been detected in soil, surface water, groundwater and other environmental media, and these substances have chronic toxicity and accumulated toxicity and other hazards, which have already brought great harm to the human body and the ecosystem. Threats, such as the production of resistant bacteria or viruses. The...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
Inventor 赵慧敏刘猛高升全燮
Owner DALIAN UNIV OF TECH
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