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Kit for detecting white spot syndrome virus of prawn and detection method thereof

A technology of white spot syndrome and kits, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microbial measurement/testing, can solve the problems of specific primer design and detection kit development complexity, and achieve detection methods Fast, sensitive, high-sensitivity, and specific effects

Inactive Publication Date: 2010-04-07
SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical applications, due to the complexity of the design of specific primers and the development of its detection kits, LAMP technology has not yet been promoted in the fields of medical care, food, etc.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The DNA extraction of embodiment 1 sample to be tested

[0060] (1) Take 30 mg of the sample to be tested and place it in a 1.5 mL centrifuge tube, add 200 μL of tissue lysate, mash it; lyse at room temperature for 30 minutes;

[0061] (2) Use a desktop centrifuge at 12000 rpm for 5 minutes, take the supernatant, and place it in a 1.5mL centrifuge tube;

[0062] (3) Add 15 μL of DNA adsorption solution (contains white substance, shake well before use), let stand at room temperature for 5 minutes, and shake well 3 times during the period;

[0063] (4) Use a desktop centrifuge at 10,000 rpm / centrifuge for 20 seconds, discard the supernatant, add 200 μL of 70% ethanol, and mix upside down to fully resuspend the precipitate;

[0064] (5) Use a desktop centrifuge at 10,000 rpm / centrifuge for 30 seconds, discard the supernatant, place the precipitate in a constant temperature metal bath at 60°C and dry for 5 minutes, and open the centrifuge tube cover;

[0065] (6) Add 20μL...

Embodiment 2

[0066] The LAMP amplification of embodiment 2 prawn white spot syndrome virus

[0067] (1) According to the number of samples to be detected, set the required number of LAMP reaction tubes N, N=number of samples+1 tube of positive control+1 tube of negative control;

[0068] (2) Draw the volume of LAMP pre-reaction solution to be N×23μL, add it to a clean 1.5mL centrifuge tube, then add NμL Bst DNA polymerase, mix well, and centrifuge at 1500-2000 rpm for 10 seconds;

[0069] (3) Add 24 μL of the above mixed solution to the set N reaction tubes, and then add 30 μL of LAMP reaction stabilization solution to each of the above reaction tubes, and centrifuge at 2000 rpm for 5 seconds;

[0070] (4) Add 1 μL each of the negative control DNA, the template DNA to be tested and the positive control DNA in sequence to the above N PCR reaction tubes, cover the tubes tightly and mark them;

[0071] (5) Put it in a constant temperature metal bath and react at a constant temperature of 61°...

Embodiment 3

[0072] Embodiment 3 color detection

[0073] Take out the LAMP reaction tube, cool to room temperature, centrifuge at 2000 rpm for 5 seconds, add 1 μL of chromogenic solution in the order of negative control, sample to be tested and positive control, mix gently, and directly observe the color change with the naked eye; it is recommended Observe against a black background.

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PUM

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Abstract

The invention discloses a kit for detecting white spot syndrome virus (WSSV) of prawn. The kit comprises DNA extractant of a sample to be detected, LAMP pre-reaction liquid, Bst DNA polymerase, LAMP reaction stabilizing solution, indicator, positive control DNA and negative control DNA. The method also discloses a method for detecting WSSV by utilizing the kit. The kit of the invention has strong specificity and can be applied to scientific research and WSSV detection of cultivation site. The method of the invention is low in cost, and is rapid and sensitive, and the detection result can be judged directly by naked eyes.

Description

technical field [0001] The invention relates to the field of virus detection, in particular to a kit and a detection method for detecting prawn white spot syndrome virus by using a loop-mediated isothermal amplification (Loop-mediated isothermal amplification, LAMP) technique. Background technique [0002] Shrimp white spot syndrome virus (White Spot Syndrome Virus, WSSV) is by far the most serious shrimp virus, which can cause large-scale outbreaks of farmed shrimp; its infected host range is very wide, including Litopenaeus vannamei, A variety of cultured species such as Penaeus monodon, Penaeus chinensis, Penaeus japonicus and Penaeus villosa have caused huge economic losses to the shrimp farming industry. Due to its strong infectivity and high fatality rate, people must focus on early prevention and control when dealing with it; the International Veterinary Office (OIE), the Food and Agriculture Organization of the United Nations (FAO) and the Asia-Pacific Aquaculture De...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 赵哲任春华胡超群张吕平罗鹏江晓
Owner SOUTH CHINA SEA INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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