A kind of ssDNA nucleic acid aptamer and application thereof

A nucleic acid aptamer, peptide nucleic acid technology, applied in the field of genetic resource detection, can solve the problems of lack of effective prevention and detection methods for neuronecrosis virus, and achieve the effects of easy synthesis and preservation, high affinity and high specificity

Active Publication Date: 2020-12-22
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to: overcome the existing problem of lack of effective prevention and detection means for nerve necrosis virus, provide a ssDNA nucleic acid aptamer and its application

Method used

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  • A kind of ssDNA nucleic acid aptamer and application thereof
  • A kind of ssDNA nucleic acid aptamer and application thereof
  • A kind of ssDNA nucleic acid aptamer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0024] The ssDNA nucleic acid aptamer sequence of the present invention comprises any one or more of the DNA fragments shown in SEQ ID NO: 1-3.

[0025] The ssDNA nucleic acid aptamer of the present invention is mainly screened by the following methods:

[0026] (1) Synthesize a random single-stranded DNA library and primers shown in the following sequences:

[0027] The sequence of the random library Library50 is shown in SEQ ID NO: 4; the sequences of the primers are shown in SEQ ID NO: 5-7.

[0028] (2) Cell-SELEX screening:

[0029] i. Culture GB cells in L15 medium containing 10% fetal bovine serum to 95% of the bottom of the culture bottle, add grouper neuronecrosis virus (GNNV) and infect for 48 hours, wash the cells with PBS buffer 3-5 times and prepare for use for filtering.

[0030] ii. Dissolve 10nmol random library in screening buffer, bathe in constant temperature water at 95°C for 5min, then quickly insert into ice, and bathe in ice for 10min.

[0031] iii. A...

experiment example

[0039] Experimental example The specific binding of aptamers to RGNNV-infected GB cells was obtained by flow cytometry detection and screening

[0040] The RGNNV-infected GB cells were blown down with a gun, centrifuged at 300 g to remove the supernatant, and washed 3 times with 1 mL of PBS. Dissolve 300 nM fluorescein isothiocyanate (FITC)-labeled aptamer (SEQ ID NO: 1-3) in 1 mL of binding buffer, and then incubate with the above-treated cells on ice for 30 min, 300 g Centrifuge to remove the supernatant, wash with 1mL PBS for 3 times, and finally mix the cells in 400ul PBS for flow cytometry detection. The aptamer combined with normal GB cells was used as a control. This result confirms that the aptamer has high specificity and affinity to RGNNV-infected GB cells ( figure 1 ).

[0041] Experimental example Fluorescence microscopy detection of the specific binding of carboxytetramethylrhodamine (TAMRA)-labeled nucleic acid aptamers to RGNNV-infected GB cells in this examp...

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Abstract

The invention discloses an ssDNA aptamer and applications of the ssDNA aptamer. The ssDNA aptamer comprises any one or more sequences shown as SEQ ID NO:1-3. The ssDNA aptamer has high specificity and high appetency for grouper brain cells infected with the nervous necrosis virus, and can be applied to the rapid detection and treatment scheme of the grouper nervous necrosis virus.

Description

technical field [0001] The invention belongs to the technical field of genetic resource detection, and in particular relates to an ssDNA nucleic acid aptamer that can be used to detect grouper neuronecrosis virus. Background technique [0002] Grouper is one of the most valuable economic fish in marine culture, and the supply exceeds demand in domestic and foreign markets. Its meat is tender and delicious, and its body color is colorful and auspicious. In recent years, the scale of grouper cage culture in the southern provinces of my country has rapidly expanded, and various diseases of grouper have also shown a trend of rapid rise, which has brought huge economic losses to the breeding owners and seriously restricted the development of grouper aquaculture. . Fish viral neuronecrosis, also known as vacuolar encephalopathy and retinopathy, is a kind of fish epidemic infectious disease worldwide (except Africa), and is an important fish disease. The disease is very harmful t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/115G01N33/569
CPCC12N15/115C12N2310/16G01N33/56983
Inventor 秦启伟周伶俐李鹏飞魏世娜
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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