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Method of establishing double-detection sensor for antibiotics based on aptamers

A technology for establishing methods and aptamers, which can be used in biochemical equipment and methods, biological testing, microbial determination/inspection, etc., and can solve the problems of high instrument cost and low detection sensitivity

Pending Publication Date: 2020-10-09
BEIJING UNIV OF CHEM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional detection methods of antibiotics include microbial detection, chromatographic detection and enzyme-linked immunoassay, etc., but these detection methods have certain defects, such as low detection sensitivity or high instrument cost, etc.

Method used

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  • Method of establishing double-detection sensor for antibiotics based on aptamers
  • Method of establishing double-detection sensor for antibiotics based on aptamers
  • Method of establishing double-detection sensor for antibiotics based on aptamers

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Synthesize the following sequence, the shown chloramphenicol nucleic acid aptamer and kanamycin aptamer

[0061] 1.CAP-aptamer:

[0062] Chloramphenicol Aptamer (Apt-CAP):

[0063] 5'-AGCAGCACAGAGGTCAGATGACTTCAGTGAGTTGTCCCACGGTCGGCGAGTCGGTGGTAGCCTATGCGTGCTACCGTGAA-3'

[0064] Kanamycin aptamer (Apt-KANA):

[0065] 5'-CACCTAATACGACTCACTATAGCGGATCCGTGTCCAAGTGGTCTTGAGGTTCTGGCTCGAACAAGCTTGC-3'

[0066] Apt-CAP corresponds to the above quotation (C1): 5'-AGCAGCACAGAGGTCAGATG-3'

[0067] Apt-CAP corresponds to the following quote (C2): 5'-CCTATGCGTGCTACCGTGAA-3'

[0068] Apt-Kana corresponds to the upper citation (K1): 5’-CACCTAATACGACTCACTATA-3’

[0069] Apt-Kana corresponds to the following quotation (K2): 5'-CTGGCTCGAACAAGCTTGC-3'

Embodiment 2

[0070] Embodiment 2: the establishment of antibiotic double detection system

[0071] The buffer used in the experiment:

[0072] F1 buffer solution: Tris (Tris) 10 mM, sodium chloride (NaCl) 100 mM, ethylenediaminetetraacetic acid disodium (EDTA Na2) 1 mM, pH 8.0.

[0073] F2 buffer: Tris (Tris) 20mM, sodium chloride (NaCl) 200mM, ethylenediaminetetraacetic acid disodium (EDTA Na2) 1mM, Triton X-100 0.02%, pH 7.8 .

[0074] 1×B buffer solution: Tris (Tris) 50mM, Magnesium chloride (MgCl2) 1mM, Sodium chloride (NaCl) 200mM, Potassium chloride (KCl) 5mM, Triton X-100) 0.02% , pH 7.4.

[0075] 2×B buffer: Tris (Tris) 100mM, Magnesium chloride (MgCl2) 2mM, Sodium chloride (NaCl) 400mM, Potassium chloride (KCl) 10mM, Triton X-100) 0.02% , pH 7.4.

[0076] 2.1 Dual detector feasibility verification experiment

[0077] 2.1.1 Predict the secondary structure of the two aptamers by mfold simulation respectively, and check the data to determine the sequence of the complementary re...

Embodiment 3

[0093] Embodiment 3. The mensuration of detector linear interval and detection limit

[0094] Add chloramphenicol and antibiotics with gradient final concentration to the reaction system as the experimental group, and add sterile water as the blank group. The standard curve was drawn with the antibiotic concentration as the abscissa and the 2^-ΔΔCt values ​​of the experimental group and the blank group as the ordinate. The standard curve of chloramphenicol concentration and 2^-ΔΔCt value is y=0.0125X+1.0135, correlation coefficient R 2 =0.9867, the linear detection range is from 0ng / ml to 20ng / ml. From 10 groups of blank groups without chloramphenicol, calculate the standard deviation SD of the fluorescence values ​​of 10 groups, according to the standard curve formula y=0.0125X+1.0135 obtained above, the value calculated by 3SD / k is the minimum detection limit . The standard deviation SD of the blank group was 0.047, which was brought into the standard curve, and the minim...

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Abstract

The invention discloses a method of establishing a double-detection sensor for antibiotics based on aptamers, belonging to the field of detection of substances such as antibiotics. The aptamer sensorcapable of simultaneously detecting two antibiotics, namely chloramphenicol and kanamycin, is established, so experiment cost is reduced as much as possible, and rapid and high-sensitivity detection of antibiotic residues is realized. According to the invention, chloramphenicol and kanamycin aptamers are taken as examples; an auxiliary short chain with one end modified by biotin is used for forming a partially-complementary composite chain structure which is fixed on magnetic beads; after target substance antibiotics are added, the aptamers are separated from the magnetic beads into a solutionthrough high affinity between the antibiotics and the aptamers, the content of the corresponding aptamer is determined by using a real-time fluorescent quantitative PCR method, and an aptamer biological detector for joint detection of the antibiotics is established. By means of the high-specificity affinity effect between the antibiotics and the aptamers, the contents of free aptamers obtained through competition are quantitatively analyzed through the q-PCR method, and therefore, the contents of the two antibiotics are obtained.

Description

technical field [0001] The invention belongs to the field of substance detection such as antibiotics, and in particular relates to a method for detecting two kinds of antibiotics, chloramphenicol and kanamycin, in a biosensor based on an aptamer. At present, most of the sensors that use aptamers to detect antibiotics detect a single target substance. This method can provide a new idea for the establishment of dual-detection aptasensors and even multi-detection sensors. Background technique [0002] Chloramphenicol (CAP) is a class of broad-spectrum antibiotics, and the effect is very significant, strong bactericidal. However, because chloramphenicol has serious adverse reactions: inhibition of bone marrow hematopoietic function, causing aplastic anemia, the current Food and Agriculture Organization of the United Nations / World Health Organization Food Additives Expert Committee recommends prohibiting the use of chloramphenicol for food animals. The Ministry of Agriculture of...

Claims

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Application Information

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IPC IPC(8): G01N33/53C12Q1/6851
CPCG01N33/5308C12Q1/6851C12Q2531/113C12Q2525/205C12Q2563/107
Inventor 李灏杜晓彦胡鸿炜丁于敬高子涵
Owner BEIJING UNIV OF CHEM TECH
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