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ssDNA nucleic acid aptamer and application thereof in quick detection of vibrio alginolyticus

A technology of nucleic acid aptamer and Vibrio alginolyticus, which is applied in the direction of DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve the problems of detection and diagnosis of Vibrio alginolyticus, and achieve short preparation cycle and small molecular weight , high affinity and specificity

Active Publication Date: 2018-05-15
GUANGXI ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention is to provide a high specificity, high sensitivity, non-immunogenicity, stable and easy to modify, easy to synthesize and store ssDNA nucleic acid aptamer for detection of aquatic pathogen Vibrio alginolyticus, to at least solve the problem The problem that some biological detection technologies cannot quickly and accurately detect and diagnose Vibrio alginolyticus on the spot

Method used

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  • ssDNA nucleic acid aptamer and application thereof in quick detection of vibrio alginolyticus
  • ssDNA nucleic acid aptamer and application thereof in quick detection of vibrio alginolyticus
  • ssDNA nucleic acid aptamer and application thereof in quick detection of vibrio alginolyticus

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Embodiment 1

[0037] The preparation method of embodiment 1ssDNA nucleic acid aptamer is as follows:

[0038] Step 1: Synthesize a random ssDNA library and primers shown in the sequence below

[0039] Random library Library50:

[0040] 5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3';

[0041] 5' primer (FITC marker): 5'-FITC-GACGCTTACTCAGGTGTGACTCG-3';

[0042] 5' primer (Biotin marker): 5'-Biotin-GACGCTTACTCAGGTGTGACTCG-3';

[0043] 5' primer (TAMRA marker): 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

[0044] Step 2: Dissolve 10nmol of the above random library in 500μl PBS, heat it in a constant temperature water bath at 92°C for 5 minutes, then quickly insert it into ice, and put it in ice bath for 10 minutes. Put the treated random library and Vibrio alginolyticus live bacteria on ice Incubate for 1 hour; after the incubation and binding are completed, remove the supernatant by centrifugation, wash the viable Vibrio alginolyticus with 10 mL of PBS, bathe in a constant temperature wa...

Embodiment 2

[0054] The preparation method of embodiment 2 ssDNA nucleic acid aptamer is as follows:

[0055] Step 1: Synthesize a random ssDNA library and primers shown in the sequence below

[0056] Random library Library50:

[0057] 5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3';

[0058] 5' primer (FITC marker): 5'-FITC-GACGCTTACTCAGGTGTGACTCG-3';

[0059] 5' primer (Biotin marker): 5'-Biotin-GACGCTTACTCAGGTGTGACTCG-3';

[0060] 5' primer (TAMRA marker): 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

[0061] 3' primer: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3' (SEQ ID NO: 3);

[0062] Step 2: Dissolve 10nmol of the above random library in 500μl PBS, heat it in a constant temperature water bath at 92°C for 5 minutes, then quickly insert it into ice, and put it in ice bath for 10 minutes. Put the treated random library and Vibrio alginolyticus live bacteria on ice Incubate for 1 hour; after the incubation and binding are completed, remove the supernatant by centrifugation, wash the viabl...

Embodiment 3

[0078] In the embodiment of the present invention, the ssDNA nucleic acid aptamer obtained in Example 2 was used to assemble an ELISA kit to form an ELISA detection kit for detecting the aquatic pathogen Vibrio alginolyticus.

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Abstract

The invention discloses a ssDNA nucleic acid aptamer capable of specifically recognizing vibrio alginolyticus, a screening method, a detection method and application thereof. A nucleotide sequence ofthe ssDNA nucleic acid aptamer is 5'-CTTCTATCTACATTTCTTTTTCGTCAATTTTTATTCCCTGGCCCACCCTA-3'(SEQ ID NO:1) or 5'-GACGCTTACTCAGGTGTGACTCG-CTTCTATCTACATTTCTTTTTCGTCAATTTTTATTCCCTGGCCCACCCTA-CGAAGGACGCAGATGAAGTCTC-3'(SEQ ID NO:2). The ssDNA nucleic acid aptamer has specificity and high sensitivity to vibrio alginolyticus and has no immunogenicity. The ssDNA nucleic acid aptamer has the advantages of stable structure, easiness in modification and convenience for synthesis and storage and can be applied in quick and accurate detection and diagnosis of vibrio alginolyticus.

Description

technical field [0001] The invention relates to an ssDNA nucleic acid aptamer and its screening method, detection method and application, in particular to an ssDNA nucleic acid aptamer and its application in rapid detection of Vibrio alginolyticus. Background technique [0002] As a big aquaculture country, my country accounts for 70% of the world's total aquaculture production. However, due to the outbreak and prevalence of bacterial pathogens in recent years, my country's aquaculture industry has suffered huge economic losses. Especially in Guangxi and other coastal areas of South China, Vibrio alginolyticus causes bacterial fish diseases in seawater cultured fish. The fish diseases caused by it have rapid onset, high mortality, and wide prevalence, which have serious implications for the development of aquaculture industry in South China. threaten. At present, the diagnosis of fish bacterial diseases in the world mainly adopts traditional observation methods, immunologi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/569
CPCC12N15/115C12N2310/16C12N2330/31G01N33/56911G01N2333/28
Inventor 李鹏飞秦启伟余庆陈波
Owner GUANGXI ACAD OF SCI
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