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Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application thereof

A technology for enrofloxacin and ciprofloxacin, applied in the field of rapid and simultaneous detection of aptamers for enrofloxacin and ciprofloxacin

Active Publication Date: 2020-04-14
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, an aptamer can only be combined with a specific target substance for detection, and due to the strong flexibility of the aptamer, it can only be combined under specific conditions.
In the prior art, there is no detection method and kit that can simultaneously detect the two target substances of enrofloxacin and ciprofloxacin rapidly and efficiently

Method used

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  • Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application thereof
  • Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application thereof
  • Aptamer and kit for detecting enrofloxacin and ciprofloxacin and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1 Materials and methods

[0040] 1) Main reagents and equipment

[0041] Table 1 Reagents and instruments

[0042]

[0043] Table 2 Aptamer sequence

[0044]

[0045] Table 3 Binding buffer configuration

[0046]

[0047]

Embodiment 2

[0048] Example 2 Design and Characterization of Aptamers

[0049] 2.1 Aptamer sequence design

[0050] Sequence alignment of ENR-60 and CIP-98 was performed using DNAMAN software, and ENR-60 and CIP-98 were simulated using the online structure simulation software Mc-Flod (http: / / www.major.iric.ca / MC-pipeline / ) The secondary structure and tertiary structure of the aptamer are found, the central binding site is found, the original sequence is cut and spliced ​​to obtain a new aptamer sequence, and the most suitable structure is selected by online scoring software.

[0051] 2.2 Molecular docking simulation

[0052] According to the screened three aptamer sequences, Autodock was used to simulate docking, and the docking results analyzed by software were used to conduct a preliminary analysis of the binding possibility of the three aptamers.

[0053] 2.3 Determination of aptamer dissociation constant

[0054] The Kd value was determined by equilibrium adsorption experiment. Mix ...

Embodiment 3

[0059] Preparation and characterization of embodiment 3 colloidal gold

[0060] 3.1 Preparation of colloidal gold

[0061] This experiment uses the classic sodium citrate reduction method to prepare colloidal gold. The specific method is: heat 100ml of 0.01% chloroauric acid solution to boil and continue to stir, then quickly add 2.4ml of 1% sodium citrate solution to it, and continue to heat and boil to the solution Stop heating until the color changes from colorless to blue-purple and finally into a stable wine red, continue stirring to room temperature, centrifuge at 14000r / m for 10min, concentrate 10 times, and set aside.

[0062] 3.2 Characterization of colloidal gold

[0063] Use a TEM transmission electron microscope to scan the concentrated colloidal gold solution to observe its particle size and shape. At the same time, use a UV spectrophotometer to scan its full wavelength of 400-700nm, and calculate its particle size and concentration through the absorbance values ...

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Abstract

The invention provides an aptamer and a kit for rapidly and simultaneously detecting enrofloxacin and ciprofloxacin and an application of the aptamer and the kit. Based on the existing enrofloxacin and ciprofloxacin aptamers with the lengths of 60-mer and 98-mer respectively, an aptamer with the length of 37-mer is obtained by carrying out computer simulation and target simulation butt joint on the secondary structures and tertiary structures of the existing aptamers, finding out binding sites of the aptamers on the basis and carrying out shearing and splicing, and experiments prove that the aptamer only has high binding specificity on enrofloxacin and ciprofloxacin. An aptamer nanogold detection method is established for the two existing aptamers at the same time, and the aptamer nanogolddetection method can be used for detecting enrofloxacin and ciprofloxacin in an actual sample.

Description

technical field [0001] The invention belongs to the technical field of biomolecular detection, and in particular relates to an aptamer, a kit and an application thereof for rapid and simultaneous detection of enrofloxacin and ciprofloxacin. Background technique [0002] Enrofloxacin (ENR) is a third-generation quinolone antibacterial drug, also known as ethyl ciprofloxacin, which is a kind of synthetic broad-spectrum antibacterial drug. In recent years, it has been widely used in diseases of aquatic animals, especially bacteria. in the prevention and treatment of diseases. However, in recent years, due to the low price and obvious antibacterial effect of sandacins, the abuse and misuse of sandacins are very serious, leading to the emergence of super bacteria. The metabolite ciprofloxacin (ciprofloxacin, CIP) is generated, and finally accumulates in the human body along with the food chain, and then induces adverse reactions in the human body. The adverse reactions often cau...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53G01N21/64
CPCC12N15/115G01N33/9446G01N21/6428G01N21/643C12N2310/16G01N2021/6432
Inventor 林洪沙隽伊隋建新安然王赛曹立民
Owner OCEAN UNIV OF CHINA
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