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Specific nucleic acid aptamer and application

A nucleic acid aptamer and species-specific technology, which can be used in DNA/RNA fragments, measuring devices, instruments, etc., and can solve the problems of long time, cumbersome operation steps, and complicated operations.

Inactive Publication Date: 2017-07-07
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional detection methods of Escherichia coli O157:H7 mainly include selective culture medium separation and biochemical methods, etc. Most of these methods are cumbersome and time-consuming
With the development of science and technology, some new detection methods have appeared, such as PCR-based molecular biology methods, immunological methods, flow cytometry and gene chip methods, etc. These methods also have expensive instruments and difficult operation. Complicated, time-consuming and other disadvantages

Method used

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  • Specific nucleic acid aptamer and application
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  • Specific nucleic acid aptamer and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Screening of nucleic acid aptamers specifically binding to enterohemorrhagic Escherichia coli O157:H7 by whole-bacteria subtractive SELEX technology

[0025] (1) Synthesis of random single-stranded DNA (ssDNA) library and primers

[0026] Random ssDNA library references are as follows:

[0027] 5'-GCAATGGTACGGTACTTCC-N45-CAAAAGTGCACGCTACTTTGCTAA-3', the middle random region is 45 nucleotides, both ends are fixed regions, the total sequence length is 88 nucleotides, and the library capacity is 4 45 . The primer sequences used for screening are:

[0028] Primer Ⅰ: 5'-GCAATGGTACGGTACTTCC-3'

[0029] Primer Ⅱ: 5'-TTAGCAAAGTAGCGTGCACTTTTG-3'

[0030] Primer Ⅲ: 5'-GCTAAGCGGGTGGGACTTCCTAGTCCCACCCGCTTAGCAAAGTAGCGTGCACTTTTG-3'

[0031] Among them, Primer I needs FITC fluorescent group to be modified during the preparation of the secondary library. In the constructed fluorescence detection method, aptamer A46 needs to be modified with biotin.

[0032] The above nucleic aci...

Embodiment 2

[0057] Fluorochemical analysis to monitor the screening process

[0058] (1) Preparation of fluorescent primary library and secondary library

[0059] 1) The initial random ssDNA library labeled with FITC fluorescence was directly synthesized by Shanghai Sangon Biotechnology Co., Ltd.;

[0060] 2) The FITC fluorescently labeled secondary ssDNA library was obtained by gel-cutting recovery and purification experiments, and the specific steps were as follows:

[0061] ①Using the ssDAN-target bacteria obtained in each round of screening as a template, the FITC-labeled Primer Ⅰ synthesized by Sangon as the upstream primer, and other primers and PCR conditions unchanged, refer to the PCR amplification system and PCR amplification system of the first round of screening in Example 1. condition;

[0062] ② Store the amplified product in the dark, and prepare the FITC-labeled ssDNA library according to the steps for preparing the secondary library in Example 1;

[0063] ③The FITC flu...

Embodiment 3

[0071] Cloning and Sequencing

[0072] (1) After 7 rounds of screening, the sixth round of screening product with the highest binding efficiency to the target bacteria was selected as a template, and the upstream primer Primer I and the downstream primer Primer II were used for PCR amplification. The amplification system and conditions were referred to in Example 1. PCR program;

[0073] (2) Connect the PCR product to the cloning vector pMD19-T, select blue and white spots, extract the plasmid after cell culture, and send the plasmid to Shanghai Sangon Biotechnology Co., Ltd. for sequencing;

[0074] (3) Use Chromas and DNAMAN software to perform homology and secondary structure prediction on the sequencing results, and finally obtain the sequence of the nucleic acid aptamer (named A46) specifically binding to Escherichia coli O157:H7 (sequence list 1) and predicted secondary structure diagram (see figure 2 ). Sequence length of aptamer A46 specifically binding to Escheri...

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Abstract

The invention provides sepecific nucleic acid aptamer. The nucleotide sequence of the specific nucleic acid aptamer is a sequence shown in the sequence table <400>1, the nucleic acid aptamer is screened through a whole bacterium reduction SELEX technique, is low in molecular weight and easy to synthesize and modify, can recognize enterohemorrhagic escherichia coli O157:H7 in a high-specific mode and be combined with the enterohemorrhagic escherichia coli O157:H7 in a high-affinity mode, does not specifically combined with other bacteria, can be used for detecting the enterohemorrhagic escherichia coli O157:H7 and has the important significant for effectively preventing and controlling infection of the enterohemorrhagic escherichia coli O157:H7.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a specific nucleic acid aptamer and its application. Background technique [0002] Enterohemorrhagic Escherichia coli O157:H7 (Enterohemorrhagic Escherichia coli, EHEC is a flagellated, non-spore-forming, mobile Gram-negative bacterium, which is a major type of enterohemorrhagic Escherichia coli. Since 1982, it was first introduced in Since the United States was confirmed and isolated, it has spread widely around the world.Escherichia coli O157:H7 can mainly cause human diarrhea, hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura, etc., with a mortality rate of up to 30 %.Escherichia coli O157:H7 is widely separated in nature, and its pathogenicity is stronger after infection, and only dozens of live bacteria can cause gastrointestinal infection.In my country, Escherichia coli O157:H7 is the existing anti-national One of the pathogens that pose a ma...

Claims

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Application Information

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IPC IPC(8): C12N15/115G01N33/569
Inventor 谌志强邱志刚李君文金敏杨栋刘伟丽王景峰江建华
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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