Method for extracting DNA of citrus canker by water bath precipitation

A technology of citrus canker fungus and extraction method, which is applied in the field of DNA water bath precipitation extraction of citrus canker canker sores, can solve problems such as difficulty in judging infected plants, complicated biochemical identification of citrus canker disease, etc., and achieves the effect of low experimental cost

Inactive Publication Date: 2012-10-03
GANNAN NORMAL UNIV
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Problems solved by technology

[0004] At present, most of the quarantine of citrus canker is based on field diagnosis, which can be judged by naked eyes under normal circumstances, but it is difficult to judge the infected plants with no obvious symptoms or no symptoms, and it is easy to be confused with other diseases. There are many types and varieties of bacteria, and the biochemical identification of citrus canker is very complicated and difficult

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  • Method for extracting DNA of citrus canker by water bath precipitation
  • Method for extracting DNA of citrus canker by water bath precipitation

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Embodiment 1

[0021] Embodiment 1: use the method described in the present invention to the extraction of canker sores citrus DNA:

[0022] 1. Weigh 0.12 g of fresh bacteria into a 1.5 mL centrifuge tube, add 1 mL of washing buffer, shake on the mixer for 30 s, then centrifuge at 6,000 rpm for 2 min to precipitate the bacterial cells, and discard the supernatant.

[0023] The wash buffer consists of: 50 mmol / L Tris, pH 7.7, 25 mmol / L EDTA, 0.1% PVP.

[0024] 2. Add 50 μL of lysis buffer to the washed sample, shake to suspend, and treat it in a water bath at 65 °C for 5-10 min to complete the reaction.

[0025] The composition of the lysis buffer is: 50 mmol / L Tris, pH 8.0, 25 mmol / L EDTA, 3.0% SDS, 1.2% PVP.

[0026] 3. Add 400 μL of extraction buffer preheated at 65 ℃, vortex and mix well, add an equal volume of saturated phenol:chloroform:isoamyl alcohol (25:24:1) solution for extraction, and centrifuge at 10,000 rpm for 5 min to denature the For protein precipitation, take the supernat...

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Abstract

The invention discloses a method for extracting DNA (Deoxyribonucleic Acid) of citrus canker by water bath precipitation. The method comprises the following steps: adding a washing buffer into a fresh thallus for washing; carrying out water bath by using a crashing buffer; extracting the buffer and a mixed solution of saturated phenol, chloroform and isoamyl alcohol; washing and precipitating ethanol; drying and adding sterilizing ddH2O for dissolving and precipitating; and preserving the precipitation at the temperature of -20 DEG C. According to the method disclosed by the invention, a template DNA of the citrus canker can be extracted quickly and effectively and the simpleness and quickness in operation are realized. The method dispenses with a special instrument, and thus the experiment cost is relatively-low. A DNA sample prepared by the method disclosed by the invention can meet the requirement of molecular diagnosis on the citrus canker by a PCR (Polymerase Chain Reaction) method can be met. Therefore, the invention constructs a method for quickly extracting the template DNA of the citrus canker by using a water bath method. The method provides a base and technical reserve for early-stage diagnosis and predicts detection applying the PCR technology.

Description

technical field [0001] The invention relates to a DNA extraction method, in particular to a DNA water-bath precipitation extraction method of canker sore citrus. Background technique [0002] Citrus canker is caused by Xanthomonas rugosa Xanthomonas axonopodis pv. citri ) caused by a quarantine disease at home and abroad, seriously affecting the safe production and foreign trade of citrus, and was included in the "List of Imported Plant Quarantine Pests of the People's Republic of China" published in 2007. [0003] The disease can be transmitted over long distances through citrus materials such as seedlings, scions and fruits that are infected with bacteria, and the occurrence is more serious in varieties such as snow orange, sweet orange, navel orange and Sijiyou. The pathogen that causes the disease survives the winter in the lesions of leaves, branches and fruits, and overflows from the diseased parts when the conditions are suitable in the next spring, spreads by wind...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 易龙陶珍珍卢占军
Owner GANNAN NORMAL UNIV
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