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Detection method of kanamycin residue based on nucleic acid aptamer

A technology of nucleic acid aptamer and kanamycin, which is applied in the field of analytical chemistry, can solve the problems of complex operation, large error in results, and long measurement time, and achieve the effect of simple, fast and high sensitivity

Inactive Publication Date: 2013-10-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Microbial detection method is widely used, its advantage is low cost, general laboratory can operate, but the measurement time is long, the result error is large, and the operation is complicated, so it cannot meet the needs of high-throughput and high-sensitivity quantitative detection
The instrumental inspection method uses the molecular structure and physical and chemical properties of antibiotics for separation and qualitative and quantitative determination. It has high sensitivity and good accuracy, but the equipment is often expensive, and there are high requirements for experimenters and high requirements for sample pretreatment. Analysis is time-consuming and not easy to promote

Method used

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  • Detection method of kanamycin residue based on nucleic acid aptamer
  • Detection method of kanamycin residue based on nucleic acid aptamer
  • Detection method of kanamycin residue based on nucleic acid aptamer

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Embodiment 1. The determination of kanamycin standard solution absorbance-concentration standard curve

[0028] Kanamycin standard solutions of different concentrations were added to the aggregated AuNPs solution prepared above, the reaction system was 500 μL, and reacted at room temperature for 5 min. Determination of the UV-visible spectrum, obtained as figure 2 Shown A 527nm The relationship curve with the concentration of kanamycin, in the range of 1-500 nM concentration of kanamycin, A 527nm It varies with the concentration of kanamycin, and in the two concentration ranges of 1-8 nM and 100-500 nM, A 527nm There is a linear relationship with the concentration, and the linear regression equation is y=0.0079x+1.7267, R 2 =0.9856 and y=9e-5x+1.8852, R 2 =0.9958, where y is A 527nm , x is the concentration of kanamycin (nM).

Embodiment 2

[0029] Example 2. Detection of Kanamycin Residues in Milk Samples

[0030] Here, milk samples containing 0-500 nM kanamycin residues were obtained by adding standard concentrations of kanamycin to milk to prepare artificially polluted milk. Add 20% acetic acid dropwise to the milk sample, adjust the pH to 4.6, and precipitate the casein in a water bath at 45°C for 10 minutes, then centrifuge at 10,000 r / min for 25 minutes to remove coagulated protein and fat, and finally filter with a 0.22 μm filter membrane. The pH of the obtained sample was then adjusted to neutral to obtain a pretreated milk sample. The pretreated milk samples were added to the aggregated AuNPs solution prepared above, the reaction system was 500 μL, and reacted at room temperature for 5 min. The UV-Vis spectrum was measured to obtain image 3 Shown A 527nm The relationship curve with the concentration of kanamycin in the milk sample. Similarly, in the two ranges of kanamycin concentration of 1-8 nM and...

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Abstract

The invention discloses a detection method of a kanamycin residue based on a nucleic acid aptamer, and belongs to the technical field of analytic chemistry. The method comprises the steps that by using a sulfydryl covalent modification mode, ssDNA (single-stranded Deoxyribonucleic Acid) complementary with the two ends of a kanamycin aptamer is modified on the surfaces of AuNPs (Gold Nanoparticles) respectively; two kinds of functionalized AuNPs are obtained; the kanamycin aptamer is added and pairwise bound with ssDNA on the surfaces of the functionalized AuNPs; spatial distances among the AuNPs are closed; the AuNPs are mediated to be aggregated; after the AuNPs are mixed with kanamycin samples with different concentrations, specific binding between kanamycin and the aptamer allows an aggregation to be disaggregated, which is manifested by a change of an AuNPs characteristic absorption peak in an ultraviolet-visible spectrum; and the kanamycin residue is detected by using a relation between a change of the absorbency at a 527-nanometer part and the concentration of the kanamycin. The method is good in repeatability and stability, easy to operate and high in sensitivity, and can be used for directly determining the kanamycin residues in samples such as pretreated milk.

Description

technical field [0001] The invention is a nucleic acid aptamer-based detection method for kanamycin residues, in particular a detection method for kanamycin residues in milk, which belongs to the technical field of analytical chemistry. Background technique [0002] Kanamycin belongs to aminoglycoside antibiotics, which is a kind of strong water-soluble alkaline antibiotics extracted from the culture solution of Streptomyces or Micromonas or semi-synthetically extracted from natural products. Negative bacilli have a large range of action, strong and long-lasting action, but adverse reactions of this drug are more common. Some of these are irreversible toxicities, including nephrotoxicity, ototoxicity, neuromuscular blockade, hematopoietic toxicity, and anaphylaxis. Kanamycin residues in food of animal origin can cause damage to the human body to varying degrees, and the residues of kanamycin in food should be strictly controlled. Therefore, establishing an effective method...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/31
Inventor 周楠迪张娟田亚平
Owner JIANGNAN UNIV
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