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Aeromonas hydrophila aptamer, and screening method and application thereof

A technology of Aeromonas hydrophila and aptamer, applied in the field of Aeromonas hydrophila aptamer and its screening, can solve the problems of time-consuming, high cost and cumbersome detection process

Inactive Publication Date: 2013-12-25
JIMEI UNIV
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

At present, the detection of Aeromonas hydrophila has always been a thorny problem. The traditional chemical pathological detection process is cumbersome and time-consuming; enzyme-linked immunosorbent immunoassay and multiplex PCR technology are too expensive (2. Wang Li. Fish Aeromonas hydrophila Several detection methods of bacteria [J]. Inland Fisheries, 2006, (2): 22-23.)

Method used

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  • Aeromonas hydrophila aptamer, and screening method and application thereof
  • Aeromonas hydrophila aptamer, and screening method and application thereof
  • Aeromonas hydrophila aptamer, and screening method and application thereof

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Embodiment Construction

[0023] 1. Synthesis of ssDNA library and primers

[0024] Referring to the method disclosed in the literature (7. Liu Fengwei, Lan Xiaopeng. In vitro screening of Pseudomonas aeruginosa aptamer research and preliminary application [D]. Fuzhou Fujian Medical University, 2007.), the random ssDNA library used for SELEX screening and primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0025] The library used was: 5'-GGG AGC TCA GAA TAA ACG CTC AA-N35-TT CGA CAT GAG GCC CGG ATC-3'.

[0026] The primers used are:

[0027] Primer Ⅰ: 5′-GGG AGC TCA GAA TAA ACG CTC AA-3′;

[0028] Primer II: 5′-GAT CCG GGC CTC ATG TCG AA-3′;

[0029] Primer Ⅲ: 5′-Digoxigenin-GGG AGC TCA GAA TAA ACG CTC AA-3′;

[0030] Primer IV: 5'-biotin-GAT CCG GGC CTC ATG TCG AA-3'.

[0031] After the above primers were synthesized, they were diluted with TE buffer (pH 8.0) commonly used in molecular biology to a concentration of 10 μmol, and stored at -20°C for future use....

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Abstract

The invention relates to an aeromonas hydrophila aptamer, and a screening method and application thereof, and provides an aeromonas hydrophila aptamer, and a screening method and application of the aeromonas hydrophila aptamer. The screening method of the aeromonas hydrophila aptamer specifically comprises the following steps: constructing and synthesizing ssDNA libraries of a to-be-screened aeromonas hydrophila aptamer and designing and synthesizing a corresponding primer; taking a proper amount of ssDNA libraries to be combined with aeromonas hydrophila, separating obtained ssDNA which is combined with the aeromonas hydrophila, performing PCT amplification by taking the ssDNA as a template, and performing SELEX screening on an amplification product again until obtaining a corresponding aptamer. According to the invention, the SELEX technology is adopted to screen out a high-appetency oligonucleotide aptamer of pathogenetic aeromonas hydrophila, and the aptamer can be widely applied to detection and analysis of aeromonas hydrophila.

Description

technical field [0001] The invention relates to common pathogenic bacteria in aquaculture—Aeromonas hydrophila, in particular to the Aeromonas hydrophila aptamer and its screening method and application. Background technique [0002] Aeromonas hydrophila ( Aeromonas hydrophila) Widely distributed in nature, it is the primary pathogenic bacterium of various aquatic animals and can produce a variety of pathogenic factors. It is pathogenic to aquatic animals, livestock and humans, and can cause sepsis in various animals and human diarrhea. , has brought threats to human health, and also caused relatively large economic losses to the breeding industry (1. Zhang Cuijuan, Yu Zhouliang, Zhao Baohua, He Hongxuan. Research progress on Aeromonas hydrophila [J]. China Veterinary Journal, 2008 , 42(7):46-50.). Therefore, for the pollution of Aeromonas hydrophila, which is an indicator bacterium of water source pollution, the detection strength still needs to be further strengthened. ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/68C12Q1/04C12R1/01
Inventor 李元跃王雷段博文陈融斌黎中宝
Owner JIMEI UNIV
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