Oligonucleotides aptamer special for distinguishing fumonisin B1

A fumonisin, specific recognition technology, applied in the biological field, can solve the problems of high cost, complicated operation, low sensitivity, etc., and achieve the effects of high purity and efficiency, simple preparation method and low cost

Active Publication Date: 2013-04-03
JIANGNAN UNIV
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Problems solved by technology

Thin-layer chromatography is easy to operate, suitable for personnel without special training, and low in cost, without the need for expensive instruments; but its steps are cumbersome and its sensitivity is low
The HPLC method has high accuracy and strong sensitivity, and can be used as a quantitative detection method. However, the pretreatment process of this method is cumbersome and the operation is complicated. During the experiment, fumonisin needs to be derivatized before the column, and usually requires professionals to carry out Operation and equipment are expensive, the detection process is time-consuming and expensive, and it is not suitable for promotion at the grassroots level and on-site rapid detection of a large number of samples
The ELISA method has strong specificity, high sensitivity, good accuracy, easy operation, and is suitable for the detection of large quantities of samples; however, the sensitivity of the detection and the stability of the kit are still far from practical applications. Preparation of tedious, time-consuming and expensive antibodies, and the stability of the prepared antibodies between batches is not as good as oligonucleotides, and the storage conditions of antibodies are also stricter than oligonucleotides

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  • Oligonucleotides aptamer special for distinguishing fumonisin B1
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  • Oligonucleotides aptamer special for distinguishing fumonisin B1

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Embodiment 1

[0019] Example 1: Competitive SELEX screening of fumonisin B1 specific binding oligonucleotide aptamers

[0020] 1. In vitro chemical synthesis of initial random single-stranded DNA (ssDNA) library and primers (completed by IDT, USA)

[0021] The sequence is as follows: 5'-AGCAGCACAGAGGTCAGATG(40N)CCTATGCGTGCTACCGTGAA-3'(40N represents 40 random nucleotides);

[0022] Upstream primer: 5′-AGCAGCACAGAGGTCAGATG-3′

[0023] Downstream primer: 5′-TTCACGGTAGCACGCATAGG-3′

[0024] 5′ phosphorylation downstream primer: 5′-P-TTCACGGTAGCACGCATAGG-3′

[0025] The random ssDNA library and primers were made into 100 μM stock solution with TE buffer and stored at -20°C for later use.

[0026] 2. Conditions for PCR amplification and Lambda exonuclease digestion to prepare single-strand sub-libraries

[0027] The synthetic random single-stranded library (ssDNA) was diluted as a PCR template to amplify the phosphorylated double-stranded DNA (dsDNA) product, and the influencing factors of L...

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Abstract

The invention provides a group of single-stranded DNA nucleic acid aptamers of fumonisin B1. Each aptamer is obtained by carrying out screening, amplification, sequencing, and analysis on affinity and specificity on a random original single-stranded DNA library with the combination of an SELEX (systematic evolution of ligands by exponential enrichment) technology and a magnetic bead with fumonisin B fixed on the surface. As a special efficient distinguishing aptamer of fumonisin B, the group of DNA aptamers provides a new choice for developing a method for replacing the existing methods which are used for detecting fumonisin B by depending on an antibody, and can be used for analyzing and detecting or separating the fumonisin B in the environment of enriched food. The nucleic acid aptamer is small in molecular weight, low in chemical synthesis cost, easy to mark, strong in affinity and specificity, reversible in degeneration renaturation, high in speed, and suitable for repeated use, room-temperature transportation and long-term storage.

Description

technical field [0001] The present invention relates to a group of oligonucleotide aptamers that specifically recognize fumonisin B1 and a preparation method thereof, in particular to the preparation of an aptamer using SELEX technology (exponentially enriched ligand system evolution technology) in molecular biology technology. An oligonucleotide aptamer specifically binding to fumonisin B1 belongs to the field of biotechnology. Background technique [0002] Fumonisins are water-soluble secondary metabolites produced by Fusarium moniliforme under certain humidity and temperature conditions. They are widely distributed around the world and mainly pollute food crops and feed, especially corn and its products. Seriously, it can also exist in some products that use grains as raw materials, such as noodles, beer, condiments, etc. Fumonisin has high thermal stability and is not easy to be destroyed. It has various toxicity and carcinogenicity to humans and animals, such as neurot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12Q1/68C07C219/06C07C213/10G01N21/64
Inventor 王周平陈秀娟段诺吴世嘉
Owner JIANGNAN UNIV
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