Method for quickly extracting DNA (deoxyribonucleic acid) of huanglongbing pathogen

A technology of Huanglongbing bacteria and extraction method, applied in the field of molecular biology, can solve the problems of endangering personal safety and high test cost, and achieve the effects of increasing yield, ensuring personal safety, and saving test cost

Active Publication Date: 2014-01-22
POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no matter which PCR method is used for detection, it needs to use the DNA of Huanglongbing bacteria as a template. First, the DNA of Huanglongbing bacteria must be extracte

Method used

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  • Method for quickly extracting DNA (deoxyribonucleic acid) of huanglongbing pathogen
  • Method for quickly extracting DNA (deoxyribonucleic acid) of huanglongbing pathogen
  • Method for quickly extracting DNA (deoxyribonucleic acid) of huanglongbing pathogen

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Embodiment 1

[0023] The samples to be tested are citrus leaves with typical symptoms of Huanglongbing collected from the Institute of Pomology, Pudang Academy of Agricultural Sciences, Jin'an District, Fuzhou City, from which the DNA of Huanglongbing bacteria was extracted. The specific operation is as follows:

[0024] (1) Take 0.4 g of the leaf midrib, cut it into pieces, put it into a 1.5 mL centrifuge tube, freeze and dry it, and grind it into powder with a grinder;

[0025] (2) Add 500 μL 0.5 M NaOH and an appropriate amount of quartz sand, place on a shaker at 200 rpm and mix thoroughly for more than 1 hour;

[0026] (3) Place in a boiling water bath for 30 s, take it out, add 500 μL of 0.5 M Tris-HCl with pH 8.0, invert and mix several times, and centrifuge at 12,000 rpm for 10 min;

[0027] (4) Take the supernatant, add 2 times the volume of absolute ethanol and 1 / 10 volume of 3 M pH 5.2 sodium acetate to precipitate nucleic acid, and place it at -20 °C for more than 0.5 h;

[002...

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Abstract

The invention discloses a method for quickly extracting the DNA (deoxyribonucleic acid) of huanglongbing pathogen. The method comprises the following steps: sequentially performing freeze-drying and grinding on orange leaves, roots and periwinkle leaves which are infected with huanglongbing, adding a proper amount of NaOH and a proper amount of quartz to separate the DNA of the huanglongbing pathogen through secondary grinding, performing high-speed centrifugation and absolute ethyl alcohol precipitation. The method is simple, safe, quick and low in cost; the extracted DNA solution can be directly used as a template for nest type PCR (polymerase chain reaction) and quantitative PCR detection; the method is suitable for extraction of the DNA of the huanglongbing pathogen in various tissues such as the orange leaves, the roots and the periwinkle leaves which are infected with the huanglongbing; furthermore, according to the extracting method, high-corrosivity reagents such as Tris saturated phenol and chloroform are not needed; the safety of people can be effectively guaranteed.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and specifically relates to a method for rapidly extracting Huanglongbing bacteria DNA, which is suitable for extracting Huanglongbing bacteria DNA from various tissues such as citrus leaves, roots, periwinkle leaves and the like infected with Huanglongbing. Background technique [0002] Citrus huanglongbing, also known as yellow shoot disease, has occurred and spread in more than 40 countries and regions in the world, including Asia, Africa and America. Among the 19 provinces that plant citrus in my country, 11 provinces (municipalities directly under the central government) have suffered from Huanglongbing, and the affected area accounts for more than 80% of the total citrus cultivation area. The pathogenic bacteria of this disease is a bacterium of the genus Phloembacterium, which can be divided into 3 strains, the Asian strain ( Candidatus Liberobacter asiaticus), African strains...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12R1/01
Inventor 林雄杰范国成胡菡青蔡子坚阮传清刘波
Owner POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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