Aminoglycoside antibiotic aptamer as well as affinity column, preparation method and application thereof

An aminoglycoside and antibiotic technology, applied in the fields of agriculture, food and environmental testing, can solve problems such as single target, achieve the effect of saving samples, high affinity and high purification efficiency

Pending Publication Date: 2022-04-22
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the aptamer affinity columns reported at home and abroad are mai

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Aminoglycoside antibiotic aptamer as well as affinity column, preparation method and application thereof
  • Aminoglycoside antibiotic aptamer as well as affinity column, preparation method and application thereof
  • Aminoglycoside antibiotic aptamer as well as affinity column, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1 Preparation of aptamer affinity column using amino-modified aminoglycoside antibiotic aptamer

[0081] 1. Preparation of NHS-modified agarose

[0082] ①Wash 10mL of agarose gel with 4FF water and drain it, wash it with 30% and 70% acetone with 5 times the volume of the agarose gel in sequence in a sand core funnel, and finally wash it with 100% dioxane with 5 times the volume of the agarose gel The ring was washed 5 times, drained, and transferred to a 100 mL Erlenmeyer flask with a stopper, 10 mL of dioxane, 100 μL of allyl glycidyl ether and 300 μL of boron trifluoride diethyl ether were added, and reacted in a water-bath shaker at 140 rpm at 35°C for 45 minutes. The reacted matrix was washed successively with 30% and 70% acetone of 5 times the volume of the agarose gel, and finally washed with a large amount of deionized water to obtain an activated matrix.

[0083] ②Carboxylation of thioglycolic acid: take 10mL of activated matrix, add 1.20mL of thioglyco...

Embodiment 2

[0100] The optimization of the working condition of embodiment 2 aminoglycoside antibiotic aptamer affinity column

[0101] 1. Sample solution

[0102] Because the chemical structure of aminoglycoside antibiotics contains -OH and -NH 2 , Yi and H + Therefore, the pH value of the sample solution will have a certain impact on the existing form of the compound, as well as the retention and separation on the AGs-AAC, thereby affecting the recovery rate. In the present invention, the retention of sample solutions with different pH values ​​on AGs-AAC was investigated. Prepare 9 parts of binding buffer containing 10 mM Tris HCl, 120 mM NaCl, 5 mM KCl and 1 mM MgCl 24M sodium hydroxide solution was used to adjust the pH values ​​to 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, and 9, and the binding buffers with different pH values ​​were used as diluents to prepare the concentration of each antibiotic. The 500 ng / mL mixed AGs solution (including AMI, TOB, KANB and KANA) was loaded, and the el...

Embodiment 3

[0111] Example 3 Purification of aminoglycoside antibiotics in milk samples by using aminoglycoside antibiotic aptamer affinity column and its detection

[0112] In this example, aminoglycoside antibiotic standard substances were quantitatively added to the milk sample, and then the aminoglycoside antibiotic aptamer affinity column prepared in Example 1 was used for purification, and after purification, ultra-high performance liquid chromatography triple quadrupole mass spectrometry Instrument detection, determination of recovery.

[0113] Extract aminoglycoside antibiotics from milk samples as follows:

[0114] 1. Milk sample processing

[0115] 1) Measure 5 mL of milk sample and add binding buffer to a centrifuge tube (the binding buffer contains 10 mM TrisHCl, 120 mM NaCl, 5 mM KCl and 1 mM MgCl 2 aqueous solution) to 50 mL, and mix well.

[0116] 2) Extract by shaking for 10 minutes, and centrifuge at 8000 r / min for 10 minutes.

[0117] 3) Take the supernatant in anoth...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides an aminoglycoside antibiotic aptamer, an affinity column of the aminoglycoside antibiotic aptamer, a preparation method of the affinity column and application of the affinity column. The nucleic acid sequence of the nucleic acid aptamer for the aminoglycoside antibiotics is shown as SEQ ID NO.1, an affinity column takes N-hydroxysuccinimide modified agarose as a carrier, and then the nucleic acid aptamer capable of recognizing the aminoglycoside antibiotics with high affinity and high specificity is covalently coupled with the carrier, so that the nucleic acid aptamer for recognizing the aminoglycoside antibiotics with high affinity and high specificity is obtained. And filling the affinity column with the coupled product as a filler. The affinity column provided by the invention can be repeatedly used for at least 23 times, has good purification and enrichment effects on four aminoglycoside antibiotics including kanamycin A, kanamycin B, tobramycin and amikacin, and is mainly used for purification and enrichment of aminoglycoside antibiotics in feeds, foods, environmental samples and other complex samples; the method is beneficial to high performance liquid chromatography and rapid detection of the antibiotics in a sample in the later period, and has a wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of agriculture, food and environmental detection, in particular to an aminoglycoside antibiotic aptamer, its affinity column, its preparation method and application. Background technique [0002] Aminoglycoside antibiotics (AGs) are a class of broad-spectrum antibiotics, mostly polar compounds, easily soluble in water, and their molecules are formed by linking an aminocyclic alcohol and one or more aminosugar molecules through glycosidic bonds. Most of AGs are produced by different species of Streptomyces and Micromonospora, such as Kanamycin, Neomycin, Streptomycin, Gentamicin, Tobramycin and Spectinomycin, etc., and some It is artificial and semi-synthetic, such as netilmicin, amikacin and arbekacin. As an important antibacterial drug, AGs mainly play a bactericidal role by inhibiting bacterial protein synthesis and destroying the integrity of bacterial cell membranes. It is mainly used clinically to trea...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): G01N30/02G01N30/14G01N30/72
CPCG01N30/02G01N30/14G01N30/72
Inventor 栾云霞王楠
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap