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Method for detecting pathogen of sugarcane ratoon stunting disease

A technology for pathogenic bacteria and sugarcane, which is applied in the field of rapid detection of molecular biology, can solve the problems of long time consumption, complicated operation and low specificity, and achieves the effects of simple operation, wide popularity and high specificity.

Inactive Publication Date: 2011-03-09
FUJIAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the defects of cumbersome operation and low specificity and time-consuming of current sugarcane dwarf disease pathogenic bacteria detection technology, and to develop a rapid and accurate detection method of molecular biology for detecting sugarcane perennial dwarf disease

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Select the sugarcane strain 1 suspected of containing dwarf disease pathogenic bacteria in the perennial root cultivation sugarcane, take the sugarcane stem at the third section from the root, wash the surface impurities, and peel off the skin. Wipe and disinfect with 70% ethanol.

[0035] 2. Cut the sterilized sugarcane into small pieces, put them into a mortar, add 100ml of liquid nitrogen to quick-freeze and grind to powder.

[0036] 3. Put 10 grams of powder into a sterilized 50ml centrifuge tube, add 20ml TE buffer solution, and heat in a water bath at 80°C for 60min. In a centrifuge at room temperature, centrifuge at a centrifugal force of 12000×g for 10 min. Take 20ml of the supernatant to another sterilized 50ml centrifuge tube.

[0037] 4. Add 20ml of β-mercaptoethanol chloroform solution, and centrifuge at a centrifugal force of 12000×g for 10 minutes in a normal temperature centrifuge. Take 15ml of the supernatant to another sterilized 50ml centrifuge t...

Embodiment 2

[0046]1. Select the perennial sugarcane strain 2 that is suspected to contain the pathogenic bacteria of dwarf disease, and get the sugarcane stem at the third section from the root, clean the surface impurities, and peel off the skin. Wipe and disinfect with 70% ethanol.

[0047] 2. Cut the sterilized sugarcane into small pieces, put them into a mortar, add 100ml of liquid nitrogen to quick-freeze and grind to powder.

[0048] 3. Put 10 grams of powder into a sterilized 50ml centrifuge tube, add 20ml TE buffer solution, and heat in a water bath at 80°C for 60min. In a centrifuge at room temperature, centrifuge at a centrifugal force of 12000×g for 10 min. Take 20ml of the supernatant to another sterilized 50ml centrifuge tube.

[0049] 4. Add 20ml of β-mercaptoethanol chloroform solution, and centrifuge at a centrifugal force of 12000×g for 10 minutes in a normal temperature centrifuge. Take 15ml of the supernatant to another sterilized 50ml centrifuge tube.

[0050] 5. A...

Embodiment 3

[0058] 1. Select the perennial sugarcane strain 3 that is suspected to contain the pathogenic bacteria of dwarf disease, and get the sugarcane stem at the third section from the root, clean the surface impurities, and peel off the skin. Wipe and disinfect with 70% ethanol.

[0059] 2. Cut the sterilized sugarcane into small pieces, put them into a mortar, add 100ml of liquid nitrogen to quick-freeze and grind to powder.

[0060] 3. Put 10 grams of powder into a sterilized 50ml centrifuge tube, add 20ml TE buffer solution, and heat in a water bath at 80°C for 60min. In a centrifuge at room temperature, centrifuge at a centrifugal force of 12000×g for 10 min. Take 20ml of the supernatant to another sterilized 50ml centrifuge tube.

[0061] 4. Add 20ml of β-mercaptoethanol chloroform solution, and centrifuge at a centrifugal force of 12000×g for 10 minutes in a normal temperature centrifuge. Take 15ml of the supernatant to another sterilized 50ml centrifuge tube.

[0062] 5. ...

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PUM

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Abstract

The invention relates to a rapid detection method of the pathogen of the sugarcane ratoon stunting disease. The method is characterized in that a sugarcane sample is frozen quickly with liquid nitrogen to perform grinding treatment, four specific primers are designed according to the ITS sequence of the pathogen of the sugarcane ratoon stunting disease, the loop-mediated isothermal amplification of the target fragment of the pathogen is performed; and the reaction product of the loop-mediated isothermal amplification is detected and identified, fluorescent dye GlodView is added in the reaction tube, and the judgement is made through visual inspection under the ultraviolet light of a money genuineness testing pencil. The detection method of the invention has reliability, high sensitivity, short detection time and low cost, and can diagnose the sugarcane ratoon stunting disease accurately and rapidly. The method does not require other special instruments, is convenient to operate and is particularly suitable for the field rapid and accurate detection of the sugarcane ratoon stunting disease.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a rapid detection method of molecular biology of sugarcane dwarf disease pathogenic bacteria. Background technique [0002] Sugarcane dwarf disease is one of the important diseases affecting sugarcane yield, and its pathogenic bacteria are Leifsonia xyli subsp. xyli (Lxx), its pathogenic mechanism is due to pathogenic bacteria Lxx parasitizing on sugarcane ( Saccharum officinale ) in the xylem, secrete and produce gelatinous substances, which block the xylem ducts of sugarcane, resulting in the inability of nutrients to be transported upwards, resulting in dwarfing of sugarcane and stunted growth. The growth process of sugarcane is often mistaken for insufficient soil fertility, poor management and other reasons, resulting in misjudgment . The infection rate of dwarf disease pathogens in sugarcane grown on perennial roots can reach 30%, resulting in a significant reduc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 黄镇黄祖新陈由强许利萍陈如凯
Owner FUJIAN NORMAL UNIV
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