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RT-PCR detection method of nervous necrosis virus

A technology of RT-PCR and nerve necrosis, applied in the direction of microbial-based methods, microbial measurement/inspection, biochemical equipment and methods, etc., can solve expensive and other problems

Inactive Publication Date: 2012-01-11
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these methods require the use of expensive equipment and reagents

Method used

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  • RT-PCR detection method of nervous necrosis virus
  • RT-PCR detection method of nervous necrosis virus
  • RT-PCR detection method of nervous necrosis virus

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Embodiment Construction

[0062] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

[0063] The RT-PCR detection method of the nerve necrosis virus comprises the following steps:

[0064] 1. RT-PCR primer design

[0065] According to the obtained NNV outer membrane protein gene sequence, use the software PrimerPremier 5 to design 5 primers for establishing the detection method, and the primer sequences are shown in the table below:

[0066]

[0067] 2. Construction of recombinant plasmid pSP-NNV containing the detection target fragment and preparation of small RNA fragments as positive control

[0068] In vitro construction of recombinant plasmid pSP-NNV for in vitro transcription, and then in vitro transcription to obtain small RNA fragments, the specific steps are as follows:

[0069] 1) Construction of pSP-NNV plasmid

[0070] Use NNV-longR as a reverse primer to obtain the target fragment to be detected by RT-PCR from the vi...

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Abstract

An RT-PCR detection method of a nervous necrosis virus relates to the field of virus detection. The invention provides an RT-PCR detection method of a nervous necrosis virus, an RT-PCR detection kit of nervous necrosis virus and a preparation method thereof. The RT-PCR detection method of the nervous necrosis virus comprises steps of: designing of an RT-PCR primer; construction of a recombinant plasmid pSP-NNV containing a detection target fragment, and preparation of a small RNA fragment as a positive control; an experiment of primer singularity and an experiment of sensitivity detection; and a recovery rate experiment, and a method validity experiment. The kit comprises a kit body and detection reagents, wherein the detection reagents comprise a lysate, an adsorption solution, a washing solution, redistilled water, an RT-PCR mixed reaction solution, an NNV- PCR reaction solution, a positive control, a negative control, an RNA extraction and an RT reaction tube. The preparation method of the kit comprises steps of: preparing the detection reagents; and putting the detection reagents in the kit body.

Description

technical field [0001] The invention relates to a virus detection method, in particular to a RT-PCR detection method for nerve necrosis virus (Nervous necrosis virus, NNV). Background technique [0002] Neuroencephalopathy and retinopathy is a viral neuronecrosis caused by Nervous necrosis virus (NNV), which is characterized by necrosis and vacuolation of the brain and retina, swimming abnormalities and body changes in fish. Black ([1] Mori K, Nakai T, Muroga K, Arimoto M, Mushiake K, Furusawa I: Properties of a new virus belonging to nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 1992, 187(1 ): 368-371; [2] Lin C-C, Lin JH-Y, Chen M-S, Yang H-L: An oral nervous necrosis virus vaccine that induces protective immunity in larvae of grouper(Epinephelus coioides). Aquaculture 2007, 268(0044-8486 ): 265-273). NNV has caused a large number of marine teleost juveniles and juveniles to die worldwide, and the number of infected teleos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 陈新华敖敬群母尹楠
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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