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Aptamer for specifically recognizing GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and application of aptamer

A nucleic acid aptamer, a technology for oval pomfret, which is applied in the direction of microbial determination/test, chemical library, combinatorial chemistry, etc., can solve the problems of inability to detect and diagnose oval pomfret-derived nerve necrosis virus, and achieves convenient in vitro Chemical synthesis, high affinity and specificity, good reproducibility

Active Publication Date: 2019-01-04
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 detecting neuronecrosis virus derived from pompano pomfret, to at least solve the problem The problem that some biological detection techniques cannot quickly and accurately detect and diagnose the neuronecrosis virus of pomfret ovata on the spot

Method used

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  • Aptamer for specifically recognizing GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and application of aptamer
  • Aptamer for specifically recognizing GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and application of aptamer
  • Aptamer for specifically recognizing GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and application of aptamer

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The preparation method of embodiment 1ssDNA nucleic acid aptamer is as follows:

[0035] Step 1: Synthesize the single-stranded DNA library and primers shown in the following sequences:

[0036] Random library Library50:

[0037] 5'-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT

[0038] 5' primer: 5'-FAM-GTCTGAAGTAGACGCAGGAG-3';

[0039] 3' Primer: 5'-Biotin-ACGCTTACTCAGGTGTGACT-3';

[0040] Step 2: Dissolve 10 nmol of the above random library in 500 μl PBS, place in a constant temperature water bath at 92°C for 5 minutes, then quickly ice-bath for 10 minutes, take the treated random library and incubate GTONNV-infected cells on ice for 1 hour; wait for incubation After the combination is completed, centrifuge and remove the supernatant, take 10mL of PBS to wash the GTONNV infected cells, and in a constant temperature water bath at 92°C for 10min, centrifuge at 12000g for 1-20min, and collect the supernatant, which is To specifically recognize the ssDNA nucleic acid...

Embodiment 2

[0052] The preparation method of embodiment 2 ssDNA nucleic acid aptamer is as follows:

[0053] Step 1: Synthesize the single-stranded DNA library and primers shown in the following sequences:

[0054] Random library Library50:

[0055] 5'-GTCTGAAGTAGACGCAGGAG(50N)AGTCACACCTGAGTAAGCGT

[0056] 5' primer: 5'-FAM-GTCTGAAGTAGACGCAGGAG-3';

[0057] 3' Primer: 5'-Biotin-ACGCTTACTCAGGTGTGACT-3';

[0058] Step 2: Dissolve 10 nmol of the above random library in 500 μl PBS, place in a constant temperature water bath at 92°C for 5 minutes, then quickly ice-bath for 10 minutes, take the treated random library and incubate GTONNV-infected cells on ice for 1 hour; wait for incubation After the combination is completed, centrifuge and remove the supernatant, take 10mL of PBS to wash the GTONNV infected cells, and in a constant temperature water bath at 92°C for 10min, centrifuge at 12000g for 1-20min, and collect the supernatant, which is To specifically recognize the ssDNA nucleic aci...

Embodiment 3

[0082] The secondary structure of the nucleic acid aptamer was predicted online by MFOLD software.

[0083] The secondary structure prediction results of the nucleic acid aptamer of SEQ ID NO:1 are as follows image 3 As shown, the nucleic acid aptamer forms a special stem-loop structure and hairpin structure.

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Abstract

The invention discloses an ssDNA aptamer capable of specifically recognizing a GTONNV (Guangxi trachinotus ovatus nervous necrosis virus) and an application of the aptamer. The nucleotide sequence ofthe ssDNA aptamer is 5'-GTCTGAAGTAGACGCAGGAGCCTTTCGTGTTTCATTAGTGTGTTTCCATTGGGCGGCTCGGGGCAAAAGGAGTCACACCTGAGTAAGCGT-3'(SEQ ID NO: 1) or 5'-CCTTTCGTGTTTCATTAGTGTGTTTCCATTGGGCGGCTCGGGGCAAAAGG-3'(SEQ ID NO: 2). The ssDNA aptamer has specificity and high sensitivity to GTONNV infected cells and has no immunogenicity. The ssDNA aptamer has high specificity and high affinity, and is free of cytotoxicity,stable, easy to modify and convenient to synthesize and preserve, and the GTONNV infected cells can be detected and diagnosed rapidly and accurately.

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 nucleic acid aptamer capable of specifically recognizing cells infected with neuronecrosis virus from pompano pomfret and its application. Background technique [0002] As a big country in aquaculture, my country's aquaculture volume accounts for 70% of the world's total aquatic product farming. Guangxi is a large aquaculture province in my country, and the main cultured species include pomfret trevally, grass carp, channel catfish, grouper, prawns, oysters, pearl oyster and various edible plants. However, with the acceleration of urbanization, industrialization, and large-scale farming, the aquaculture environment in Guangxi is deteriorating day by day, and various diseases frequently break out, causing huge economic losses. In 2017, the viral neuronecrosis fish disease of pomfret ovata in offshore cage culture ...

Claims

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Application Information

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IPC IPC(8): C12N15/115C12Q1/6806C40B50/06G01N33/569
CPCC12N15/115C12N2310/16C12Q1/6806C40B50/06G01N33/56983C12Q2531/113
Inventor 李鹏飞余庆刘明珠李菲吴思婷肖贺贺
Owner GUANGXI ACAD OF SCI
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