Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers

A technology of enzyme-linked immunosorbent adsorption and nucleic acid aptamer, which is applied in the direction of measuring devices, biological tests, material inspection products, etc., can solve the problems of long time-consuming preparation of antibodies, quality differences between batches, cumbersome operations, etc., and achieve easy operation , short time-consuming, high-sensitivity effect

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

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Problems solved by technology

However, the traditional antibody-based ELISA detection technology has some shortcomings, such as the need for strict low-temperature storage conditions for antibodies, long time-consuming antibody preparation, high cos

Method used

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  • Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers
  • Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers
  • Aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers

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Embodiment 1

[0024] Three nucleic acid aptamers (synthesized by ThermoFisherScientific) for ELISA detection of grouper iridescent virus infection were synthesized, and the sequences were as follows:

[0025] Sequence 1:

[0026] 5'-GACGCTTACTCAGGTGTGACTCGTATGTCCATGGCCGCATATTGGGAAGGTTGGTTTGGACTATGTGGAAGTTCGAAGGACGCAGATGAAGTCTC-3'

[0027] Sequence 2:

[0028] 5'-GACGCTTACTCAGGTGTGACTCGTTTCGTGTTATGCTCCTCTTTATTGTCAGCTGAGTTTCTGCAGTGCGAAGGACGCAGATGAAGTCTC-3'

[0029] Sequence 3:

[0030] 5'-GACGCTTACTCAGGTGTGACTCGTATTCGGGTTATTGCTCCTCTTTATTGTCACCTGGATGTATGATCGTGTAGCGAAGGACGCAGATGAAGTCTC-3'

[0031] The above-mentioned nucleic acid aptamers are modified with biotin tags at the 5' end or 3' end.

[0032] The method for the detection of grouper iridescent virus infection by enzyme-linked immunosorbent assay technology based on nucleic acid aptamer comprises the following steps:

[0033] 1. Refolding of nucleic acid aptamers: biotin-labeled nucleic acid aptamers were denatured at 94°C for 10 mi...

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Abstract

The invention discloses an aptamer-based enzyme-linked immunosorbent assay method for detecting iridovirus infection of groupers. The method comprises the following steps: incubating and combining a biotin-labeled aptamer and to-be-detected cells or tissues; cleaning a to-be-detected sample, adding horse radish peroxidase-labeled streptavidin, cleaning the to-be-detected sample after incubation completion, adding a TMB developing solution in a horse radish peroxidase developing kit for carrying out a developing reaction, and determining whether the to-be-detected cell or tissue sample is infected by grouper iridovirus according to light absorption value change. Compared with the existing antibody-based ELISA technology, the aptamer-based ELISA technology disclosed by the invention has the advantages that the method is high in specificity and sensitivity and convenient to operate, is suitable for large-scale rapid detection of iridovirus infection of cultured fishes in aquaculture farms and has good application prospects in the detection field of iridovirus infection.

Description

Technical field: [0001] The invention belongs to the field of grouper iridescent virus detection, and in particular relates to a method for detecting grouper iridescent virus infection by an enzyme-linked immunosorbent technology based on nucleic acid aptamers. Background technique: [0002] Grouper is one of the most valuable mariculture fish in the coastal provinces of southern my country and Southeast Asian countries, with extremely high economic value. However, the outbreak and prevalence of grouper iridovirus (SGIV) in grouper in recent years has seriously threatened the mariculture of grouper and caused huge economic losses. After the grouper is infected by SGIV, there will be necrosis and swelling of tissues and organs such as black body, liver, spleen and kidney, which will eventually lead to the death of the fish. For high-density cultured grouper, a rapid and easy-to-operate technical method that can detect grouper iridescent virus infection in the early stage is ...

Claims

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Application Information

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IPC IPC(8): G01N33/58G01N33/569G01N33/543
CPCG01N33/581G01N33/543G01N33/56983
Inventor 秦启伟李鹏飞魏世娜周伶俐
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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