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Isothermal amplification detection kit for scallop acute viral necrobiotic virus (AVNV) nucleic acid and detection method thereof

A technology for detecting kits and viral nucleic acids, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection. It can solve the problems of difficult detection technology, difficulty in on-site detection, and long detection time. To achieve the effects of proceduralization and standardization, safety of operators and the environment, and high practical value

Inactive Publication Date: 2010-01-27
YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some of these detection methods require cumbersome material preparation, complicated operation, high cost, some have high technical requirements, long detection time, and some methods have low sensitivity, low specificity and low accuracy.
Therefore, it is very difficult for operators to master the key detection technologies, and it is difficult to use them for on-site detection, which limits the application of these technologies in on-site detection and quarantine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Test kit of the present invention is made up of following (4 samples):

[0038] (1) Grinding solution, 1 tube, 800 μ L of SEMP sampling solution is installed inside. The SEMP sampling solution is characterized in that it is composed of 10 parts of 1M Tris-HCl (Tris-HCl, pH8.0), 0.5M ethylenediaminetetra 1 part of disodium acetate (EDTA), 10 parts of 10% sodium dodecyl sulfate (SDS), 0.01 part of mercaptoethanol, 20 parts of balanced phenol, and distilled water to 100 parts.

[0039] (2) Nucleic acid extraction solution A, 1 tube, containing 400 μL of 4M sodium acetate (NaAc, pH 5.2).

[0040](3) Nucleic acid extraction solution B, 1 tube, filled with 800 μL absolute ethanol.

[0041] (4) Nucleic acid extraction solution C, 1 tube, filled with 800 μL of 70% absolute ethanol.

[0042] (5) TE buffer, 1 tube, containing 200 μL of TE buffer (10 mM Tris-HCl and 1 mM EDTA, pH 8.0).

[0043] (6) LAMP reaction solution, 1 tube, containing 100 μL LAMP reaction solution, charac...

Embodiment 2

[0056] Scallop acute viral necrosis virus LAMP detection method of the present invention

[0057] Use the test kit in embodiment 1, carry out according to the following steps:

[0058] (1) Take about 0.1 g of the mantle tissue of Chlamys farreri and put it in a centrifuge tube, add 200 μL of grinding solution, and grind the sample fully with a grinding rod until it becomes a slurry.

[0059] (2) Centrifuge the ground sample at 10,000 r / min for 2 min, and take 100 μL of the supernatant and place it in a new centrifuge tube.

[0060] (3) Add 100 μL of nucleic acid extraction solution A, mix up and down, then add 200 μL of nucleic acid extraction solution B (pre-cooled at -20°C) and mix thoroughly by turning upside down, centrifuge at 12000r / min for 10min, carefully discard the supernatant, and keep the precipitate .

[0061] (4) Add 100 μL of nucleic acid extraction solution C to wash the precipitate, then centrifuge at 12000 r / min for 5 min, carefully discard the supernatant,...

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Abstract

The invention relates to an isothermal amplification detection kit for scallop acute viral necrobiotic virus (AVNV) nucleic acid and a detection method thereof. The detection kit comprises regents, such as SEMP sample liquid, nucleic acid extraction liquid, LAMP reaction liquid, color development agent, AVNV positive control nucleic acid and the like and various kinds of apparatuses. The detection method uses four primers designed by the sequence of conservative region of scallop acute viral necrobiotic virus (AVNV) gene to quantitatively detect the specific nucleic acid fragment of the scallop acute viral necrobiotic virus (AVNV). The method has the characteristics of convenient and quick detection, high sensitivity and good specificity. Particularly the method is convenient for the spot rapid determination. By the invention, only a water bath is needed, in 2 hours, the virion with less than 30 copies can be detected in the scallop and the farming water body. The method can be used for tracking and detecting the virus in each period in the process of farming scallop. And the method is hopeful to replace the methods, such as a histopathological method, an electronic microscopy, an immunological method, a PCR detection method and the like.

Description

Technical field: [0001] The invention belongs to the technical field of shellfish pathogen detection, in particular to a scallop acute viral necrosis virus (AVNV, Acute Viral Necrosis Virus) nucleic acid isothermal amplification detection kit and a detection method thereof. Background technique [0002] Since the 1990s, the large-scale death of Chlamys farreri has broken out year after year in the breeding sea area of ​​northern my country (the cumulative mortality rate is as high as more than 90%), which has caused huge economic losses to the scallop farming industry and seriously hindered the development of this industry. Continuous development. AVNV is the direct pathogen causing mass mortality of Chlamys farreri in summer. At present, there is no effective treatment for AVNV. The promotion of healthy scallop farming technology is the most effective preventive measure, which mainly relies on early rapid detection. Therefore, research and development of fast, accurate, se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
Inventor 王崇明任伟成蔡玉勇黄倢张庆利
Owner YELLOW SEA FISHERIES RES INST CHINESE ACAD OF FISHERIES SCI
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