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Visualized rapid detection kit for carp dropsy virus and detection method thereof

A technology for detecting kits and viruses, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., can solve the lack of high-sensitivity detection kits for carp edema virus, which cannot meet the needs of disease quarantine and prevention work and other problems, to overcome the long detection time, reduce background influence, and easy identification

Pending Publication Date: 2017-08-18
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Carp edema virus is the cause of a new infectious disease in my country, and there is a lack of highly sensitive detection kits for carp edema virus in the world, which can no longer meet the quarantine and prevention of diseases caused by the virus

Method used

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  • Visualized rapid detection kit for carp dropsy virus and detection method thereof
  • Visualized rapid detection kit for carp dropsy virus and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Visual rapid detection kit for carp edema virus

[0048] Carp edema virus visual rapid detection kit includes virus DNA extraction reagents and reaction reagents. The kit only needs a conventional centrifuge and a water bath to complete nucleic acid extraction and detection, and the entire detection process only takes 2 hours, and the detection product is closed for observation. It will not pollute the environment. In addition, the test result can be judged by observing the color change with the naked eye, which is very practical.

[0049] Wherein, the viral DNA extraction reagent comprises the following components: 80mM Tris, 50mM EDTA, 500mM NaCl, 1.5% by mass SDS, 0.01% by volume lysate A of pH 8.0 of β-mercaptoethanol; Tris saturated phenol, pH8.0 ; sodium acetate aqueous solution, concentration 3mol / L; absolute ethanol; lotion A containing 75% absolute ethanol configured with DEPC water; DEPC water.

[0050] The reaction reagent contains a nucleic acid p...

Embodiment 2

[0057] Example 2 The method for detection using the carp edema virus visualization rapid detection kit described in Example 1

[0058] (1) DNA extraction:

[0059] Take carp spleen or heart tissue 30-80mg in a 2mL centrifuge tube, grind it with a grinding rod on ice, add 600μL lysate A, continue grinding, add 600μL Tris saturated phenol with a pH value of 8.0, shake vigorously, and centrifuge at 11000g for 10min. Take the supernatant and repeat the phenol extraction; take the supernatant, add 0.1 times the volume of sodium acetate aqueous solution with a concentration of 3mol / L, mix well, add two times the volume of ice-cold absolute ethanol, mix well and let stand at low temperature Centrifuge at 15,000 g for 5 min for 10 min, discard the supernatant, wash the pellet twice with washing solution A containing 75% absolute ethanol prepared in DEPC water, dry at room temperature for 5-10 min, resuspend in 30 μL DEPC water, and store at -80°C for future use. , Lysis solution A co...

Embodiment 3

[0065] Example 3 Kit Specific Detection

[0066] (1) DNA extraction:

[0067] Take carp edema virus-positive carp tissue samples of 30-80 mg in a 2 mL centrifuge tube, grind on ice with a grinding rod, add 600 μL of lysate A, continue to grind fully, add 600 μL of Tris saturated phenol with a pH value of 8.0, shake vigorously, and centrifuge at 11,000 g 10min, take the supernatant, and repeat the phenol extraction; take the supernatant, add 0.1 times the volume of sodium acetate aqueous solution with a concentration of 3mol / L, mix well, add two times the volume of ice-cold absolute ethanol, mix well and then lower the temperature Let stand for 10 minutes, centrifuge at 15,000 g for 5 minutes, discard the supernatant, wash the precipitate twice with washing solution A containing 75% absolute ethanol prepared in DEPC water, dry at room temperature for 5-10 minutes, resuspend in 30 μL DEPC water, and store at -80°C for later use.

[0068] (2) Rapid amplification of carp edema vi...

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Abstract

The invention discloses a visualized rapid detection kit for carp dropsy virus and a detection method thereof. The visualized rapid detection kit for the carp dropsy virus includes a virus DNA extraction reagent and a reaction reagent, the reaction reagent contains a nucleic acid primer set used for detecting amplification of the carp dropsy virus, and the primer set contains a primer CEV-F3, a primer CEV-B3, a primer CEV-BFIP, a primer CEV-BIP, a primer CEV-LpF and a primer CEV-LpB. The rapid detection kit establishes a set of optimized isothermal amplification reaction system, the qualitative detection of the carp dropsy virus is thus simpler, faster, high in specificity and high in sensitivity, and the kit is the first kit used for detecting the carp dropsy virus, the problem that there is no detection method for the carp dropsy virus is solved, and has high scientific and economic value.

Description

technical field [0001] The invention belongs to the field of rapid detection of target gene fragments, and in particular relates to a visual rapid detection kit of carp edema virus and a detection method thereof. Background technique [0002] Carp edema virus (Carp edema virus, CEV) is the pathogen that causes carp edema disease and koisleepy disease (Koisleepy disease, KSD), seriously endangers the healthy breeding of carp and koi, and the mortality rate that disease causes is as high as 80-100%. Carp edema disease is caused by carp edema virus belonging to the Poxviridae family. The disease has spread rapidly around the world and was first discovered in China in 2016. During the occurrence of the disease, the sick fish were unconscious under the water surface or lying on the bottom of the pool. The main symptoms were enophthalmos, gill hyperplasia and anal ulcerative inflammation, and they died within 2 weeks after the onset. [0003] Carp edema virus is the pathogen of e...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119
Inventor 潘晓艺沈锦玉蔺凌云袁雪梅徐洋姚嘉赟尹文林郝贵杰
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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