Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers

A nucleic acid aptamer and nerve necrosis technology, which is applied in the direction of measuring devices, biological testing, material inspection products, etc., can solve the problems of not having many molecular biology, high requirements for instrument platforms, and little experimental experience, so as to achieve low cost and high detection efficiency. The effect of low cost and easy detection and operation

Inactive Publication Date: 2016-07-20
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most of these methods have problems such as time-consuming, cumbersome operation, high requirements on the instrument platform, and poor stability.
Especially not suitable for grouper farms where there are not many molecular biology, protein and other instruments and equipment, and the staff have little experimental experience

Method used

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  • Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers
  • Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers
  • Aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers

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Embodiment 1

[0036] 1. Synthesis of nucleic acid aptamer of grouper neuronecrosis virus

[0037] Synthesize the nucleic acid aptamer shown in the following sequence (synthesized by ThermoFisherScientific):

[0038] Sequence 1:

[0039] 5'-GACGCTTACTCAGGTGTGACTCGTGTCATTTGTTTTGTTTGTGATTGTTTTTAATCTTAATTTTGATTGGTTCCGAAGGACGCAGATGAAGTCTC-3'

[0040] Sequence 2:

[0041] 5'-GACGCTTACTCAGGTGTGACTCGTGTGTGTCTATTGTCTGTGGTTCATGTCAAGCTTATTTTCCACACACGGTCGAAGGACGCAGATGAAGTCTC-3'

[0042] Sequence 3:

[0043] 5'-GACGCTTACTCAGGTGTGACTCGTTCTTTTTATTAGTTGATTTTTTTGATTTTGGCAGCTACTGCTTTGGGGGTCGAAGGACGCAGATGAAGTCTC-3'

[0044] The 5' end of the above nucleic acid aptamer was modified with biotin.

[0045] 2. Immobilization of nucleic acid aptamers: wash the well plate (96 wells) pre-coated with streptavidin with PBS, then add 0.2 nmol aptamers to the wells, incubate for 1 hour, and wash off excess aptamers with PBS. Ligand;

[0046] 3. Blocking: add 100 μl blocking solution 0.3% BSA, block, and wash with ...

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Abstract

The invention discloses an aptamer-based Sandwich ELASA method for detecting nervous necrosis virus infection of groupers. The method comprises the following steps: fixing a biotin-labeled aptamer onto a pore plate pre-coated with streptavidin; capturing CP proteins in a to-be-detected sample by the fixed aptamer, and combining the CP proteins by the biotin-labeled aptamer; adding horse radish peroxidase-streptavidin for incubating and combining, developing by a horse radish peroxidase developing kit, and analyzing whether NNV virus infection exists according to light absorption value change. According to the invention, by utilizing the characteristics of high affinity and high specificity of the aptamer, a Sandwich ELASA detection method based on aptamer detection is established and can be used for detecting nervous necrosis viruses during grouper culture. Compared with the traditional ELISA and other detection methods, the method disclosed by the invention has the characteristics of high detection speed, high sensitivity and specificity, low cost and high simplicity in operation.

Description

Technical field: [0001] The invention belongs to the field of detection of grouper nerve necrosis virus, and in particular relates to a method for detecting grouper nerve necrosis virus infection based on nucleic acid aptamer-based SandwichELASA. Background technique: [0002] Fish viral neuronecrosis is a kind of fish epidemic infectious disease worldwide (except Africa), which has caused huge losses to the mariculture industry of various countries, and is an important fish disease. The disease, also known as vacuolar encephalopathy and retinopathy, is very harmful to larvae and young fish. In severe cases, the mortality rate can reach 100% within a week, and it also has a high lethality rate to adult fish. In recent years, neuronecrosis caused by nerve necrosis virus (NNV) has been widespread in our country, seriously affecting the aquaculture industry in our country, especially the development of grouper aquaculture. However, most of the reports on this disease focus on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/569G01N33/543
CPCG01N33/581G01N33/543G01N33/56983
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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