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Method for detecting flounder viral nervous necrosis

A technology of nerve necrosis and detection method, applied in the direction of microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of unsatisfactory specificity and stability, less application, difficulty in NNV virus antibody, etc., and achieve high practicality value effect

Inactive Publication Date: 2009-06-24
TIANJIN AGRICULTURE COLLEGE
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  • Abstract
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Problems solved by technology

However, its specificity and stability are far from meeting the needs of experiments.
It is difficult to prepare antibodies against NNV virus
Moreover, the specificity of ELISA is often not stable enough for various reasons, so these methods are rarely used in actual detection.

Method used

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  • Method for detecting flounder viral nervous necrosis
  • Method for detecting flounder viral nervous necrosis

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Embodiment Construction

[0032] The steps of the method for detecting Paralichthys olivaceus viral neuronecrosis are as follows:

[0033] 1. Take about 100mg of fish eyes and brain tissue in a 1.5ml centrifuge tube, add 1ml Ttizol extract, and quickly place it in the ice box and homogenize with a glass homogenizer (about 1min);

[0034] 2. Add 200ul chloroform to the static platform, mix well, and room temperature for 5 minutes;

[0035]3. Centrifuge at 12000r / min for 10min at 4℃;

[0036] 4. Take 400ul of the upper water phase in a new centrifuge tube. Add an equal volume of isopropanol, mix well and let it stand for 5 minutes;

[0037] 5. Centrifuge at 12000r / min for 10min at 4℃, discard the supernatant.

[0038] 6. Add 700ul of pre-cooled 70% alcohol and gently pipet, ℃, 12000r / min for 10min, discard the supernatant;

[0039] 7. Repeat the previous step once to ventilate and dry the ultra-quiet platform;

[0040] 8. Add 20ul DEPC sterilized double distilled water to dissolve to obtain PCR template;

[...

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Abstract

The invention relates to a method for detecting paralichthys olivaceus viral nervous necrosis. A set of nested RT-PCR primer is designed in a PONNV capsid protein highly conservative region. A nested RT-PCR reaction system is established by specificity and sensitivity experiment, and the nested RT-PCR reaction system is optimized. The method for detecting the paralichthys olivaceus viral nervous necrosis provides a method which is simple, fast and flexible for operating, has good specificity, can be used for tracing and monitoring virus and monitoring water quality during paralichthys olivaceus breeding process and has very high practical value.

Description

Technical field [0001] The technology belongs to the invention of the diagnosis and detection of aquatic animal diseases, and particularly relates to the detection method of Paralichthys olivaceus viral nervous necrosis (PONNV). Background technique [0002] Fish viral neuronecrosis is one of the major infectious diseases that have seriously harmed marine and freshwater fish in recent years and caused outbreaks worldwide. At present, more than 40 species of fish, including Anguillae, Codelliformes, Perciformes, Flounderformes, and Fukuiformes, are known to be infected by neuronecrosis viruses. The viruses can be transmitted through both vertical and horizontal channels. Larvae and juveniles are susceptible to infection, with a fatality rate of more than 90%, or even 100%, causing huge economic losses to seawater industrial aquaculture enterprises. The infectious disease is listed as an important fish disease by the International OIE (OIE) (OIE, 2000). Antibiotics and chemical dru...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68
Inventor 毛海涛刘爽白东清孙金生
Owner TIANJIN AGRICULTURE COLLEGE
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