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Method for analyzing aflatoxin B1 by fluorescence anisotropy of sensitive aptamer

A kind of aflatoxin and anisotropic technology, applied in the direction of material analysis, material analysis, fluorescence/phosphorescence, etc. through optical means, can solve the problems of poor antibody stability, high antibody preparation cost, cumbersome and time-consuming operation, etc., to achieve Ease of high-throughput analysis, easy introduction of labels, convenient storage and transportation

Active Publication Date: 2019-08-06
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

Chromatography and mass spectrometry often require expensive instruments and equipment, and the operation is cumbersome and time-consuming
Immunoassay needs to use immune antibody to identify AFB1. Immunoassay sensing is relatively fast, but the preparation cost of antibody is high, the stability of antibody is not good, and it is easy to inactivate

Method used

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  • Method for analyzing aflatoxin B1 by fluorescence anisotropy of sensitive aptamer
  • Method for analyzing aflatoxin B1 by fluorescence anisotropy of sensitive aptamer
  • Method for analyzing aflatoxin B1 by fluorescence anisotropy of sensitive aptamer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Embodiment 1, the preparation of nucleic acid aptamer and complementary nucleic acid sequence

[0055] 1. Preparation of nucleic acid aptamers

[0056] A nucleic acid aptamer that can specifically bind aflatoxin B1 (as shown in sequence 1 in the sequence listing) is artificially synthesized, and the 5' end of the nucleic acid aptamer is labeled with fluorescein (FAM).

[0057] 2. Screening and preparation of complementary nucleic acid sequences

[0058] 1. For the nucleic acid aptamer prepared in step 1, design the following single-stranded DNA molecules that can complementarily bind to the nucleic acid aptamer:

[0059] Ⅰ: 5'-AGA GAC AAC ACG TGC A-3' (sequence 2 of the sequence listing);

[0060] Ⅱ: 5'-GAG ACA ACA CGT GCA-3' (sequence 3 of the sequence listing);

[0061] Ⅲ: 5'-AGA CAA CAC GTG CA-3' (sequence 4 of the sequence listing);

[0062] Ⅳ: 5'-GAC AAC ACG TGC A-3' (sequence 5 of the sequence listing);

[0063] V: 5'-ACA ACA CGT GCA-3' (sequence 6 of the seq...

Embodiment 2

[0068] Example 2. Establishment of a method for detecting aflatoxin B1 using fluorescein-labeled nucleic acid aptamers and streptavidin-labeled single-stranded DNA molecules

[0069] 1. Mix the FAM-labeled nucleic acid aptamer prepared in Example 1, the single-stranded DNA molecule shown in sequence 4 marked by streptavidin, and aflatoxin B1 in 100 μl of reaction buffer solution, and incubate at 4°C for 60 min . The concentration of the FAM-labeled nucleic acid aptamer in the reaction system is 1nM, the concentration of the streptavidin-labeled single-stranded DNA molecule in the reaction system is 20nM, and the aflatoxin B1 in the reaction system is set at different concentrations (each Concentration set 2 replicates). At the same time, set a blank control without adding the sample to be tested.

[0070] 2. After the reaction in step 1, the fluorescence anisotropy (fluorescence polarization) value was measured using a multifunctional microplate reader (Synergy H1 Microplate...

Embodiment 3

[0074] Embodiment 3, the impact of streptavidin labeling on detection sensitivity

[0075] 1. The FAM-labeled nucleic acid aptamer prepared in Example 1, only the single-stranded DNA molecule (not labeled with streptavidin) shown in sequence 4 using biotin-labeled reaction with aflatoxin B1 in 100 μl Mix in the buffer solution and incubate at 4°C for 60 min. The concentration of FAM-labeled nucleic acid aptamer in the reaction system was 1nM, and the concentration of only biotin-labeled single-stranded DNA molecules (without streptavidin labeling) in the reaction system was 20nM. Yellow in the reaction system Different concentrations of Aspergillus toxin B1 were set (two replicates were set for each concentration). At the same time, set a blank control without adding the sample to be tested.

[0076] 2. After the reaction in step 1, the fluorescence anisotropy (fluorescence polarization) value was measured using a multifunctional microplate reader (Synergy H1 Microplate read...

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Abstract

The invention discloses a method for analyzing aflatoxin B1 by fluorescence anisotropy of a sensitive aptamer. The method protected by the invention comprises the steps of: subjecting the aptamer marked by means of a fluorochrome, single-chain DNA molecules marked by means of streptavidin and a to-be-tested sample to co-reaction, and realizing detection of aflatoxin B1 in the to-be-tested sample through determining a fluorescence anisotropic value of a reaction system, wherein the aptamer can be specifically bound to the aflatoxin B1, and the single-chain DNA molecules can form a double-chainstructure with the aptamer through reverse complementation; and placing the fluorochrome and the streptavidin on the same end of the double-chain structure after the single-chain DNA molecules form the double-chain structure with the aptamer through reverse complementation, wherein the fluorochrome and the streptavidin are closed to each other spatially. The method has the advantages of being sensitive, simple and rapid, having good repeatability, being easy in high-throughput analysis, having low sample consumption and the like, the materials used are easy to prepare, the synthesis cost is low, the stability is good, the storage and transportation are convenient, the life of the shelf is long, and the method has high sensitivity and good selectivity.

Description

technical field [0001] The invention relates to a method for analyzing aflatoxin B1 by fluorescence anisotropy of a sensitive aptamer. Background technique [0002] Aflatoxin B1 (aflatoxin B1, AFB1) is a highly toxic mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus. Grain, feed, fruit, and dried fruit are easily contaminated by aflatoxin B1. , pose a great threat to human and animal health. AFB1 induces mutations, suppresses immunity, and causes diseases such as cancer. AFB1 is listed as a Group 1 carcinogen by the Cancer Research Institute of the World Health Organization. Many countries in the world have put forward strict limit standards for the content of aflatoxin B1 in food. Sensitive and rapid detection of AFB1 has a wide range of needs in food safety analysis, environmental analysis, quality control, import and export trade and other fields. Commonly used methods for the detection of aflatoxin B1 mainly include chromatography, chromatography-m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64C12N15/115
CPCC12N15/115C12N2310/16G01N21/6428G01N2021/6439
Inventor 赵强李亚飘
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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