A group of nucleic acid aptamers that specifically recognize three marine toxins

A nucleic acid aptamer and aptamer technology, applied in recombinant DNA technology, DNA preparation, DNA/RNA fragments, etc., can solve the problems of changing the natural conformation of toxins, affecting the efficiency of screening, aptamer performance, etc., to achieve the convenience of chemical Modification, low cost, good repeatability

Active Publication Date: 2020-07-28
JIANGNAN UNIV
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Problems solved by technology

However, there are steric hindrance effects in the immobilization method, the linker molecules used will change the natural conformation of the toxin, and the beads also have non-specific adsorption of oligonucleotides, which will affect the efficiency of screening and the performance of the obtained aptamers

Method used

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  • A group of nucleic acid aptamers that specifically recognize three marine toxins
  • A group of nucleic acid aptamers that specifically recognize three marine toxins
  • A group of nucleic acid aptamers that specifically recognize three marine toxins

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Embodiment Construction

[0016] 1. Preparation and characterization of MRGO

[0017] Simultaneous reduction of graphene oxide (Graphene Oxide, GO) and FeCl under alkaline conditions using glucose as a reducing agent using a one-pot method 3 ·6H 2 O to synthesize MRGO. The prepared MRGO was characterized by transmission electron microscopy, ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy and magnetization. The results showed that MRGO was successfully synthesized and had good magnetic properties. The adsorption effect of MRGO on 80nt ssDNA was measured by fluorescence method, and the results showed that when the mass ratio of MRGO / ssDNA was 300, MRGO could completely adsorb free ssDNA.

[0018] 2. Screening of three marine toxin aptamers

[0019] 2.1 Synthesis of initial random ssDNA library and primers

[0020] A random ssDNA library with a length of 80 nt was synthesized by Integrated DNA Technologies in the United States, including a 20 nt primer region at both ends and...

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Abstract

The invention provides a set of single stranded DNA (ssDNA) aptamers DA-01 and TTX-27 which can simultaneously recognize domoic acid (DA) and tetrodotoxin (TTX), including one ssDNA aptamer DA-06 which specifically recognizes DA, one ssDNA aptamer TTX-07 which can specifically recognize TTX, and one ssDNA aptamer STX-41 which can specifically recognize saxitoxin (STX). According to the invention,magnetic reduced graphene oxide (MRGO) is adopted to assist in separated systematic evolution of ligands by exponential enrichment (SELEX), properties of MRGO of being capable of adsorbing free ssDNA,but not adsorbing ssDNA combined with target molecules are utilized, and magnetic separation enrichment characteristics of MRGO are further used to separate ssDNA with affinity and without affinity to target toxins; primary structure homology and secondary structure similarity on obtained ssDNA are analyzed after 16 repeated rounds of incubation, separation, amplification and in-vitro screening for single-strand preparation, then the affinity and the specificity are measured, and finally one set of the aptamers with high affinity and specificity is obtained. The set of the aptamers has broadapplication prospects in the aspects of analysis, detection, separation, enrichment, removal and purification for DA, TTX and STX in aquatic products.

Description

technical field [0001] The invention belongs to the field of food safety biotechnology, and relates to the screening of a group of single-stranded DNA nucleic acid aptamers capable of simultaneously recognizing domoic acid and tetrodotoxin by utilizing the exponential enrichment ligand system evolution technology assisted by magnetic reduction graphene oxide and its specificity. One single-stranded DNA nucleic acid aptamer that specifically recognizes domoic acid, tetrodotoxin, and saxitoxin. Background technique [0002] Marine toxins mainly come from toxic algae, which are passed to aquatic animals through the food chain and accumulate in their bodies. In recent years, with the intensification of marine red tides and lake cyanobacteria outbreaks, food poisoning incidents caused by eating toxic aquatic products have gradually increased. There are many kinds of marine toxins, which can be divided into two categories: hydrophilic and lipophilic according to their solubility....

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115C12N15/10
Inventor 王周平顾华杰段诺吴世嘉叶华马鹏飞邹颖
Owner JIANGNAN UNIV
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