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Oligonucleotide aptamer for specifically recognizing histamine and application thereof

A technology of oligonucleotides and aptamers, which is applied in the field of food safety biology, can solve the problems of time-consuming and laborious, poor stability of recognition molecules, and difficult preparations, and achieve the effects of sensitive detection, short screening cycle, and convenient synthesis

Active Publication Date: 2019-04-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the emerging biosensor methods with recognition elements as the core, such as enzyme biosensor method, immune biosensor method, and molecularly imprinted membrane biosensor method, although their sensitivity has been improved, the recognition molecules have poor stability, difficult preparation, and finding suitable functional units. The process of eluting the body and template molecules with the solvent is time-consuming and labor-intensive

Method used

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  • Oligonucleotide aptamer for specifically recognizing histamine and application thereof
  • Oligonucleotide aptamer for specifically recognizing histamine and application thereof
  • Oligonucleotide aptamer for specifically recognizing histamine and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Screening of aptamers

[0022] figure 1 The flow chart of using Capture-SELEX to screen aptamers that can specifically recognize histamine specifically includes the following steps:

[0023] (1) Library hybridization: design a complementary chain CO that is complementary to some bases in the primer region of the library and perform biotin-labeling, and hybridize the ssDNA library to the complementary chain CO;

[0024] (2) Library immobilization: the ssDNA library was immobilized on the surface of the magnetic beads by specific binding of biotin labeled on the complementary chain CO and streptavidin on the magnetic beads, and the non-specifically bound ssDNA was removed by repeated washing;

[0025] (3) Anti-screening incubation: During the screening process, in the 4th, 7th, 9th, 12th, 14th, 15th, 16th, and 17th rounds, the structural analogues of histamine, tryptamine, tyramine, phenethylamine, dopamine hydrochloride, L-Histidine and L-Tryptophan are pre-...

Embodiment 2

[0032] Example 2: Cloning and sequencing

[0033] A total of 39 aptamer sequences were obtained by cloning and sequencing. Using DNAMAN, Mfold software analysis, according to its primary structure, secondary structure, etc., it is divided into 7 families, and representative sequences are selected from each family, and a total of 8 candidate aptamers are selected for characterization analyze. The 8 sequences were marked as HIM-1, HIM-2, HIM-3, HIM-4, HIM-5, HIM-6, HIM-7, HIM-8.

Embodiment 3

[0034] Example 3: Affinity Analysis

[0035] The above 8 candidate aptamers were synthesized and their 5' ends were biotinylated, diluted with TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.4) to prepare a 10μM solution, and stored at -20°C for future use. Affinity, specificity, pH stability determination steps are as follows;

[0036] First, add 200 μL of avidin to the microwell plate, and coat overnight at 4°C; wash the plate, add a certain concentration of BSA solution to block at 37°C, 150rpm for 2 hours; wash the plate, biotinylated complementary chain at an appropriate concentration at 37 Bind at 150 rpm for 90 min at ℃; wash the plate and add aptamer to it (experimental group aptamer and target were pre-incubated at 37 °C at 150 rpm for 1 h, blank group with binding buffer (50 mM Tris-HCl, 5 mM KCl , 100mM NaCl, 1mM MgCl 2 , pH7.4) instead of the target) and incubated at 37°C, 150rpm for 90min; wash the plate, and bind avidin-HRP for 1h at 37°C, 150rpm. Wash the plate, add...

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Abstract

The invention discloses an oligonucleotide aptamer for specifically recognizing histamine. the nucleotide sequence of the aptamer HIM-3 is SEQ ID NO.1; the terminals 5' and 3' of the aptamer mark oneor more of FAM, a mercapto group, FITC and biotin for analysis and detection of micromolecular histamines. The aptamer of the invention can be screened in vitro, has a short screening period, is convenient to synthesize, easily marks various functional groups and reporter molecules, has stable properties and can be stored and used for a long time.

Description

technical field [0001] The present invention relates to the field of food safety biotechnology, in particular to an oligonucleotide aptamer that can specifically recognize the small molecular compound histamine by using the SELEX technology in the molecular biology technology, and provide the aptamer for the nucleic acid aptamer. The application of ligands in the detection of histamine in food provides scientific basis and theoretical basis. Background technique [0002] Biogenic amines are a class of low-molecular nitrogen-containing organic compounds formed by the amination of aldehydes and ketones or the decarboxylation of amino acids. There are many types of biogenic amines, including putrescine, cadaverine, spermine, spermidine, tyramine, phenylethylamine, histamine, tryptamine, etc. At present, in terms of the toxic effects of biogenic amines on the human body that appear in food safety issues, histamine has the greatest impact and is the most toxic. Slight poisoning...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/5308
Inventor 段诺朱肖音吴世嘉王周平齐硕
Owner JIANGNAN UNIV
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