Single-stranded DNA aptamer specifically recognizing tobramycin and application thereof

A tobramycin and aptamer technology, applied in the field of single-stranded DNA aptamers, can solve the problems of affinity, sensitivity reduction, affinity reduction, destruction of secondary structure, etc., and achieve high sensitivity, high affinity and strong specificity Effect

Active Publication Date: 2018-11-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the process of truncation, the secondary structure of the original sequence is often destroyed, and the affinity and sensitivity will be reduced.
[0004] The tobramycin aptamer ap32 with a length of 79 nucleotides was screened by magnetic beads SELEX in the previous research of our laboratory. Starting from the aptamer ap32, the sequence of ap32 was truncated through secondary structure analysis and rational design Optimized to obtain an aptamer ap32-34nt with a length of only 34 nucleotides, but the affinity of the aptamer ap32-34nt was reduced

Method used

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  • Single-stranded DNA aptamer specifically recognizing tobramycin and application thereof
  • Single-stranded DNA aptamer specifically recognizing tobramycin and application thereof
  • Single-stranded DNA aptamer specifically recognizing tobramycin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Tobramycin ssDNA aptamer truncation

[0025] The aptamer ap32-34nt (Kd=58.92nmol·L) obtained in previous research in the laboratory -1 , its nucleotide sequence is shown in SEQ ID NO.4, which is 5'-CGTCGACGGATCCATGGCACGTTATAGGTCGACG-3') on the basis of the secondary structure (for the aptamer ap32-34nt, refer to the large paper "Tobramycin-specific single-stranded DNA Screening of aptamers and their sequence optimization and application research" Zhang Yuhong, Jiangnan University), truncated and optimized, and obtained several sets of aptamer sequences, as shown in Table 1:

[0026] Table 1 Aptamers and corresponding nucleotide sequences

[0027] Aptamer

Nucleotide sequence (SEQ ID NO.1~3)

ap32-15nt

5'-GACTAGGCACTAGTC-3'

ap32-13nt

5'-GACGGGCACCGTC-3'

ap32-15nt-2

5'-GACGTGGCACACGTC-3'

[0028] To analyze the stem-loop structure, the dissociation constant (K d ) determination, the specific steps are as follo...

Embodiment 2

[0042] Example 2: Aptamer ap32-15nt electrochemical method to detect tobramycin

[0043] The designed hairpin structure sequence is (SEQ ID NO.5): 5'-AAAAAAGACTAGGCACTAGTCAAAAAAACCCCCGATCCTAGTCTTTCCC-3'; the italic part is the truncated aptamer sequence.

[0044] The designed signal transduction probe sequence is (SEQ ID NO.6): 5'-GCGAAAAAAGCG-(CH 2 ) 6 -HS-3', the 3' end of the probe is modified with a sulfhydryl group to self-assemble onto the gold electrode surface.

[0045] The designed primer sequence is (SQE ID NO.7): 5'-AAAGACTAGGA-3'

[0046] (1) Sequence design of hairpin structure (Hp), signal transduction probe, and primer; and preparation concentrations were 10 μmol L -1 , 1 μmol L -1 , 10 μmol L -1 .

[0047] (2) Trichlorohexaammine ruthenium ([Ru(NH 3 ) 6 ] 3+ ) solution: use 10mmol·L -1 Tris-HCl prepared at a concentration of 10 μmol L -1 .

[0048] (3) The hairpin structure (Hp) in step (1) was heated at 95° C. for 5 minutes and placed in a hot wate...

Embodiment 3

[0056] Embodiment 3: the detection of tobramycin by gold gel colorimetric kit

[0057] (1) Solution A: AuNPs were prepared by trisodium citrate reduction method and concentrated 5 times;

[0058] (2) Solution B: tobramycin aptamer ap32-15nt, prepared at a concentration of 150nmol L -1 ;

[0059] (3) Liquid C: NaCl solution, the preparation concentration is 120mmol L -1 ;

[0060] (4) Take 50 μL of solution A in step (1) and 100 μL of solution B in step (2) and react for 1 hour at room temperature in the dark;

[0061] (5) Add different concentrations of tobramycin to step (4) successively, and react in the dark for 50 minutes;

[0062] (6) Add 50 μL of solution C in step (3) to the solution obtained in step (5), observe the color change of each centrifuge tube, perform spectral characterization with a spectrophotometer, and draw a graph of the relationship between the absorbance of the AuNPs solution and the concentration of tobramycin.

[0063] In the presence of differe...

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Abstract

The invention discloses a single-stranded DNA aptamer specifically recognizing tobramycin and application thereof, and belongs to the technical field of biology. On the basis of ap32-34nt, by removingsome of terminal base pairs and unpaired bases and shortening the stem region length of a secondary structure, a tobramycin aptamer ap32-15nt with the greatly shortened sequence length and the improved affinity is obtained; the aptamer ap32-15nt is applied to construction of various tobramycin detection methods and detection reagents; the aptamer has the advantages of high affinity, high specificity, a stable structure and the like; and the constructed detection methods have the advantages of a short detection period, high sensitivity, low cost, strong specificity and the like.

Description

technical field [0001] The invention relates to a single-stranded DNA aptamer specifically recognizing tobramycin and its application, belonging to the field of biotechnology. Background technique [0002] Tobramycin belongs to aminoglycoside antibiotics and is a kind of broad-spectrum antibacterial drug. It has good antibacterial effect and is suitable for treating various infection symptoms of respiratory, digestive, urinary and reproductive systems. It is widely used in animals and aquaculture. Since tobramycin is a shared drug for humans and animals, in addition to direct damage to the human body due to its toxic and side effects, tobramycin residues in food and the environment will eventually affect human health and safety. Traditional antibiotic residue detection methods include microbial detection and instrumental analysis, including gas chromatography (GC), high performance liquid chromatography (HPLC), liquid-mass spectrometry (HPLC-MS), liquid chromatography-tandem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115C12N2310/16G01N33/53
Inventor 周楠迪聂晶晶张玉红韩旭艳田亚平
Owner JIANGNAN UNIV
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