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Aeromonas hydrophila aptamer, and screening method and application thereof

A technology of Aeromonas hydrophila and aptamer, which can be applied in biochemical equipment and methods, DNA/RNA fragments, microorganism determination/inspection, etc., and can solve the problems of high cost, time-consuming, and cumbersome detection process.

Inactive Publication Date: 2013-12-25
JIMEI UNIV
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  • Abstract
  • Description
  • Claims
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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. Research and preliminary application of in vitro screening of Pseudomonas aeruginosa aptamers [D]. Fuzhou Fujian Medical University, 2007.), random ssDNA library 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 were:

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

[0032] 2. ...

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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 Aeromonas hydrophila, a common pathogenic bacteria in aquaculture, in particular to an aptamer of Aeromonas hydrophila and a screening method and application thereof. Background technique [0002] Aeromonas hydrophila ( Aeromonas hydrophila) Widely distributed in nature, it is the primary pathogen of a variety of aquatic animals, can produce a variety of pathogenic factors, is pathogenic to aquatic animals, livestock and humans, and can cause septicemia in a variety of animals and human diarrhea , posing a threat to human health, but also causing great economic losses to the aquaculture industry (1. Zhang Cuijuan, Yu Zhouliang, Zhao Baohua, He Hongxuan. Research progress on Aeromonas hydrophila [J]. Chinese Veterinary Journal, 2008 , 42(7):46-50.). Therefore, the detection of Aeromonas hydrophila, which is an indicator of water pollution, still needs to be further strengthened. At present, the detection of Aeromonas hydrophi...

Claims

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Application Information

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