Method for detecting dsRNA virus of edible fungus

A detection method and edible fungus technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of no effective prevention and control methods for viral diseases, backward research on edible fungus viruses, etc., and achieve short detection time, easy judgment, and test conditions simple effect

Active Publication Date: 2016-12-14
INST OF EDIBLE FUNGI FUJIAN ACAD OF AGRI SCI
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  • Abstract
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  • Application Information

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Problems solved by technology

Since the research on edible fungus viruses is still relatively backward, there is no effective prevention and treatment method for viral diseases.

Method used

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  • Method for detecting dsRNA virus of edible fungus

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preparation example Construction

[0041] (1) Preparation of test samples: Homogenize fresh or frozen edible fungus samples (mycelia or fruiting bodies) to be tested to obtain a homogenate, and centrifuge the homogenate at a speed of 12000-14000 rpm / min for 1 -2 min, take the supernatant for later use;

[0042] The specific operation of the homogenate is as follows: put 0.5 g of the edible fungus sample into a grinder, add 200 μL of TE buffer solution and a small amount of quartz sand, and quickly grind the homogenate to obtain a homogenate.

[0043] Or put 0.1 g of the edible fungus sample into a 2 mL centrifuge tube containing three steel balls with a diameter of 4 mm, add 50 μL of TE buffer, and place it in a FastPrep nucleic acid extraction instrument for homogenization at a homogenization speed of 4.5K for 15 to 30 s .

[0044] (2) Point film: Cut a film of appropriate size according to the number of samples. The film is nitrocellulose film, nylon film or PVDF film, and divide the film into different squa...

Embodiment 1

[0053] Taking the detection of Lentinus edodes virus as an example, the steps of using dsRNA monoclonal antibody combined with dot blot detection are as follows:

[0054] 1. Cultivation and collection of mushroom strain mycelium

[0055] In the ultra-clean workbench, the mushroom strains were transferred to a 9cm PDA plate, cultured at a constant temperature of 25°C for 15 days, and then the mushroom hyphae were scraped, and used directly for detection of poisonous conditions or stored at -20°C for later use.

[0056] 2. Dot blot detection

[0057] (1) Preparation of test samples: put 0.1 g of frozen mushroom mycelium with Lentinus edodes virus disease into a 2 mL centrifuge tube containing 3 steel balls with a diameter of 4 mm, add 50 μL of TE buffer in the FastPrep nucleic acid extractor Homogenate (speed 4.5K, time 15 to 30 s), centrifuge at 12000 rpm / min for 1 min, and take the supernatant for later use.

[0058] Take 0.5 g mushroom fruiting body and put it into the grin...

Embodiment 2

[0068] Taking the detection of Agaricus bismuth virus as an example, the steps of using dsRNA monoclonal antibody combined with dot blot detection are as follows:

[0069] (1) Preparation of test samples: Put 0.5 g of the fresh fruiting body of Agaricus bisporus to be tested into a grinder, add 200 μL TE buffer solution and a small amount of quartz sand, and quickly grind and homogenize to obtain a homogenate, which is prepared with Centrifuge at 14000 rpm / min for 2 min, and take the supernatant for later use.

[0070] (2) Dot film: Cut a nylon film of appropriate size according to the number of samples, divide the film into different squares with a pencil line, and use it to distinguish the position of different samples (the size of each square is about 0.5 cm × 0.5 cm), take 3 μL of the centrifuged supernatant after the above homogenization was spotted on the membrane, and the membrane was dried in an oven at 40°C.

[0071] (3) Membrane sealing: According to the size of the...

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Abstract

The present invention discloses a method for detecting dsRNA virus of edible fungus, the dsRNA virus of edible fungus can be detected by use of dsRNA monoclonal antibody J2 by dot hybridization, and the method is as follows: an edible fungus detection sample is prepared, the edible fungus detection sample is applied to a hybridization membrane, a closing liquid is used to fast close redundant nonspecific DNA binding sites on the hybridization membrane, the hybridization membrane is respectively incubated with the dsRNA monoclonal antibody J2 and goat anti-mouse IgG for color development, color changes are viewed by naked eyes, and toxic situations of the edible fungus can be determined. A plurality of samples can be detected by the method simultaneously, and the method has the advantages of convenient operation, fast and convenient operation, no need of special instruments and equipment, clear positive signals, easy judgment and the like.

Description

technical field [0001] The invention relates to a method for detecting edible fungus viruses, in particular to a method for detecting edible fungus dsRNA viruses by dot hybridization using dsRNA monoclonal antibody J2. Background technique [0002] With the large-scale development of edible fungus cultivation, the occurrence of diseases has become more and more common, causing considerable losses to producers. Viral diseases are a relatively important type of diseases that endanger the production of edible fungi. Viral diseases are different from bacterial and fungal diseases, and their symptoms are latent, and symptoms may not appear until a certain stage in production. Therefore, edible fungus viruses have constituted a potential hazard to the production of major edible fungi such as Agaricus bisporus, Lentinula edodes, Pleurotusotreatus, and Flammulina velutipes. [0003] There is no effective control method for viral diseases at present, and the use of non-toxic strain...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56983G01N33/577
Inventor 肖冬来杨菁张迪黄小菁
Owner INST OF EDIBLE FUNGI FUJIAN ACAD OF AGRI SCI
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