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Process for extracting potato virus RNA

A technology of potato virus and extraction method, which is applied in the direction of chemical instruments and methods, preparation of sugar derivatives, sugar derivatives, etc., can solve the problems of cumbersome operation process, high cost of RNA extraction, and difficult to be widely used, and achieve high purity, Effects of cost reduction and simplification of extraction steps

Inactive Publication Date: 2004-02-11
NANKAI UNIV +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the extraction process of this method is simpler and more economical than the guanidinium isothiocyanate method, the operation process is still very cumbersome, that is, glassware needs to be baked at 180°C for at least 8 hours, and centrifuge tubes and micropipettes that are not resistant to high temperatures Tips and experimental water are treated with DEPC
Therefore, the cost of RNA extraction is still high, and it is difficult to widely use in production
The above reasons make the current detection of potato virus by RT-PCR method limited.

Method used

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  • Process for extracting potato virus RNA
  • Process for extracting potato virus RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] 1. Weigh 0.15 g of potato leaf tissue provided by Tianjin Vegetables with potato virus Y, put it into an ice-precooled sterilized homogenizer, and grind it into a homogenate.

[0017] 2. Add 1ml RNA extraction buffer (Tris-HCl pH8.0 100mmol / L, NaCl100mmol / L, EDTA 10mmol / L) and 8μl β-mercaptoethanol to the homogenate, shake and mix in a centrifuge tube.

[0018] 3. Add 1ml of water-saturated phenol, 1ml of chloroform / isoamyl alcohol (49:1, v / v), mix thoroughly, place in ice bath for 10 minutes, and mix 2-3 times during the process.

[0019] 4. Centrifuge in a desktop high-speed centrifuge at 10,000 rpm for 15 minutes at room temperature. Aspirate the water phase, add 0.1 volume of NaAc (3mol / L, pH5.2) and an equal volume of isopropanol to the water phase, and mix well. Precipitate at -20°C for 3 hours.

[0020] 5. Centrifuge in a desktop high-speed centrifuge at 11,000 rpm for 20 minutes at room temperature, then discard the supernatant. Add 600 μl of ice-precooled 70...

Embodiment 2

[0029] Using the potato leaves with Potato S virus provided by Tianjin Vegetables as material, the Potato virus RNA was extracted by the simple method of the operation steps in Example 1, and the Potato virus RNA was extracted by the classic phenol extraction method as a control, and extracted by the above two methods Potato virus RNA was used as template for RT-PCR reaction. Simultaneously extract the viral RNA of non-infected tissue with parsimony method as negative control, RT-PCR result is as follows ( figure 2 ): figure 2 Potato S virus amplification product electrophoresis results. The four lanes in the figure are:

[0030] 1: Molecular weight standard;

[0031] 2: Potato virus S virus RNA was extracted by the classic phenol extraction method, and RT-PCR reaction was carried out using it as a template to obtain the target fragment specifically amplified by the virus;

[0032] 3: The new method extracts the potato S virus RNA, and uses it as a template to carry out ...

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Abstract

The process of extracting potato virus RNA includes grinding potato stem or leaf tissue in ice cooled sterilized homogenizer or mortar into homogeneous slurry, adding extracting buffering liquid and beta-mercaptoethanol to mixing homogeneously, adding water saturated phenol and chloroform or isoamylol, centrifugation at room temperature, adding NaAc and isopropropanol, precipitation at -20 deg.c for 3 hr, washing the precipitate with ethanol solution of 70 % concentration, dissolving with bidistilled bacteria-free water and other steps. The said process can obtain RNA with complete sequence and no degradation, and may be used in large-scale RT-PCR test of potato virus.

Description

Technical field: [0001] The invention relates to a method for extracting potato virus RNA. Background technique: [0002] At present, ELISA is the main method for detecting potato virus in production. However, the detection of potato virus by RT-PCR method is the preferred diagnostic method for RNA virus at home and abroad, which has the advantages of rapidity, sensitivity and reliability. The key to the detection of potato virus by RT-PCR is the extraction technology of potato virus RNA. In order to detect the presence of virus in potato tissue on a large scale in production practice, the RNA extraction technology used must have the characteristics of simplicity, reliability and low cost. [0003] There are two main routes for extracting plant RNA. (1) Guanidine isothiocyanate method. This method is suitable for RNase-rich tissues or tissues where nucleoplasm is not easily separated, but it is expensive and not suitable for production applications. (2) Phenol method. A...

Claims

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Application Information

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IPC IPC(8): C07H1/08C07H21/02
Inventor 杜荣骞李德森罗智敏俞键祁骥
Owner NANKAI UNIV
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