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Kit for quickly extracting double-stranded RNA of mycovirus and application of kit

A fungal virus and kit technology, which is applied in the field of kits for rapidly extracting viral double-stranded RNA, can solve the problems of large initial sample amount, many operation steps, low double-stranded RNA adsorption efficiency, etc., and achieves less initial sample amount , the effect of saving operation time

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this type of method has been proved to have a good effect on the extraction of double-stranded RNA of fungi and plant viruses, there are still the following problems: first, because the adsorption efficiency of cellulose powder to double-stranded RNA is not high, in order to obtain enough double-stranded RNA RNA is used for follow-up experiments, and such methods often require more biological materials for the extraction of double-stranded RNA
However, these improvements still have not fundamentally solved the problems of traditional methods with many steps, large initial samples, and limited number of processed samples.
In addition, since the improved method does not add additional steps to remove impurities such as proteins, it is bound to adversely affect the quality of double-stranded RNA

Method used

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  • Kit for quickly extracting double-stranded RNA of mycovirus and application of kit
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  • Kit for quickly extracting double-stranded RNA of mycovirus and application of kit

Examples

Experimental program
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Effect test

Embodiment 1

[0076] Embodiment 1 kit preparation (assembly) embodiment

[0077] 1. Cell lysate preparation:

[0078] a. Sodium lauryl sulfate 2% by weight / volume;

[0079] b. Polyvinylpyrrolidone - 404% by weight / volume;

[0080] c. Sodium chloride 0.5mol / L;

[0081] d. Tris hydrochloride 50mmol / L of pH8.0;

[0082] e.sodium edetate 25mmol / L at pH 8.0;

[0083] f. Mercaptoethanol 1% by volume / volume;

[0084] After accurately weighing medicines such as sodium lauryl sulfate, polyvinylpyrrolidone, and sodium chloride, add 1M trishydrochloride pH8.0 and 0.5M pH8. 0 sodium ethylenediaminetetraacetic acid solution to the final concentration of reagent 1 above, with deionized water as the solvent to dissolve the above drugs and then dilute to the required volume. Add mercaptoethanol to 1% by volume / volume before use;

[0085] 2. Preparation of nucleic acid adsorption solution:

[0086] a. Guanidine isothiocyanate 50% by weight / volume;

[0087] b. Potassium chloride 1.5mol / L;

[0088] ...

Embodiment 2

[0116] Embodiment 2 uses the kit of the present invention and application thereof to extract viral double-stranded RNA (application example 1) from the mycelia of R.

[0117]In this example, the pear tree ring spot (Botryosphaerin.dothidae) pathogen (EW2322) comes from the pear orchard of the Fruit Tree and Tea Research Institute of Hubei Academy of Agricultural Sciences, Jiangxia District, Wuhan City, Hubei Province, and is obtained from the branches of Jinshui No. 1 pear, and is now preserved in Huazhong Agricultural University National Fruit Tree Germplasm Resources Indoor Conservation Center, the method of isolating the ringworm of pear tree is a routine method reported, and these methods can be obtained from phytopathology, fruit tree pathology textbooks, operation manuals or public publications Guidance, separation of pear ringworm bacteria is not limited to the region, and all orchards with pear tree cultivation can be isolated, and the implementation of the present inve...

Embodiment 3

[0134] Embodiment 3 uses kit of the present invention to extract virus double-stranded RNA (application example 2) from Sclerotinia Ep-1PNA367 bacterial strain

[0135] (1) Sclerotinia sclerotiorum Ep-1PNA367 strain was isolated from Huazhong Agricultural University in 2009 from rape field sclerotinia plants, see literature: Huiquan Liu, Yanping Fu, Daohong Jiang, Guoqing Li, Jun Xie, Youliang Peng, Xianhong Yi, and Said A.Ghabrial. A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses. JOURNAL OF VIROLOGY, Feb. 2009, 1981-1991, Patent Document: Patent Application No. 201110321570.7, Isolation of Sclerotinia is a general method, generally can be isolated from the fields of rapeseed cultivation, there is no restriction on regionality, in addition, the test verification of the present invention is not limited to the above-mentioned Sclerotinia strains.

[0136] (2) Cultivation and collection of mycelium: inoculate the bacterial strain of...

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Abstract

The invention particularly relates to a kit for quickly extracting double-stranded RNA of a mycovirus and application of the kit. The kit comprises a cell lysis buffer, a nucleic acid adsorption solution, a nucleic acid cleaning solution and a double-stranded RNA agarose gel electrophoresis reagent. According to the invention, a new buffer solution system is adopted, a silicon film is used to replace cellulose powder to be used as an adsorption medium, organic reagents such as phenol are not required, through the reactions of the cell lysis buffer and the nucleic acid adsorption solution, impurities such as proteins, most DNA and polyose are removed in a chemical precipitation manner, and finally, by adjusting adsorption conditions through alcohol, the double-stranded RNA is effectively combined with the silicon film and the purified double-stranded RNA is obtained through elution. Through the adoption of the kit, high-quality double-stranded RNA can be prepared from a hypha sample with the weight as low as 0.3 g, and the whole extraction process is conducted within one hour; steps of organic reagent extraction and supernatant suction in a conventional method are eliminated, and all operations related to column-passing and the like are completed through centrifugation. The kit is suitable for high flux treatment of a large number of samples.

Description

technical field [0001] The invention belongs to the technical field of kit preparation, in particular to a kit for rapidly extracting viral double-strand RNA (double-strand RNA, dsRNA) from fungi and an application method thereof. Background technique [0002] During the multiplication process, single-stranded RNA viruses need to replicate their own nucleic acids, and then assemble to form new virus particles to complete the systemic infection of the host. Double-stranded RNA is a special class of RNA produced by viruses during this replication process. Its presence often indicates viral infection and replication. Therefore, viral double-stranded RNA has become an important basis for judging and identifying unknown single-stranded RNA viruses. In addition, since the double-stranded RNA directly contains the genome sequence of the virus, it is also of great significance to the molecular biology research of the virus. Therefore, the extraction of double-stranded RNA is part...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12R1/92
Inventor 杨帆洪霓王国平
Owner HUAZHONG AGRI UNIV
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