Broad-spectrum nucleic acid aptamer capable of specifically identifying lipopolysaccharides and directed screening method of broad-spectrum nucleic acid aptamer

Abstract

The invention provides a broad-spectrum nucleic acid aptamer capable of identifying lipopolysaccharides of different Gram-negative bacterium sources. The broad-spectrum nucleic acid aptamer is obtained by directed screening on the basis of the capture-SELEX technology of label-free target molecules and immobilized nucleic acid libraries. A directed screening method of the broad-spectrum nucleic acid aptamer includes: using mouse salmonella typhi lipopolysaccharides as the unique target, using a biotin-avidin effect to fix random oligonucleotide libraries to Fe3O4 magnetic nano particles wrapped by avidin through biotinylated short complementary chains, performing incubation, separation, amplification, digestion single chain preparation, cloning sequencing and the like, adding complete active bacteria of salmonella and escherichia coli to serve as directed molecules to perform directed screening when the libraries are enriched to a certain degree, and one broad-spectrum nucleic acid aptamer capable of identifying four kinds of lipopolysaccharides is obtained through 15 rounds of repeated screening. The nucleic acid aptamer is applicable to the analysis and detection, separation and enriching, toxicity neutralizing and the like of the lipopolysaccharides in aspects of drinking water, food or clinical medicine and the like.

Description

technical field [0001] The present invention relates to the fields of food safety and detection, clinical medicine, etc., and particularly relates to the directional screening of a broad-spectrum nucleic acid aptamer of lipopolysaccharide using SELEX technology (exponential enrichment ligand system evolution technology) in molecular biology technology, It can specifically recognize lipopolysaccharides from 4 bacterial sources including Salmonella typhimurium, Salmonella enteritidis, Escherichia coli and Pseudomonas aeruginosa. It provides scientific basis and theoretical basis for the application of concentration and toxicity neutralization. Background technique [0002] Lipopolysaccharide (LPS), also known as endotoxin (Endotoxin), is the main component of the outer membrane of the cell wall of Gram-negative bacteria. It is mainly composed of three parts: Lipid A, core polysaccharide and O-specific polysaccharide repeating unit. Among them, Lipid A is the active and toxic ...

AI Technical Summary

Problems solved by technology

At present, most screening technologies are developed on the basis of immobilized targets. Since the immobilization of targets requires chemical modification, which changes the natural structure of the target substance, the aptamers screened have low specificity and low affinity. Not high, practical application is limited

Although capillary electrophoresis-SELEX technology (CE-SELEX) makes the target and the library both free and maintains the natural structure of the target, this method is only suitable for the screening of macromolecules. In addition, macromolecules may have multiple binding sites. , so it is difficult to screen for nucleic acid aptamers with specific binding sites

Capture-SELEX technology is a kind of library immobilization and target free SELEX technology developed by immobilizing oligonucleotide libraries on magnetic beads, agarose particles and other substrates through non-covalent equilibrium. Advantages, make up for CE-SELEX's inability to screen small molecules, but there are also screening uncertainties

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