Method for detecting residual kanamycin

A kanamycin and detection method technology, applied in the field of analytical chemistry, can solve problems such as toxicity and ingestion allergy, and achieve the effect of sensitive detection and high sensitivity

Inactive Publication Date: 2016-06-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Like other aminoglycoside antibiotics, kanamycin residues in animal foods may cause allergies to ingestors, and long-term consumption of foods with excessive kanamycin residues can also lead to toxic side effects such as ear and kidney toxicity

Method used

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  • Method for detecting residual kanamycin
  • Method for detecting residual kanamycin
  • Method for detecting residual kanamycin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1. Optimization of detection conditions for kanamycin residues based on aptamer-triggered ExoIII cycle digestion

[0029] In order to detect kanamycin more sensitively, we optimized the detection conditions and studied the influence of K-aptamer concentration and ExoIII concentration on the detection of kanamycin.

[0030] Incubate the ssDNA-modified electrodes obtained in step (2) of the above method with 100 μL of K-aptamer hybridization solutions containing 0.1 nM, 0.25 nM, 0.5 nM, 0.75 nM, 1 nM, and 10 nM, respectively, at 90° C. for 5 minutes, and then slowly cool to room temperature. Then soak it in 50 μL of binding solution and incubate for 10 minutes, then rinse with washing solution. Finally, soak the electrode in 50μL containing 1U·μL -1 ExoIII was reacted in 1×NEB solution at 37°C for 1 hour, washed again with washing solution, and then scanned by square wave voltammetry. The measured scan results are as figure 2 As shown in A and 2B, with the inc...

Embodiment 2

[0032] Example 2. Kanamycin residue detection specificity map based on aptamer-triggered ExoIII cycle digestion

[0033]Incubate the modified electrodes obtained in step (3) of the above method with 50 μL of binding solutions containing 500 pM of different antibiotics for 10 minutes, then wash with washing solution, and then soak the electrodes in 50 μL of 1U·μL -1 ExoIII was reacted in 1×NEB solution at 37°C for 1 hour, washed again with washing solution, and scanned by square wave voltammetry. The measured scan results are as image 3 A, As shown in 3B, the assay has good selectivity for kanamycin.

Embodiment 3

[0034] Embodiment 3. The mensuration of electrochemical signal-concentration standard curve of different concentration kanamycin standard solution

[0035] Incubate the modified electrodes obtained in step (3) of the above method with 50 μL of kanamycin standard solutions of different concentrations for 10 minutes, and then wash them with washing solution, and then soak the electrodes in 50 μL of 1U·μL -1 ExoIII was reacted in 1×NEB solution at 37°C for 1 hour, washed again with washing solution, and scanned by square wave voltammetry. The measured scan results are as Figure 4 As shown in A and 4B, in the range of 1-500pM kanamycin concentration, the peak value of the electrochemical signal increases with the increase of the kanamycin concentration, and both meet y=-2.7163-1.2117×lgx(R 2 =0.996), where y is the peak value of the electrochemical signal (μA), and x is the concentration of kanamycin (pM).

[0036] Add standard concentration kanamycin to milk to prepare artific...

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PUM

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Abstract

The invention discloses a method for detecting residual kanamycin and application thereof, and belongs to the technical field of analysis chemistry, wherein the detection method is based on aptamer initiated cyclic enzyme digestion of exonuclease III (Exo III). According the method, mercapto self-assembly is utilized, single stranded DNA (ssDNA) that is complementary with kanamycin aptamer (K-aptamer) is grafted on the surface of a gold electrode, then a little amount of K-aptamer is added, and the K-aptamer is matched with ssDNA to form a little amount of double stranded DNA (dsDNA). Then Exo III is added, the ssDNA in dsDNA can be specifically digested by Exo III, thus the residual aptamers are released, the released aptamers can combine with ssDNA to form dsDNA, another enzyme digestion is triggered then, and after several circulations, the modified ssDNA is completely digested by enzymes. Kanamycin exists in the system, through the combination between kanamycin and aptamers, the circulation enzyme digestion is inhibited, thus ssDNA is preserved, and the amount of preserved ssDNA is related with the concentration of kanamycin. An electro-analysis method is used to absorb electrical signal molecules namely hexaammine ruthenium (RuHeX) through static electricity absorption so as to quantitatively detect residual kanamycin; and the method has high sensitivity and can sensitively detect residual kanamycin in milk.

Description

technical field [0001] The invention relates to a method for detecting kanamycin based on aptamer-triggered exonuclease III (ExoIII) circular digestion, in particular to a method for detecting kanamycin residue in milk, belonging to the field of analytical chemistry. Background technique [0002] Kanamycin is an aminoglycoside broad-spectrum antibiotic used to treat Gram-negative and Gram-positive infections, and is one of the most commonly used anti-infective drugs in clinical practice. Like other aminoglycoside antibiotics, kanamycin residues in animal foods may cause allergies to ingestors, and long-term consumption of foods with excessive kanamycin residues can also lead to toxic side effects such as ear and kidney toxicity. The European Community stipulates that the maximum residue limit of kanamycin in milk is 0.15μg / g (about 318.5nM). Therefore, it is necessary to establish an effective method for the detection of kanamycin so as to strengthen the monitoring of antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/48
Inventor 许媛媛苗晋锋李夏青
Owner NANJING AGRICULTURAL UNIVERSITY
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