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Rapid detection kit of infectious pancreas necrosis virus and detection method thereof

A pancreas necrosis virus and detection kit technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of rapid detection of pancreas necrosis virus that have not yet been seen, and achieves short detection time, high sensitivity, Highly specific effects

Inactive Publication Date: 2011-01-26
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Abstract
  • Description
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Problems solved by technology

According to the search, there is no report on the rapid detection of infectious pancreatic necrosis virus using loop-mediated isothermal amplification technology

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The loop-mediated isothermal amplification rapid detection kit for infectious pancreatic necrosis virus was prepared according to the following recipe.

[0044] Reagent A: 1.0 μL Bst DNA polymerase (8U / μL);

[0045] Reagent B: 1.0μL reverse transcriptase (200U / μL);

[0046] Reagent C: 2.5 μL reaction buffer containing 200 mmol / L tris-hydrochloric acid (Tris-HCl) pH8.8, 100 mmol / L potassium chloride (KCl), 100 mmol / L ammonium sulfate ((NH4 ) 2 SO 4 ), 80mmol / L magnesium sulfate (MgSO 4 ) and 1% Triton X-100 (Triton X-100);

[0047] Reagent D: Loop-mediated isothermal amplification mixture containing 4.0 μL 2.5mmol / L dNTP, 2.0 μL 10mol / L betaine, 0.5 μL 200 U / μL RNase inhibitor and 5 μL molecular biology grade ultrapure water, totaling 11.5 µL;

[0048] Reagent E: Primer Mix containing 2 μL 20 μmol / L forward inner primer, 2 μL 20 μmol / L reverse inner primer, 1 μL 5 μmol / L forward outer primer, 1 μL 5 μmol / L reverse outer primer and 1 μL 20 μmol / L loop Primers; 7 μL...

Embodiment 2

[0065] The loop-mediated isothermal amplification rapid detection kit for infectious pancreatic necrosis virus was prepared according to the following recipe.

[0066] Reagent A, reagent B, reagent C, and reagent E are the same as in Example 1.

[0067] Reagent D: Loop-mediated isothermal amplification mixture containing 4.0 μL 2.5mmol / L dNTP, 2.0 μL 10mol / L betaine, 0.5 μL 200 U / μL RNase inhibitor and 6 μL molecular biology grade ultrapure water; a total of 12.5 μL.

[0068] Reagent E: Same as Example 1.

[0069] Follow the procedure below for testing:

[0070] (1) Extraction of sample RNA:

[0071] The sample is a sample of turbot imported from a certain country, and its appearance is normal.

[0072] The traditional TRIZOL method was used to extract the RNA of the turbot sample, referring to the Molecular Cloning Experiment Guide (Second Edition). The specific steps are to take 50 mg turbot brain, kidney, spleen and other tissues for homogenization, add 600 μL TRIZOL l...

Embodiment 3

[0079] The loop-mediated isothermal amplification rapid detection kit for infectious pancreatic necrosis virus was prepared according to the same recipe as in Example 2.

[0080] Follow the procedure below for testing:

[0081] (1) Extraction of sample RNA:

[0082] The sample is perch collected from a farm, and its appearance is normal.

[0083] Get 50mg perch liver tissue, the extraction method of the RNA of sample is the same as embodiment 2. Determination of RNAOD of perch samples with a nucleic acid analyzer 260 / OD 280 1.8, adjust the RNA concentration to 100ng / μL.

[0084] (2) Carrying out the loop-mediated isothermal amplification reaction of infectious pancreatic necrosis virus:

[0085] Take a 0.2 mL polypropylene plastic tube, add 1.0 μL of RNA of the perch sample extracted in step (1), and then add other reaction components as in Example 2.

[0086] Place the above plastic tube in a constant temperature water bath at 65°C for 50 minutes to carry out the loop-...

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Abstract

The invention discloses a fast detecting kit of an infectious pancreatic necrosis virus and a detecting method thereof. The invention comprises the kit formed by a Bst DNA polyase, a reverse transcriptase, a reaction buffering liquor, a loop-mediated isothermal amplification mixed solution, a primer mixed solution and a fluorescent color-developing agent and a detecting operational procedure for detecting the infectious pancreatic necrosis virus in fish samples by using the same, which releases standardization to the fast detection of the infectious pancreatic necrosis virus and provides a scientific basis for the fish healthy breeding and the improvement international fish trade. The invention has advantages of good specificity, high sensitivity, easy and simple to handle and high efficiency or the like, is widely used in site instant detection in aqua farms and the import and export fish trade, provides technical supports for relative basic research and has an extensive use prospect.

Description

technical field [0001] The invention belongs to the detection technology of aquatic animal pathogens, in particular to a kit and a detection method for rapid detection of fish infectious pancreatic necrosis virus by adopting a loop-mediated isothermal amplification technology. Background technique [0002] Infectious pancreatic necrosis virus (IPNV) belongs to the family of double-segment RNA viruses and the genus of aquatic double-segment RNA viruses. It can cause acute infectious diseases of salmon fry and juvenile fish. virus disease. IPNV has caused serious harm to the aquaculture industry and has spread to Canada, Chile, Denmark, the United Kingdom, France, Germany, Italy, Japan, South Korea, Norway, Scotland, Sweden, the United States, China and other countries. Susceptible fish include more than ten species of common farmed fish such as rainbow trout, Atlantic salmon, yellowtail, turbot, European yellow cover flounder, halibut and Atlantic cod. Affected fish have ab...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/70
Inventor 岳志芹李琼梁成珠朱来华邓明俊张太翔辛学谦刘荭肖西志凌宗帅郑小龙张健
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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