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ssDNA aptamer for specifically identifying metronidazole and application thereof

A metronidazole and aptamer technology, applied in the field of ssDNA aptamers, can solve the problems of high price, non-immunogenicity, production and application limitations, etc.

Active Publication Date: 2020-01-14
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are some ELISA kit products for the detection of metronidazole residues on the market, but the price is expensive, and the types of enzyme-labeled antibodies are limited, and because metronidazole is a small molecule compound, it is not immunogenic itself, and production and application problems exist. More restrictions
These have largely limited the application of immunoassay methods in the detection of metronidazole

Method used

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  • ssDNA aptamer for specifically identifying metronidazole and application thereof
  • ssDNA aptamer for specifically identifying metronidazole and application thereof
  • ssDNA aptamer for specifically identifying metronidazole and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1: Construction of random ssDNA library and its primers

[0069] (a) Construct a random ssDNA library with a length of 79 bases:

[0070] 5'-TAGGGAATTC GTCGACGGAT CC-N35-CTGCAGGTCG ACGCATGCGC CG-3', wherein N represents any one of the bases A, T, C, G.

[0071] (b) Synthesize the forward primer:

[0072] Forward primer 1: 5′-TAGGGAATTC GTCGACGGAT-3′;

[0073] Forward primer 2: 5′-FAM-TAGGGAATTC GTCGACGGAT-3′;

[0074] (c) Synthetic reverse primer:

[0075] Reverse primer 1: 5'-CGGCGCATGC GTCGACCTG-3';

[0076] Reverse primer 2: 5'-biotin-CGGCGCATGCGTCGACCTG-3'.

Embodiment 2

[0077] Example 2: In vitro screening of nucleic acid aptamers

[0078] In order to screen out ssDNA aptamers with high affinity and specificity to metronidazole, a total of 10 rounds of nucleic acid aptamers were screened.

[0079] (a) The 25 μL PCR amplification system is shown in Table 1.

[0080] Table 1

[0081]

[0082] Amplification conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s; annealing at 55°C for 30 s; extension at 72°C for 15 s; extension at 72°C for 5 min; 29 cycles.

[0083] (b) Main steps of in vitro screening: After washing the streptavidin-coupled magnetic beads with PBS buffer for 5 times, dissolve 200 μL of the initial library PCR product in 200 μL of binding buffer, and then add streptavidin The magnetic beads coupled with protein were gently shaken at room temperature for 2 h, then placed on a magnetic separator, the supernatant was removed, and the magnetic beads were washed 5 times with binding buffer, and then 300 mmo...

Embodiment 3

[0086] Example 3: Screened ssDNA clones and sequencing

[0087] ssDNA clone sequencing: The ssDNA obtained after the final round of screening was amplified by PCR with forward primer 1 and reverse primer 1, and the entire amount of the amplified product was loaded on 3% agarose, and the PCR product was recovered. The purified PCR product was ligated with the pMD 18-T Vector vector according to the instructions of the T vector, and after overnight ligation at 16°C, it was transformed into Escherichia coli JM109 and cultured overnight. The correct transformants were verified by colony PCR and agarose gel, 39 positive clones were picked and their plasmids were extracted for sequence determination, and 39 aptamers with different sequences from ap1 to ap39 were obtained by sequencing.

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Abstract

A ssDNA aptamer for specifically identifying metronidazole and an application thereof belongs to the fields of biochemistry, molecular biology, analytical chemistry and combinatorial chemistry. In thepresent invention, a ssDNA initial library containing 35 random nucleotides is amplified by PCR through a magnetic beads-SELEX technology, and then immobilized on magnetic beads modified with streptavidin, added with metronidazole to separate ssDNAs with affinity by competition, and amplified for next screening. After ten rounds of screening and clone sequencing, 39 metronidazole aptamer sequences are obtained. After further affinity evaluation, four sequences with higher affinity including ap2, ap19, ap21, and ap32 are selected, wherein, the ap32 is selected as an optimal metronidazole aptamer due to the advantages of high affinity, high specificity and stable structure. The invention provides an identification element with excellent performance for metronidazole detection.

Description

technical field [0001] The invention relates to a ssDNA aptamer specifically recognizing metronidazole and an application thereof, belonging to the fields of biochemistry and molecular biology, analytical chemistry and combinatorial chemistry. Background technique [0002] Metronidazole belongs to nitroimidazole antibiotics and is a synthetic antibacterial and antiprotozoal drug with the basic structure of 5-nitroimidazole. This drug can also be used as a feed drug additive for poultry, livestock and aquaculture, and is widely used to prevent and control of bee microsporidia in beehives. However, studies have shown that metronidazole has potential risks of teratogenicity, carcinogenicity, mutagenesis and genotoxicity. Improper use of metronidazole may lead to drug residues in edible animal tissues and even pollute water sources. In 1999, the Ministry of Agriculture of my country issued Document No. 17 "Maximum Residue Limits of Veterinary Drugs in Animal Foods", which stipu...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/53
CPCC12N15/115G01N33/9446C12N2310/16C12N2330/31Y02A50/30
Inventor 周楠迪魏昊田亚平
Owner JIANGNAN UNIV
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