Loop-mediated isothermal amplification (LAMP) detection method for specificity of radopholus similis and application thereof

A technology of banana perforator nematode and detection method, which is applied in the field of molecular biology detection of plant diseases, can solve the problem that banana perforator nematode cannot be applied in the first line of production, and achieve the effects of fast detection, clear and obvious results, and strong specificity

Inactive Publication Date: 2011-08-17
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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Problems solved by technology

[0011] The present invention provides a method for detecting the banana perforator nematode in diseased plants by applying constant temperature amplification technology to the defects in the above-mentioned fields, so as to establish an early, rapid and accurate detection and diagnosis of the banana perforator nematode that does not require a PCR instrument The molecular biology method can detect the banana nematode in the diseased plant tissue, achieve the effect of early detection, early treatment and early control, and overcome the problem that the existing PCR detection of the banana nematode cannot be applied in the production line

Method used

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  • Loop-mediated isothermal amplification (LAMP) detection method for specificity of radopholus similis and application thereof
  • Loop-mediated isothermal amplification (LAMP) detection method for specificity of radopholus similis and application thereof
  • Loop-mediated isothermal amplification (LAMP) detection method for specificity of radopholus similis and application thereof

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Embodiment 1

[0074] Example 1 DNA Extraction, rDNA-ITS Amplification, Sequence Analysis and Registration of Banana Forerunculus Nematode

[0075] 1.1 Extraction of DNA from Banana perforator nematode

[0076] The DNA extraction of all samples was based on the method of Peng Deliang et al. (2003) with slight modifications. Pick a single nematode and put it into an Eppendorf tube filled with 10 μl ddH2O, freeze overnight at -2°C, take it out, grind it with a glass rod sterilized with 75% alcohol until the ice melts, add 7 μl WLB (worm lysis buffer) and 3 μl protease K (2mg / ml) solution, mix well and centrifuge at 5000rmp, freeze at -°C for more than 30 minutes, then transfer to a PCR instrument, incubate at 6°C for 60 minutes, react at 9°C for 10 minutes, take it out and centrifuge it, take the supernatant for PCR Amplify or store at -2°C for later use.

[0077] 1.2 Amplification and Sequence Analysis of rDNA-ITS of Nematode perforatus

[0078] The general primers D2A (5'-ACAAGTACCGTGAGGG...

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Abstract

The invention relates to a loop-mediated isothermal amplification (LAMP) detection method for the specificity of radopholus similis and application thereof, and belongs to the technical field of biology. In the LAMP method for the specificity of the radopholus similis, LAMP detection primers for the specificity of the radopholus similis in a reaction system are RSF3, RSB3, RSFIP, RSBIP and RSLF respectively, and the radopholus similis is detected quickly and specifically by extracting the DNA of the radopholus similis or single-head radopholus similis in diseased plant tissues and performing LAMP isothermal amplification. Therefore, the detection method is high in specificity and sensitivity and low in cost, is easy and convenient to operate, and has the high application value in aspects of the on-site quick detection of the radopholus similis and the early diagnosis of radopholus similis diseases.

Description

technical field [0001] The invention relates to molecular biology detection technology of plant diseases, in particular to a specific LAMP detection primer and a rapid molecular detection method for LAMP of banana perforator nematode Background technique [0002] Radopholus similis, also known as Radopholus similis, is a destructive alien invasive plant quarantine harmful nematode, and is one of the foreign plant quarantine objects in my country. The host range of Radopholus similis is also very wide, and there are more than 360 kinds of hosts reported so far, mainly distributed in tropical and subtropical regions. [0003] In recent years, with the increasing frequency of international trade and overseas travel, the types and quantities of various flower seedlings and germplasm materials introduced from countries or regions affected by the banana borer nematode have increased rapidly, so the nematode was introduced into China by the host plant The possibility and risk of i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 彭德良彭焕王琼
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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