Detection method of early molecule of radopholus similes thorne

A technology for detecting banana perforating nematodes and molecules is applied in the field of molecular biology detection of plant diseases, which can solve the problems of detecting banana perforating nematodes and the like, and achieve the effect of accurate detection technology.

Inactive Publication Date: 2010-08-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But banana borer nematodes were not detected from diseased plant tissue
Zhou Chunna, Xie Hui et al. (2006) use

Method used

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  • Detection method of early molecule of radopholus similes thorne
  • Detection method of early molecule of radopholus similes thorne
  • Detection method of early molecule of radopholus similes thorne

Examples

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Effect test

Embodiment 1

[0063] Example 1 DNA Extraction, rDNA-ITS Amplification, Sequence Analysis and Registration of Banana Forerunculus Nematode

[0064] 1.1 Extraction of DNA from Banana perforator nematode

[0065] 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.

[0066] 1.2 Direct extraction of the DNA of the banana nematode from the diseased plant tissue

[0067] Using t...

Embodiment 2

[0070] Example 2 Screening and Detection of Banana Perforator Nematode Specific Primers

[0071] 2.1 Design and screening of specific primers for the banana borer nematode

[0072] 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 nematode 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 specific frag...

Embodiment 3

[0094] Embodiment 3 Specific direct detection of banana perforator nematode in diseased plant tissue

[0095] 3.1 Direct detection of healthy plant tissues after artificial addition of the banana perforator nematode

[0096] 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.

[0097] PCR reaction system: 5μl 10×PCR Buffer (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 cycl...

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Abstract

The invention discloses a detection method of an early molecule of radopholus similes thorne, which belongs to the biotechnical field and relates to a radopholus similes thorne DNA extraction solution formula and an extraction method in a morbidity plant tissue, specific primers RsF1/RsR1 sequences of the radopholus similes thorne, a formula of a one-step double polymerase chain reaction (PCR) reaction solution for combining specific primers of the radopholus similes thorne and primers of D3A and D3B as well as a detection technical method of a rapid specific amplification procedure. The detection method is rapid and accurate while improving the detection sensitivity of the molecule and has high application value in the aspects of the detection on the radopholus similes thorne and early diagnosis of radopholus similes thorne disease.

Description

technical field [0001] The present invention relates to molecular biological detection technology of plant diseases, in particular to a DNA extraction solution, extraction method and early molecular detection method of banana perforator nematode Background technique [0002] Banana piercing nematode (Radopholus similis), English common name the burrowing nematode; Blackhead ortoppling disease of bananas), also known as similar piercing nematode, it is a very destructive alien invasive plant quarantine harmful nematode, and it is also the object of foreign plant quarantine in my country [58] . Banana punch nematode (Radopholus similis) is a migratory endoparasitic nematode distributed in tropical and subtropical regions of the world [5] . Its host range is also very wide, and there are more than 360 kinds of reported hosts, most of which are accidental hosts (existing near the diseased banana tree) and artificially inoculated hosts. [0003] In recent years, with my countr...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C12Q1/04
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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