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Method for detecting ochratoxin A through fluorescence anisotropy technology

An ochratoxin and anisotropic technology, applied in the field of detection of ochratoxin A, can solve the problems of high antibody preparation cost, poor antibody stability, cumbersome and time-consuming operation, etc., and achieve easy label introduction and high-throughput analysis , convenient storage and transportation

Active Publication Date: 2019-08-13
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 requires the use of immune antibodies to identify ochratoxin A. Immunoassay sensing is relatively fast, but the cost of antibody preparation is high, the stability of the antibody is not good, and it is easy to inactivate

Method used

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  • Method for detecting ochratoxin A through fluorescence anisotropy technology
  • Method for detecting ochratoxin A through fluorescence anisotropy technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Embodiment 1, the preparation of nucleic acid aptamer and complementary nucleic acid sequence

[0059] 1. Preparation of nucleic acid aptamers

[0060] A nucleic acid aptamer (as shown in sequence 1 of the sequence listing) that can specifically bind ochratoxin A was artificially synthesized, and the 5' end of the nucleic acid aptamer was labeled with fluorescein (FAM).

[0061] 2. Screening and preparation of complementary nucleic acid sequences

[0062] 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:

[0063] Ⅰ: 5'-CCA CCC ACA CCC GAT C-3' (sequence 2 of the sequence listing);

[0064] Ⅱ: 5'-CAC CCA CAC CCG ATC-3' (sequence 3 of the sequence listing);

[0065] Ⅲ: 5'-ACC CAC ACC CGA TC-3' (sequence 4 of the sequence listing);

[0066] Ⅳ: 5'-CCC ACA CCC GAT C-3' (sequence 5 of the sequence listing);

[0067] V: 5'-CCA CAC CCG ATC-3' (sequence 6 of the sequ...

Embodiment 2

[0074] Example 2. Establishment of a method for detecting ochratoxin A using fluorescein-labeled nucleic acid aptamers and streptavidin-labeled single-stranded DNA molecules

[0075] 1. Mix the FAM-labeled aptamer prepared in Example 1, the streptavidin-labeled single-stranded DNA molecule and ochratoxin A in 100 μl of reaction buffer solution, and incubate at 25° C. for 10 minutes. The concentration of the FAM-labeled nucleic acid aptamer in the reaction system is 5nM, the concentration of the streptavidin-labeled single-stranded DNA molecule in the reaction system is 10nM, and the ochratoxin A in the reaction system is set at different concentrations (per Each concentration set 2 replicates). 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 with a multi-functional microplate reader (Synergy H1 Microplate reader, Biotek, USA), the ...

Embodiment 3

[0080] Embodiment 3, the impact of streptavidin labeling on detection sensitivity

[0081] 1. The FAM-labeled nucleic acid aptamer prepared in Example 1, only the single-stranded DNA molecule shown in sequence 5 labeled with biotin (not labeled with streptavidin) was reacted with ochratoxin A in 100 μl Mix in buffer solution and incubate at 25°C for 10 min. The concentration of the FAM-labeled nucleic acid aptamer in the reaction system was 5 nM, and the concentration of only biotin-labeled single-stranded DNA molecules (not labeled with streptavidin) in the reaction system was 10 nM. The concentration of Aspergillus toxin A was 200 nM. At the same time, set a blank control without adding the sample to be tested.

[0082] 2. After the reaction in step 1, the fluorescence anisotropy (fluorescence polarization) value was measured with a multi-functional microplate reader (Synergy H1 Microplate reader, Biotek, USA), the excitation wavelength was 485nm, and the emission waveleng...

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Abstract

The invention discloses a method for detecting ochratoxin A through a fluorescence anisotropy technology. According to the method, an aptamer capable of being specifically combined with the ochratoxinA is selected and marked with a fluorochrome; a single-stranded DNA molecule which is complementary to the aptamer is designed for the aptamer, and marked with streptavidin; after the aptamer is combined with the single-stranded DNA molecule, the fluorochrome and the streptavidin are adjacent to each other in space; the prepared aptamer and the prepared single-stranded DNA molecule react with a to-be-tested sample, and by measuring the fluorescence anisotropy value of the reaction system, the ochratoxin A in the to-be-tested sample is detected. The built method has the advantages that the method is sensitive, simple, quick and good in reproducibility, high-throughput analysis is easy to achieve, and the use quantity of the sample is small; the adopted materials are easy to prepare, the synthesis cost is low, the stability is high, storage and transportation are convenient, the shelf life is long, and the sensitivity and selectivity are high.

Description

technical field [0001] The invention relates to a method for detecting ochratoxin A by using fluorescence anisotropy technology. Background technique [0002] Ochratoxin A (ochratoxin A, abbreviated as OTA) is a secondary metabolite produced by several Penicillium fungi such as Ochratoxin, which is listed as a 2B carcinogen by the International Agency for Research on Cancer. Cereals (wheat, corn, etc.), grains, grapes, coffee, etc. are often contaminated by OTA. Ingestion of OTA-contaminated food poses a serious threat to animal and human health. Sensitive and rapid detection of OTA is of great significance and demand in food safety, quality control, environmental analysis, especially on-site analysis. [0003] Commonly used methods for detecting ochratoxin A mainly include chromatography, chromatography-mass spectrometry, mass spectrometry, and immunoassay sensing. Chromatography and mass spectrometry often require expensive instruments and equipment, and the operation i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6816
CPCC12Q1/6816C12Q2561/119
Inventor 赵强李亚飘
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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