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Group of nucleic acid aptamers for specifically recognizing okadaic acid

A technology of okadaic acid and nucleic acid aptamer, applied in recombinant DNA technology, DNA/RNA fragments, measuring devices, etc., can solve the problem of poor repeatability and stability of antibodies, tedious and time-consuming process, and harsh storage conditions and other problems, to achieve the effect of easy chemical modification, good stability and small difference

Active Publication Date: 2015-05-20
JIANGNAN UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a nucleic acid aptamer sequence that can specifically recognize okadaic acid and has high affinity, so as to overcome the deficiencies in the existing detection technology of okadaic acid, especially in the immunoassay method. , Antibody preparation must rely on animals or cells, the process is cumbersome, time-consuming, expensive, poor repeatability and stability between different batches of antibodies, and storage conditions are relatively strict, etc., for the development of a new type of okadaic acid analysis and detection method lays the groundwork

Method used

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  • Group of nucleic acid aptamers for specifically recognizing okadaic acid
  • Group of nucleic acid aptamers for specifically recognizing okadaic acid
  • Group of nucleic acid aptamers for specifically recognizing okadaic acid

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Embodiment Construction

[0020] 1. Synthesis of random ssDNA library and primers

[0021] A random ssDNA library with a length of 80nt was constructed, consisting of 20nt primer regions at both ends and a 40nt random region in the middle, with the sequence: 5'-CAGCTCAGAAGCTTGATCCT-N 40 -GACTCGAAGTCGTGCATCTG-3'(N 40 represents 40 random nucleotides), synthesized by Integrated DNA Technologies, USA.

[0022] Upstream primer: 5'-CAGCTCAGAAGCTTGATCCT-3'

[0023] Downstream primer: 5'-CAGATGCACGACTTCGAGTC-3'

[0024] Downstream primer for 5' phosphorylation: 5'-P-CAGATGCACGACTTCGAGTC-3'

[0025] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

[0026] The random ssDNA library and primers were prepared with 1×TE buffer (pH 7.4) into a 100 μM stock solution and stored at -20°C for later use.

[0027] 2. Determination of the optimal amount of graphene oxide to adsorb ssDNA

[0028] Take six 2mL centrifuge tubes, add 8 μL of 10 μM carboxyfluorescein-labeled 80nt ssDNA, the mass is ...

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Abstract

The invention provides a group of single-chain DNA nucleic acid aptamers for specifically recognizing okadaic acid and belongs to the biotechnical field of food safety. A group of nucleic acid aptamers are obtained through 13 rounds of vitro screening of incubating, separating, amplifying and preparing single chains by virtue of a systematic evolution technology of ligands by exponential enrichment employing graphene oxide-assisted separation; and an aptamer OA-27 which has high affinity and high specificity to the okadaic acid is screened through affinity and specificity analysis. Through analysis, OA27-1 from which a primer zone is removed also has high affinity and specificity. The single-chain DNA nucleic acid aptamers have a wide application prospect in analysis detection of the okadaic acid in aquatic products and diagnosis and treatment of okadaic acid poisoning in clinical.

Description

technical field [0001] The invention belongs to the field of food safety biotechnology, and relates to a sequence of single-stranded DNA nucleic acid aptamer, a recognition element with high affinity and high specificity for diarrhea shellfish toxin okadaic acid. Background technique [0002] Okadaic acid (OA) is a small-molecule fat-soluble toxin, which is a secondary metabolite produced by algae of the genus Dinosaurs and Prorocentrum. Mussels, scallops, oysters and other shellfish filter-feed these toxic algae and accumulate okadaic acid in their bodies. When people eat these shellfish by mistake, it may cause poisoning, and the symptoms are diarrhea, nausea, vomiting, abdominal pain and so on. Because its main poisoning symptom is diarrhea, it is called Diarrhetic Shellfish Poisoning (DSP). Diarrheal shellfish toxins include Dinophysistoxin (DTX), Saxitoxin (Pectenotoxin, PTX), Scallop toxin (Yessotoxin, YTX) and so on. Although OA is a non-lethal toxin without strong...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/543G01N33/531
Inventor 王周平顾华杰段诺吴世嘉陈秀娟郝丽玲
Owner JIANGNAN UNIV
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