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A colorimetric method for the detection of tobramycin based on double-strand displacement and three-way DNA structure

A technology for tobramycin and structure detection, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of complicated personnel training and sample preparation, long test cycle, expensive equipment, etc., to expand the detection range , Improve the detection sensitivity, the effect of simple operation

Active Publication Date: 2022-06-24
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have some disadvantages, such as high detection limit, expensive equipment, long test cycle, personnel training and complicated sample preparation

Method used

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  • A colorimetric method for the detection of tobramycin based on double-strand displacement and three-way DNA structure
  • A colorimetric method for the detection of tobramycin based on double-strand displacement and three-way DNA structure
  • A colorimetric method for the detection of tobramycin based on double-strand displacement and three-way DNA structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Drawing of tobramycin concentration standard curve

[0037] T1 and T2 were mixed at the same concentration, denatured at 95 °C for 5 min, and then renatured at 37 °C for 120 min. 2 μL (10 μmol·L -1 ) double strands were mixed with 4 μL of tobramycin solutions of different concentrations and incubated at 37°C for 30 min. To the above mixture, add 2 μL (10 μmol·L -1 ) primer 1, 2 μL (10 μmol·L -1 ) primer 2, 5 U Nt.BstNBI nickase, 8 U Bsm DNA polymerase, and 2 μL (10 mmol·L -1 ) free deoxyribonucleoside triphosphate, 1× buffer (100 mmol L -1 NaCl, 50 mmol·L -1 Tris-HCl, 10 mmol·L -1 MgCl 2 , 0.1 mg·mL -1 BSA), incubate at 55°C for 120 min after mixing to generate a large number of reporter probes, inactivate at 75°C for 10 min, and store at 4°C for later use.

[0038] Combine 10 μL of S1, 10 μL of S2, and 10 μL of S3 (all 10 -6 mol·L -1 ) were mixed, heated at 95 °C for 5 min, and then gradually cooled to room temperature. After this, 30 μL of t...

Embodiment 2

[0040] Example 2 Determination of tobramycin content in actual water samples

[0041] In order to further verify the accuracy of this method in the determination of tobramycin content in actual samples, Taihu Lake water without pretreatment was selected to dilute tobramycin to different concentrations. The reaction was carried out in exactly the same way as the standard sample of tobramycin, and the absorbance value of the solution at 420 nm was read by the UV-vis spectrophotometer in the obtained reaction solution, and the concentration of tobramycin could be calculated by substituting it into the standard curve.

[0042] The specific samples and test results are shown in Table 1.

[0043] Table 1

[0044]

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Abstract

The invention discloses a colorimetric method for detecting tobramycin based on double strand replacement and three-way DNA structure, belonging to the fields of food safety, medical analysis and environmental pollution detection. The present invention first designs double-strand T1 / T2; when there is tobramycin, Bsm DNA polymerase synthesizes a fully complementary double-strand through a strong strand displacement reaction, and Nt.BstNBI nicking endonuclease cuts the recognition site on the double-strand Point; three-way DNA structure captures the reporter probe, regenerates and displaces a large number of S1 strands containing G-quadruplex-forming sequences. Thereafter, the G‑quadruplex / heme catalyzes the ABTS 2‑ / H 2 o 2 Color reaction, the content of tobramycin can be determined by using the linear relationship between the absorbance value and the concentration of tobramycin. The present invention triggers Nt.BstNBI nicking endonuclease and Bsm DNA polymerase-mediated double strand displacement reaction through aptamer capture tobramycin to generate a large number of reporter probes, and at the same time, the reporter probe triggers λ exonuclease The auxiliary loop amplification realizes the multiple amplification of the colorimetric signal, broadens the detection range and improves the detection sensitivity.

Description

technical field [0001] The invention relates to a colorimetric method for detecting tobramycin based on double strand displacement and three-way DNA structure, and belongs to the fields of food safety, medical analysis and environmental pollution detection. Background technique [0002] Aminoglycoside antibiotics are antibiotics composed of two or more amino sugars linked to a hexose ring through a glycosidic bond, and have good therapeutic effects on common bacterial diseases. Tobramycin is a broad-spectrum aminoglycoside antibiotic mainly used to treat infections caused by certain Gram-positive and aerobic Gram-negative microorganisms. The mechanism is to bind to ribosomes, thereby destroying synthetic proteins, leading to cell membrane damage and cell death. Tobramycin is widely used in animal husbandry due to its good water solubility, low cost and broad antimicrobial spectrum. However, excessive and misuse of tobramycin results in large residues in animal-derived food...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/682
CPCC12Q1/682C12Q2537/143C12Q2531/119C12Q2525/205C12Q2521/301
Inventor 周楠迪欧莹田亚平
Owner JIANGNAN UNIV
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