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ssDNA aptamer capable of identifying and combining with vibrio alginolyticus and application thereof

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

Active Publication Date: 2018-03-30
GUANGXI ACAD OF SCI
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  • 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 aptamer capable of identifying and combining with vibrio alginolyticus and application thereof
  • ssDNA aptamer capable of identifying and combining with vibrio alginolyticus and application thereof
  • ssDNA aptamer capable of identifying and combining with vibrio alginolyticus and application thereof

Examples

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Effect test

Embodiment 1

[0037] Example 1

[0038] The preparation method of ssDNA nucleic acid aptamer is as follows:

[0039] Step 1: Synthesize the random single-stranded DNA library and primers shown in the sequence below

[0040] Random library Library50:

[0041]5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3';

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

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

[0044] 5' primer (TAMRA marker): 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

[0045] Step 2: Dissolve 10 nmol of the above random library in 500 μl PBS, heat it in a constant temperature water bath at 92°C for 5 min, then quickly insert it into ice for 10 min, put the treated random library and the viable Vibrio alginolyticus on ice Incubate for 1 h; after incubation and binding, remove the supernatant by centrifugation, wash the viable Vibrio alginolyticus with 10 mL of PBS, take a 92°C constant temperature water bath for 10 min, and cen...

Embodiment 2

[0055] The preparation method of embodiment 2ssDNA nucleic acid aptamer is as follows:

[0056] Step 1: Synthesize the random single-stranded DNA library and primers shown in the sequence below

[0057] Random library Library50:

[0058] 5'-GACGCTTACTCAGGTGTGACTCG(50N)CGAAGGACGCAGATGAAGTCTC-3';

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

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

[0061] 5' primer (TAMRA marker): 5'-TAMRA-GACGCTTACTCAGGTGTGACTCG-3';

[0062] 3' primer: 5'-Biotin-GAGACTTCATCTGCGTCCTTCG-3';

[0063] Step 2: Dissolve 10 nmol of the above random library in 500 μl PBS, heat it in a constant temperature water bath at 92°C for 5 min, then quickly insert it into ice for 10 min, put the treated random library and the viable Vibrio alginolyticus on ice Incubate for 1 h; after incubation and binding, remove the supernatant by centrifugation, wash the viable Vibrio alginolyticus with 10 mL of PBS, take a 92°C ...

Embodiment 3

[0079] In the embodiment of the present invention, the ssDNA nucleic acid aptamer obtained in Example 2 is used, and the ELISA kit is assembled to form an ELISA detection kit for the detection of aquatic pathogenic bacteria Vibrio alginolyticus.

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Abstract

The invention discloses an ssDNA aptamer capable of identifying and combining with aquatic pathogenic bacteria, namely vibrio alginolyticus, and a screening method, a detection method and applicationthereof. The nucleotide sequence of the ssDNA aptamer is 5'-ATTTTGCTTTTGCCACTTAT CAACATTTGTTTTTCCGCTTTCGCACTTC-3'(SEQ ID NO:1), or 5'-GACGCTTACTCAGGTGTGACTCG-ATTTTGCTTTTGCCACTTATCAACATTTGTTTTTCCGCTTTCGCACTTC-CGAAGGACGCAGATGAAGTCTC-3'(SEQ ID NO:2). The ssDNA aptamer disclosed by the invention has specificity and high sensitivity to the vibrio alginolyticus, has the advantages of being stable in structure, easy to modify and convenient to synthesize and preserve, and can quickly and accurately detect and diagnose the vibrio alginolyticus.

Description

technical field [0001] The present invention relates to a ssDNA nucleic acid aptamer and its screening method, detection method and application, in particular to a ssDNA nucleic acid aptamer capable of identifying and binding aquatic pathogenic bacteria Vibrio alginolyticus and its screening method and detection method with application. Background technique [0002] Vibrio alginolyticus, as one of the main pathogens causing bacterial fish diseases in marine fish, mainly broke out and spread in the coastal areas of South China such as Guangxi, causing huge economic losses to the local aquaculture industry. Moreover, the fish disease caused by Vibrio alginolyticus has the characteristics of rapid onset, high mortality and wide prevalence. Once the fish die, it is difficult to carry out rapid and effective prevention and control, which poses a serious threat to the development of the aquaculture industry in South China. . At present, the international diagnosis of bacterial d...

Claims

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Application Information

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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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