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Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense

A technology of banana fusarium wilt and detection method, applied in the field of rapid detection of banana fusarium wilt No. 4 race, can solve the unfavorable problems of timely prevention and control of banana fusarium wilt, long-term and other problems, and achieve easy operation, accurate detection, and simple steps Effect

Inactive Publication Date: 2010-12-08
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Even if the No. 4 race is determined by the isolation of pathogenic bacteria and the traits on VCG or Komada’s improved medium, it will take a long time, usually 30 days or more to identify and confirm the diagnosis, which is not conducive to the timely prevention and control of banana wilt

Method used

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  • Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense
  • Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense
  • Method for detecting No.4 race of Fusarium oxysporum f sp. Cubense

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Effect test

Embodiment 1

[0036] Embodiment 1 The inventive method specificity, accuracy experiment

[0037] The specific electropherogram detection of PCR detection is carried out in 23 bacterial strains by applying the method of the present invention.

[0038] 1. Extract the DNA of the plant: proceed according to the conventional CTAB method.

[0039] 2. Electrophoresis detection of template DNA: Take 5 μL of the extracted DNA sample and mix it with loading buffer, electrophoresis with 1% agarose gel for 40 minutes, 5v / cm, and observe it under the gel imaging system. If there is a DNA band, If the DNA is successfully extracted, it can be used continuously. If there is no DNA band, it needs to be extracted again.

[0040] 3. PCR amplification: DNA amplification reaction system is as follows:

[0041] Template DNA 1 μL

[0042] 10×PCR Buffer 2.5μL

[0043] Mg 2+ (25mmoL / L) 2.5μL

[0044] dNTPs (25mmoL / L) 2μL

[0045] Primer RF4-1 (10μmoL / L) 1μL

[0046] Primer RF4-1 (10μmoL / L) 1μL

[0047] Taq...

Embodiment 2

[0055] Embodiment 2 detects different concentration pathogenic bacteria DNA with PCR method

[0056] The experimental procedure is the same as that in Example 1, and PCR is used to detect different concentrations of pathogenic bacteria DNA, and the sensitivity electrophoresis results are shown in the attached figure 2 , where, M: DL2000 Marker; 1-7: FOC4 total DNA dilution gradient (10 0 -10 -6 ); 8: clear water control. attached figure 2 Indicates: DNA extraction solution is diluted 10 4 After doubling, the specific band of 903bp can still be amplified, that is, fresh mycelium equivalent to 2ng can be detected, which illustrates the sensitivity of the method of the present invention.

Embodiment 3

[0058] (1) DNA extraction from banana plants

[0059] According to the conventional CTAB method, grind 100-200 mg of the banana tissue to be tested into powder with liquid nitrogen, add 600 μL reagent CTAB and 12 μL 2-mercaptoethanol, transfer the grinding solution into a 1.5 mL centrifuge tube, bathe in 65 ° C for 30 minutes, add 600 μL The phenol / chloroform mixture was centrifuged at room temperature and 12,000 rpm for 5 minutes, and the supernatant was taken and placed in a new centrifuge tube. Add 1 / 10 volume of 3mol / L, pH5.2 NaAc and 2 volumes of cold absolute ethanol, set at -20°C for precipitation for 20 minutes; centrifuge at 4°C and 12000rpm for 10 minutes, rinse the DNA precipitate with 70% ethanol respectively , air-dried at room temperature; dissolved in 50 μL sterile double-distilled water to obtain a DNA solution.

[0060] (2) Detection of template DNA by electrophoresis

[0061] Take 5 μL of the extracted DNA sample and mix it with loading buffer, electrophore...

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Abstract

The invention discloses a rapid detection method of No.4 race of fusarium oxysporum f.sp.cubense, including the steps of extraction of DNA of banana plants, electrophoresis detection of the template DNA, PCR amplification of DNA amplification, PCR circulation and electrophoresis detection, the invention makes use of a primer of the No.4 race of the fusarium oxysporum f.sp.cubense to carry out thePCR amplification and circulation of the DNA of the banana plants, and then the electrophoresis detection is carried out, the whole detection process consumes the time of less than 6 hours, which greatly improves the detection efficiency, the invention does not need to carry out the separation and the purification of pathogens in the sample, the steps are simple, the operation is easy, the complicated and precise instruments are unnecessary, the invention is more accurate and sensitive, so the invention is more conductive to the rapid and convenient development of a plant quarantine work.

Description

technical field [0001] The invention relates to the fields of plant protection and biotechnology, in particular to a rapid detection method for No. 4 race of Fusarium wilt fungus. Background technique [0002] Banana fusarium wilt, also known as banana Panama disease and yellow leaf disease, is a soil-borne disease caused by fusarium infection and necrosis of vascular bundles. The pathogen is Fusarium oxysporum f.sp.cubense . The pathogen invades from the injured rhizome, develops to the stem through the host vascular bundle, and further spreads to the leaves. When the plant dies, the pathogenic bacteria remains in the soil with the remains, and then spreads naturally in the banana orchard along with the water flow or the infected agricultural tools, and its chlamydospores can survive in the soil for 8-10 years. Because the bacteria has a long survival period and spreads rapidly, there is no specific drug to prevent and treat the disease, so it is easy to cause large-scale...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447C12Q1/02
Inventor 王振中廖林凤纪春艳董章勇
Owner SOUTH CHINA AGRI UNIV
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