Method for checking cucumber fusarium axysporum molecules

A technology for the detection of Fusarium wilt of cucumber and molecular detection is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection. The effect of high stability and stability, not easy to contaminate

Inactive Publication Date: 2010-04-28
INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the invention is to solve the problem that the PCR-RFLP method used in the existing cucumber wilt molecular detection method has a weak fungal general primer specificity and cumbersome methods, and the nested PCR method needs 2 steps of PCR amplification and is easy to pollute. A molecular detection method for cucumber wilt pathogen is provided

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  • Method for checking cucumber fusarium axysporum molecules
  • Method for checking cucumber fusarium axysporum molecules
  • Method for checking cucumber fusarium axysporum molecules

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specific Embodiment approach 1

[0016] Specific embodiment one: present embodiment cucumber fusarium wilt molecular detection method is realized by the following steps: one, according to the characteristics of the ribosome transcribed spacer (ITS) DNA sequence of cucumber fusarium wilt, design a Fusarium wilt tool specific amplification effect For primers Fc-1 and Fc-2; 2. Extract the genomic DNA of the infected sample, and use primers Fc-1 and Fc-2 to carry out PCR amplification of the genomic DNA; 3. Put the PCR amplification product in 1.0% of GelRed dye Use 1×TAE buffer for electrophoresis on the agarose gel. After the electrophoresis, analyze it with a gel imaging system. If a specific band appears at 315bp, the infected sample contains Fusarium wilt, and the detection is completed; the Fc in step 1 -1 is 5`-CATACCACTTGTTGCCTC-3`, Fc-2 is 5`-ATTAACGCGAGTCCCCACC-3`; PCR amplification reaction system in step 2: 25μl reaction system, 2.5μl of 10×PCR buffer (200mM Tris-HCl pH 8.4, 200mM KCl, 100mM (NH 4 ) ...

specific Embodiment approach 2

[0022] Specific embodiment two: what this embodiment is different from specific embodiment one is that the ribosome transcribed spacer (ITS) DNA sequence of cucumber fusarium wilt bacterium in step 1 is:

[0023] cataccactt gttgcctcgg cggatcagcc cgctcccggt aaaacgggac ggcccgccag

[0024] aggacccccta aactctgttt ctatatgtaa cttctgagta aaaccataaa taaatcaaaa

[0025] ctttcaacaa cggatctctt ggttctggca tcgatgaaga acgcagcaaa atgcgataag

[0026] taatgtgaat tgcagaattc agtgaatcat cgaatctttg aacgcacatt gcgcccgcca

[0027] gtattctggc gggcatgcct gttcgagcgt catttcaacc ctcaagcaca gcttggtgtt

[0028] gggactcgcgttaat. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0029] Embodiment 3: The difference between this embodiment and Embodiment 1 or 2 is that the genomic DNA of the infected sample is extracted in step 2 by freezing 20 mg of the infected sample at -70°C for 3 hours, taking it out and putting it into a sterilized mortar for grinding , add 500μl of TES buffer (100mM Tris, pH8.0, 10mM EDTA, 2% SDS, 100μg proteinase K), then transfer the solution into a centrifuge tube and incubate in a 60°C water bath for 1h, take it out and add 140μl of 5M NaCl and 65 μl of 10% CTAB, then put it in a 65°C water bath and incubate for 10 minutes, then add 700 μl of chloroform and place it at 0°C for 30 minutes, take it out and centrifuge at 4°C for 10 minutes, transfer the upper layer solution to a new sterilized centrifuge tube, and add RNA Add 5 μl of enzyme, incubate at 37°C for 1 hour, add 510 μl of isopropanol and place on ice for 30 minutes to precipitate DNA, then centrifuge at 4°C and 12,000 r / min for 10 minutes, collect the precipitate, and...

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Abstract

The invention relates to a method for checking cucumber fusarium axysporum molecules. The invention solves the problems that the specificity of fungi general primers is not strong and the method is complicated by using the PCR-RFLP method and the nested PCR method needs two steps for amplification and is easy to pollute environment in the existing methods for checking the cucumber fusarium axysporum moleculars. The method for checking the cucumber fusarium axysporum moleculars comprises the following steps: (1) primers Fc-1 and Fc-2; (2) extracting ailing sample genome DNA to perform the PCR amplification; and (3) performing electrophoresis for a PCR amplification product and analyzing the product by a gel imaging system, wherein if a special strip appears at the position of 315bp, the ailing sample is provided with the cucumber fusarium axysporum, therefore the checking step is finished. The primers Fc-1 and Fc-2 have high specificity and sensitivity; and the checking result can be obtained in 2 hours with only one step, and the method is fast, simple and not easy to pollute environment.

Description

technical field [0001] The invention relates to a method for detecting cucumber wilt pathogen. Background technique [0002] Cucumber wilt is a worldwide soil-borne fungal disease caused by the parasitism of Fusarium oxysporum (Fusarium oxysporum, also known as Fusarium oxysporum). It can occur during the whole growth period of the plant, especially in the adult stage. [0003] Cucumber Fusarium wilt is particularly serious. Once it occurs, it is difficult to eradicate, and with the increase of continuous cropping years, the incidence rate increases. It is a stubborn soil-borne disease. The diseased field generally reduces production by 20% to 30%, and the severe field can reach 50%. % to 60%, or even extinction, is the main reason for the large-scale reduction in cucumber production. Therefore, it is of great theoretical and practical value to detect and diagnose the early stage of Fusarium wilt infection early and establish a rapid and accurate early warning diagnosis me...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/77
Inventor 张淑梅赵晓宇王玉霞李晶张先成孟利强
Owner INST OF MICROBIOLOGY HEILONGJIANG ACADEMY OF SCI
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