DNA extract of radopholus similis in morbid plant tissues, extraction method and application thereof

A technology for banana perforating nematodes and plant tissue, which is applied in the field of molecular biology detection of plant diseases, can solve problems such as difficulties in effective extraction and detection, and no detection of banana perforating nematodes, etc., and achieves the effect of ensuring sensitivity and specificity

Inactive Publication Date: 2012-03-21
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] However, there is currently no report on the detection of banana borer nematodes from diseased plant tissues
For the early detection of the infestation of banana nematodes from diseased plants, in addition to the necessary condition of banana-specific primers, it must first be able to effectively extract the DNA of banana nematodes from the diseased host plants, because in the extraction process The ratio of nematode DNA to plant DNA is quite different, and protein, plant phenols and other substances in plant tissues are easy to cause nematode DNA to degrade during the extraction process. When detecting early-infected plants, since there are very few parasitic nematodes, effective extraction And detecting the DNA of the banana borer nematode was even more difficult

Method used

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  • DNA extract of radopholus similis in morbid plant tissues, extraction method and application thereof
  • DNA extract of radopholus similis in morbid plant tissues, extraction method and application thereof
  • DNA extract of radopholus similis in morbid plant tissues, extraction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 DNA Extraction, rDNA-ITS Amplification, Sequence Analysis and Registration of Banana Forerunculus Nematode

[0068] 1.1 Extraction of DNA from Banana perforator nematode

[0069] The DNA extraction of all samples was based on the method of Peng Deliang et al. (2003) with slight modifications. Pick one to 10 nematodes and put them into an Eppendorf tube containing 10 μl ddH2O, freeze overnight at -20°C, take it out, grind it with a glass rod sterilized with 75% alcohol until the ice cubes melt, add 7 μl WLB (worm lysis buffer) and 3μl proteinase K (2mg / ml) solution, mix well and centrifuge at 5000rmp, freeze at -20°C for more than 30min, then transfer to a PCR instrument, incubate at 65°C for 60min, react at 95°C for 10min, take it out and centrifuge it, take the The supernatant was subjected to PCR amplification or stored at -20°C for future use.

[0070] 1.2 Direct extraction of the DNA of the banana nematode from the diseased plant tissue

[0071] Using t...

Embodiment 2

[0074] Example 2 Screening and Detection of Banana Perforator Nematode Specific Primers

[0075] 2.1 Design and screening of specific primers for the banana borer nematode

[0076] The seven ITS sequences of the banana perforator nematode amplified by the invention unit (NCBI accession numbers: EF208207, EF208208, EF517226, EF208209, EF208206, EF517228, EF517227, see the appendix for specific sequences) and the ITS sequences downloaded from the NCBI gene bank belonging to the banana perforator The ITS sequences (NCBI accession numbers: AF375369, AF375386, AF3753687, AF375389, AF375391, AF375393) of 6 closely related species of the genus Nematode were compared and analyzed, and a pair of specific primers RsF1 / RsR1 were designed and screened out. The sequence of the upstream specific primer (RsF1) of the nematode perforatus is located at 208bp to 228bp of the ITS1 sequence, the downstream specific primer (RsR1 is located at 458bp to 479bp, and the length of the amplified specifi...

Embodiment 3

[0098] Embodiment 3 Specific direct detection of banana perforator nematode in diseased plant tissue

[0099] 3.1 Direct detection of healthy plant tissues after artificial addition of the banana perforator nematode

[0100] Add 20 banana perforator nematodes per 0.05g root system, extract DNA according to the method described in 1.2, and use it as a template for specific detection. During the PCR process, BSA (bovine serum albumin), a substance that reduces the inhibitory effect of the PCR reaction, is added, and the number of PCR cycles is increased, and the template concentration is appropriately reduced.

[0101] PCR reaction system: 5μl 10×PCRBuffer (containing Mg 2+ ), 4μl 10mM dNTP (2.5mM), 1μl 0.1% BSA, 1.5μl primer pair RsF1 / RsR1 (20uM), 0.5μl Taq DNA polymerase (5U / ul), 1μl different concentrations of template DNA, sterilized ddH 2 O to make up to 25 μl. Negative worm DNA template was used as negative control. Amplification was performed on an Eppendorf PCR cycle...

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Abstract

The invention discloses 'a DNA extract of radopholus similis in morbid plant tissues, an extraction method and application thereof', and relates to a molecular biology detection technology of plant diseases. The DNA extract contains the following components with final concentration: 200mM of Tris-HCl with the PH of 8.0, 400mM of NaCl, 10mM of ethylene diamine tetraacetic acid (EDTA) with the PH of 8.0, 3 percent (W / V) of cetyltrimethyl ammonium bromide (CTAB) and 4 percent (V / V) of beta-mercaptoethanol. The extract serving as a lysis solution of the morbid plant radopholus similis DNA can be used for perfectly extracting the DNA of a little radopholus similis of the infected plant tissues, and is not degraded by phenols, proteases, degrading enzymes and the like of the plant tissues. According to the DNA extract and the extraction method, radopholus similis infection is early monitored by adopting molecular biology means of polymerase chain reaction (PCR) and the like, so that the infection is early controlled and prevented.

Description

[0001] This application is a divisional application of an invention patent application with the original application number "201010113930.X" and the title "A Method for Early Molecular Detection of Banana Perforator Nematode". technical field [0002] The invention relates to molecular biology detection technology of plant diseases, in particular to a DNA extracting solution of the nematode nematode nematode in diseased plant tissues, an extraction method and application thereof. Background technique [0003] Banana punch nematode (Radopholus similis), English common name the burrowing nematode; Blackhead or toppling disease of bananas), also called similar punching nematode, it is a kind of extremely destructive alien invasive plant quarantine harmful nematode, and it is also the burrowing nematode of our country. object [58] . Banana punch nematode (Radopholus similis) is a migratory endoparasitic nematode distributed in tropical and subtropical regions of the world [5] ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
Inventor 彭德良王琼耿丽凤李佳彭焕张东升
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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