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Method for detecting deoxynivalenol by using GelRed-based non-labeled nucleic acid aptamer sensor and application thereof

A technology of nucleic acid aptamer and deoxynivalenol, which is applied in biochemical equipment and methods, microbial measurement/inspection, instruments, etc., can solve the problems of complicated operation and easy inactivation of antibodies, and achieve simple operation, avoid antibody cost, low cost effect

Inactive Publication Date: 2021-08-24
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the deficiencies of the prior art, the present invention provides a method and application for detecting vomitoxin based on a non-labeled nucleic acid aptamer sensor based on GelRed, which can detect vomitoxin economically, quickly, accurately and sensitively, and can effectively avoid existing detection operations Complicated, or problems such as easy inactivation of antibodies

Method used

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  • Method for detecting deoxynivalenol by using GelRed-based non-labeled nucleic acid aptamer sensor and application thereof
  • Method for detecting deoxynivalenol by using GelRed-based non-labeled nucleic acid aptamer sensor and application thereof
  • Method for detecting deoxynivalenol by using GelRed-based non-labeled nucleic acid aptamer sensor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A method for detecting vomitoxin based on the dye GelRed non-labeled nucleic acid aptamer sensor, the specific steps are as follows:

[0033] (1) Mix 5 μmol / L deoxynivalenol aptamer with the sample to be tested (containing different concentrations of deoxynivalenol standards), mix and incubate at room temperature for 16 min.

[0034] The sequence of the DON aptamer is shown in SEQ ID NO.1:

[0035] 5'-GACATATTCAGTCTGACAGCGCGCTCACAATAAAAGGTCTTGGATGAGGGAAAGCATCCCGGATGGACGAATATCGTCTAGC-3';

[0036] (2) Then add 5 μmol / L deoxynivalenol aptamer complementary chain, and hybridize at room temperature for 10 minutes.

[0037] The sequence of the complementary strand of the DON aptamer is shown in SEQ ID NO.2:

[0038] 5'-GCTAGACGATATTCGTCCATCCGGGATGCTTTTCCCTCATCCAAGACCTTTTATTGTGAGCGCGCTGTCAGACTGAATATGTC-3'.

[0039] (3) Finally, add 2×GelRed dye, mix and react for 6 minutes, and detect the fluorescence intensity on the machine.

[0040] (4) When there is no deoxynivalenol i...

Embodiment 2

[0042] Example 2: Application of Detecting Other Mycotoxins

[0043] Select other mycotoxins such as aflatoxin (AFB1) and zearalenone (ZEN), and measure these samples according to the detection method of Example 1, and observe the change of fluorescence signal. The result is as image 3 As shown, compared with the fluorescent signals caused by the three toxins, only vomitoxin (DON) caused the most obvious change in the fluorescent signal. This result proves that the method based on aptamer-free fluorescence assay for the detection of DON is highly specific and specific.

Embodiment 3

[0044] Embodiment 3: the application of detection wheat spiked sample

[0045]First, use a pulverizer to grind an appropriate amount of wheat seeds into powder. Then, 0.1 g of wheat sample, 0.02 g of PEG8000 and 1 mL of methanol were mixed in 25 mL of distilled water for 2 min. The mixture was centrifuged at 8000r / min for 15min. The supernatant was filtered with a 0.22 μm filter membrane, and the filtrate was concentrated in a 25 ml spinner bottle until no solution remained within 20 minutes, and dissolved in double distilled water to a final volume of 1 ml. According to the detection method of Example 1, the spiked sample of the wheat to be tested was measured, and the change of the fluorescent signal was observed. The results are shown in Table 1 below. Using this method to analyze wheat spiked samples containing different concentrations of vomitoxin (40, 60, 100 μmol / L), the average recovery rate of vomitoxin was between 82.7% and 122%, as Figure 4 shown. The results p...

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Abstract

Deoxynivalenol (DON) is a mycotoxin mainly generated by fusarium graminearum and fusarium oxysporum and widely exists in food crops such as wheat. The deoxynivalenol not only pollutes cereals, causes reduction of agricultural production, but also causes serious threats to human health. A non-labeled nucleic acid aptamer and a GelRed dye are combined, and quantitative detection of the deoxynivalenolis realized according to the fluorescence intensity change of the dye. According to the fluorescence analysis method, the detection limit of deoxynivalenol in a wheat sample can reach 1.06 [mu] mol / L, and the detection range is 10-100 [mu] mol / L; The method makes use of the advantages of low cost, high affinity and the like of the aptamer, has good reproducibility and the good accuracy, and the expensive antibody technology can be replaced to detect the deoxynivalenol in the wheat sample.

Description

technical field [0001] The invention belongs to the fields of drug analysis and food safety, and in particular relates to a method and application of a GelRed-based non-labeled nucleic acid aptamer sensor for detecting vomitoxin. Background technique [0002] Deoxynivalenol (DON), a mycotoxin mainly produced by Fusarium graminearum and Fusarium flavum, is widely present in food crops. DON not only poses serious threats to human health, such as loss of appetite, vomiting, and weight loss, but also contaminates grains such as wheat head blight. In view of the harmfulness and widespread existence of DON, the state stipulates that the maximum limit of DON in grains and its products is 1000 μg / kg, so the detection of DON has attracted much attention. [0003] At present, it has been widely used in detection methods of DON, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS), enzyme-linked immunosorbent assays (ELISAs), etc. LC-MS / MS has high sensitivity and accura...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12Q1/6825G01N21/64
CPCG01N33/5308C12Q1/6825G01N21/6402C12Q2563/107C12Q2565/607
Inventor 梅艳珍戴传超吴梅
Owner NANJING NORMAL UNIVERSITY
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