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Method of utilizing aptamer molecular switch to detect aflatoxin B1

An aflatoxin and DNA molecule technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of variability, poor stability, complex and expensive preparation of immune antibodies, and achieve rapid The effect of sensitive detection

Active Publication Date: 2017-07-04
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

The preparation of immune antibodies is complicated and expensive, with poor stability and easy denaturation

Method used

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  • Method of utilizing aptamer molecular switch to detect aflatoxin B1
  • Method of utilizing aptamer molecular switch to detect aflatoxin B1
  • Method of utilizing aptamer molecular switch to detect aflatoxin B1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1, the preparation of specific probe

[0034] 1. Artificially synthesize the single-stranded DNA molecule (nucleic acid aptamer) shown in Sequence 1 of the Sequence Listing.

[0035] Sequence 1 of the Sequence Listing: 5'-CGTGTTGTCTCTCTGTGTCTCG-3'.

[0036] 2. Label the fluorescent group FAM at the 5' end of the nucleic acid aptamer prepared in step 1, and label the quencher group BHQ1 at the 3' end to obtain a specific probe.

Embodiment 2

[0037] Example 2. Detection of Aflatoxin B1 Using Nucleic Aptamer Molecular Switch

[0038] Mix the specific probe, buffer solution and aflatoxin B1 prepared in Example 1 (in the initial system, the concentration of the probe is 25nM, and the concentration of aflatoxin B1 is shown in figure 1 abscissa), and then incubated at room temperature for 15 minutes, and then detected the change of fluorescence intensity of FAM (excitation wavelength 485 nm, emission wavelength 528 nm).

[0039] Two replicates were set for each concentration, and the results were averaged.

[0040] see results figure 1 . As the concentration of aflatoxin B1 increased, the fluorescence intensity decreased gradually, and the detection limit was 3.9nM.

Embodiment 3

[0041] Example 3. Selective investigation of detection of aflatoxin B1 by nucleic acid aptamer molecular switch

[0042] The specific probe prepared in Example 1, the buffer solution, and the sample to be tested were mixed (in the initial system, the concentration of the probe was 25nM, and a blank control without adding the sample to be tested was set), then incubated at room temperature for 15 minutes, and then The change of fluorescence intensity of FAM was detected (excitation wavelength 485 nm, emission wavelength 528 nm).

[0043] Group a: blank control.

[0044] Group b: the sample to be tested is aflatoxin B1, and the concentration of aflatoxin B1 in the initial system is 200 nM.

[0045] Group c: the sample to be tested is ochratoxin A, and the concentration of ochratoxin A in the initial system is 1 μM.

[0046] Group d: the sample to be tested is ochratoxin B, and the concentration of ochratoxin B in the initial system is 1 μM.

[0047] Group e: the sample to be ...

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Abstract

The invention discloses a method of utilizing an aptamer molecular switch to detect aflatoxin B1. At first, protection of an aptamer is claimed, and the sequence of the aptamer is represented by the sequence 1 in the sequence table. Then protection of a probe is claim. The probe is obtained by labeling a fluorescent group (FAM) on the 5' terminal of a single chain DNA molecule and labeling a quenching group (BHQ1) on the 3' terminal of the single chain DNA molecule. Protection of a method for detecting aflatoxin B1 is also claimed. The method comprises a step of co-incubating any one of abovementioned probes with a sample to be tested, and if the fluorescent strength is reduced after co-incubation, the sample contains aflatoxin B1. The method has the advantages that aptamer is taken as an affinity ligand and is used to detect aflatoxin B1, the advantages of aptamer can be fully exerted; the provided aptamer molecular switch can generate respond of fluorescence reduction, and the rapid and sensitive detection of aflatoxin B1 is realized therefore.

Description

technical field [0001] The invention relates to a method for detecting aflatoxin B1 by using an aptamer molecular switch. Background technique [0002] Aflatoxins are a class of highly toxic mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, and are an important class of natural pollutants. Among several aflatoxins, aflatoxin B1 is the most toxic and has hepatocarcinogenic effects on humans and animals, and is a Class 1 carcinogen. Ingestion and exposure of aflatoxins in animals and humans through consumption of aflatoxin-contaminated food can have important health effects. Sensitive and rapid detection of aflatoxins is of great significance in food safety, quality control, environmental analysis, etc., and has a wide range of needs. [0003] At present, the commonly used analytical methods for the detection of aflatoxin mainly include chromatography, mass spectrometry, immunoassay sensing and other methods. Chromatography and mass spectrometry require...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/11C12Q1/68
CPCC12N15/115C12N2310/16C12Q1/6816C12Q2525/205C12Q2563/107
Inventor 赵强王超
Owner RES CENT FOR ECO ENVIRONMENTAL SCI THE CHINESE ACAD OF SCI
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