Eureka AIR delivers breakthrough ideas for toughest innovation challenges, trusted by R&D personnel around the world.

Nucleic acid aptamer of leuco-malachite green and application thereof

A colorless malachite green, nucleic acid aptamer technology, applied in the fields of instruments, biochemical equipment and methods, analytical materials, etc., can solve the problem of identifying the lack of rapid detection of LMG, etc., to facilitate rapid detection on site, simplify Operation process, ease of preparation and effect of modification

Active Publication Date: 2022-05-13
YANBIAN UNIV
View PDF2 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on rapid detection of LMG based on ssDNA aptamer recognition at home and abroad is still blank.

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
  • Nucleic acid aptamer of leuco-malachite green and application thereof
  • Nucleic acid aptamer of leuco-malachite green and application thereof
  • Nucleic acid aptamer of leuco-malachite green and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, nucleic acid aptamer sequence and secondary structure prediction

[0063] 1.1. Screening of A5-b

[0064] The present invention adopts the magnetic bead SELEX (Mag-SELEX) method to screen the single-stranded DNA (ssDNA) aptamer against colorless malachite green (LMG), and obtains the ssDNA aptamer named A5-b.

[0065] A5-b is a single-stranded DNA with the following nucleotide sequence: 5'-CTGCGCGTCCTATGCGTGCT-3', synthesized and purified by Sangon Bioengineering Co., Ltd. (Shanghai).

[0066] 1.2. Nucleic acid aptamer sequence and secondary structure prediction

[0067] The spatial structure of aptamers will affect the binding ability to target molecules, which is of great significance for obtaining high-affinity aptamers. Studies have shown that the smaller the Gibbs free energy ΔG value, the more stable the secondary structure formed by the aptamer.

[0068] The secondary structure and Gibbs free energy (△G) of aptamer A5-b were predicted by mfold softw...

Embodiment 2

[0070] Embodiment 2, aptamer affinity determination

[0071] 2.1 Aptamer affinity analysis method

[0072] Colorless malachite green (LMG) itself has strong fluorescence, and its own fluorescence intensity will decrease when it binds to aptamers. Therefore, the fluorescent method is used to measure the fluorescence changes when aptamers bind to LMG, so as to determine their affinity. For identification, the specific steps are as follows, and the temperature for incubation at room temperature as described below is 25°C:

[0073] (1) Dissolution of aptamers. Centrifuge the aptamer A5-b at 12000r / min for 10 minutes, then wash with DPBS buffer (0.9mM CaCl 2 , 2.68mM KCl, 1.47mM KH 2 PO4, 0.5mM MgCl 2 ·6H 2 O, 136.8mM NaCl, 8.1mM NaCl 2 HPO 4 12H 2 O) Dilution Dilute A5-b to a stock solution of 100 μmol / L, vortex and mix well, and store at -20°C for future use.

[0074] (2) Aptamer-target incubation. When used, the aptamer stock solution was diluted again with DPBS buffer...

Embodiment 3

[0081] Example 3, nucleic acid aptamer specificity verification

[0082] 3.1. Aptamer Specificity Determination Method

[0083]The specificity of aptamer A5-b was verified by fluorescence analysis. The study showed that crystal violet (CV) and malachite green (MG) had almost no fluorescence, and the fluorescence intensity was enhanced after combining with the aptamer. Colorless crystal violet ( LCV) and colorless malachite green (LMG) themselves have strong fluorescence, but the fluorescence intensity decreases after combining with aptamers.

[0084] Referring to the method described in Example 2, the aptamer A5-b was determined for affinity identification with LMG, LCV, MG, and CV, and the specificity of A5-b was judged by the Kd value. Taking the aptamer concentration as the abscissa and ΔF as the ordinate (ΔF=F0-F for LMG and LCV, ΔF=F-F0 for MG and CV), draw a single-point binding saturation curve. Ex of LCV is 300nm, Em is 320-500nm; Ex of MG is 620nm, Em is 640-800nm; ...

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 discloses a leucomalachite green nucleic acid aptamer and application thereof, and belongs to the field of nucleic acid aptamers. The invention discloses a nucleic acid aptamer A5-b and a mutant thereof. The nucleic acid aptamer A5-b and the mutant thereof are single-stranded DNA of which the nucleotide sequences are respectively sequences 1-6. The nucleic acid aptamer A5-b and the mutation aptamer thereof have high specificity and affinity and can specifically recognize the LMG, so that a rapid detection method taking the LMG as a detection target can be established, the process of reducing the LMG into the MG in sample pretreatment can be avoided, the operation process is greatly simplified, and rapid on-site detection is facilitated.

Description

technical field [0001] The invention belongs to the field of nucleic acid aptamers, in particular to a colorless malachite green nucleic acid aptamer and its application. Background technique [0002] Malachite Green (Malachite Green, MG), also known as basic green, malachite green or Chinese green, belongs to triphenylmethane dyes. Malachite green is not only a dye, but also a fungicide, which has strong antifungal, antiparasitic and antiseptic effects. Due to its low price and remarkable antibacterial effect, MG is widely used in aquaculture. However, because of its high toxicity and high residue, it has been listed as a banned drug for aquaculture by countries all over the world. Malachite green will quickly metabolize into fat-soluble colorless malachite green (Leuco malachite Green, LMG) in the organism, and more than 90% of MG in the organism exists in the form of LMG. Compared with MG, LMG has a longer residual time and stronger toxicity, and can be used as the mai...

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): C12N15/115G01N33/53
CPCC12N15/115G01N33/5308C12N2310/16
Inventor 杨咏洁姜岩吴文伟刘可鑫
Owner YANBIAN UNIV
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
Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com