Aptamer molecular pair for detecting aflatoxin B1, kit and detection method thereof

A kind of aflatoxin and molecular pair technology, which is applied in the direction of measuring devices, analysis materials, material excitation analysis, etc., can solve the problems of high storage and transportation conditions of antibody reagents, sensitivity of antibody stability to temperature, high cost of immune antibody preparation, etc. , to achieve significant changes in fluorescence, high fluorescence enhancement multiples, and high detection sensitivity

Active Publication Date: 2017-08-22
RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods often require expensive instruments and complicated procedures
The analysis method using immune antibody needs to use immune antibody as a recognition reagent, and often also needs to use aflatoxin and protein-coupled antigen. The preparation cost of immune antibody is high, the reproducibility between preparation batches is poor, and the stability of antibody is sensitive to temperature. , the conditions for the storage and transportation of antibody reagents are high

Method used

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  • Aptamer molecular pair for detecting aflatoxin B1, kit and detection method thereof
  • Aptamer molecular pair for detecting aflatoxin B1, kit and detection method thereof
  • Aptamer molecular pair for detecting aflatoxin B1, kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Detection of aflatoxin B1 using fluorescein (FAM)-labeled nucleic acid aptamer AF31-5F and BHQ-1-labeled short-chain nucleic acid molecule AF31-C13B or AF31-C14B

[0042] The sequence of the FAM-labeled nucleic acid aptamer AF31-5F is SEQ ID NO.1 (5'-TGGCACGTGTTGTCTCTCTGTGTCTCGTGCC-3'), and the 5' end is marked with FAM. The sequence of the short-chain nucleic acid AF31-C14B marked by BHQ-1 is SEQ ID NO.4 (5'-GACAACACGTGCCA-3'), and the 3' end is marked with BHQ-1. The short-chain nucleic acid AF31-C13B sequence marked by BHQ-1 is SEQ ID NO.5 (5'-ACAACACGTGCCA-3'), and the 3' end is marked with BHQ-1. In binding buffer solution (10mM Tris-HCl (pH 7.5), 50mM MgCl 2 , 50mM NaCl, 0.1% Tween20), AF31-5F (50nM) was incubated with AF31-C14B (100nM) or AF31-C13B (100nM) and aflatoxin B1, and then measured the fluorescence intensity of FAM (excitation wavelength is 485nm, The emission wavelength is 528nm). With the addition of aflatoxin B1, the fluorescence intens...

Embodiment 2

[0043] Example 2: Using fluorescein (FAM)-labeled nucleic acid aptamer AF29-5F and BHQ-1-labeled short-chain nucleic acid molecule A29-C14B or A29-C13B to detect aflatoxin B1

[0044] The sequence of the FAM-labeled aptamer AF29-5F is SEQ ID NO.2 (5'-TGC ACG TGT TGT CTCTCT GTG TCT CGT GCC-3'), and the FAM label is at the 5' end of the sequence. The short-chain nucleic acid AF29-C14B sequence marked by BHQ-1 is SEQ ID NO.6 (5'-AGA CAA CAC GTG CA-3'), and the quencher BHQ-1 is marked at the 3' end of the sequence. The short-chain nucleic acid AF29-C13B sequence marked by BHQ-1 is SEQ ID NO.7 (5'-GACAACACGTGCA-3'), and the BHQ-1 mark is at the 3' end of the sequence. In binding buffer solution (10mM Tris-HCl (pH 7.5), 50mM MgCl 2 , 50mM NaCl, 0.1% Tween20), AF29-5F (50nM) was incubated with AF29-C14B (100nM) or AF29-C13B (100nM) and aflatoxin B1, and then measured the fluorescence intensity of FAM (excitation wavelength is 485nm, The emission wavelength is 528nm). With the add...

Embodiment 3

[0045] Example 3: Using fluorescein (FAM)-labeled nucleic acid aptamer AF27-5F and BHQ-1-labeled short-chain nucleic acid molecule AF27-C14B to detect aflatoxin B1

[0046] The sequence of the FAM-labeled aptamer AF27-5F is SEQ ID NO.3 (5'-TCACGTGTTGTCTCTCTGTGTCTCGTG-3'), and the FAM label is at the 5' end of the sequence. The sequence of the short-chain nucleic acid A27-C14B marked by BHQ-1 is SEQ ID NO.8 (5'-GAGACAACACGTGA-3'), and the quencher BHQ-1 is marked at the 3' end of the sequence. In binding buffer solution (10mM Tris-HCl (pH 7.5), 50mM MgCl 2 , 50mM NaCl, 0.1% Tween20), AF27-5F (50nM) was incubated with AF27-C14B (100nM) and aflatoxin B1, and then the fluorescence intensity of FAM was measured (excitation wavelength is 485nm, emission wavelength is 528nm). With the addition of aflatoxin B1, the fluorescence intensity gradually increased, and the results were as follows image 3 shown.

[0047] The results of the above examples show that the aptamer molecule pai...

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Abstract

The invention provides an aptamer molecular pair for detecting aflatoxin B1, a kit and a detection method thereof. The aptamer molecular pair is a molecular pair composed of molecules obtained from a 5' terminal of a molecule as shown in SEQ ID NO.1 through FAM marking and a 3' terminal of a molecule as shown in SEQ ID NO.4 or SEQ ID NO.5 through BHQ-1 marking, or a molecular pair composed of molecules obtained from a 5' terminal of a molecule as shown in SEQ ID NO.2 through FAM marking and a 3' terminal of a molecule as shown in SEQ ID NO.6 or SEQ ID NO.7 through BHQ-1 marking, or a molecular pair composed of molecules obtained from a 5' terminal of a molecule as shown in SEQ ID NO.3 through FAM marking and a 3' terminal of a molecule as shown in SEQ ID NO.8 through BHQ-1 marking. The product and method provided by the invention have low cost, high stability and a detection limit of 0.2nM.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to an aptamer molecule pair for detecting aflatoxin B1, a kit and a detection method thereof. Background technique [0002] Aflatoxin is a highly toxic mycotoxin that can contaminate corn, peanuts and other grains, grains, and foods. After animals and humans eat food contaminated by aflatoxin, aflatoxin can cause liver damage, cause cancer, and harm animals and humans. Human health has caused great harm, among which aflatoxin B1 (AFB1) is widely polluted and the most toxic. Detection of aflatoxin B1 is of great significance for food safety, food quality control and human health. Some commonly used detection methods mainly include thin layer chromatography analysis, enzyme-linked immunoassay, liquid chromatography analysis and other methods. These methods often require expensive instruments and complicated procedures. The analysis method using immune antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N2021/6432
Inventor 赵强李亚飘孙琳琳
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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