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Primer for detecting aphelenchoides besseyi in rice seeds and seedlings and application thereof

A technology for rice nematodes and nematodes, which is applied in the field of molecular biology, can solve the problems of shortening detection time, long time consumption, etc., and achieves the effects of high sensitivity and fast detection speed.

Inactive Publication Date: 2018-05-04
INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional quarantine method uses the Behmann funnel method to detect nematodes in rice seeds, which takes 1 to 3 days, which takes a long time. How to detect a very small amount of nematodes from a large number of seeds and single seedlings, and shorten the detection time , is a difficult point in the quarantine of D.

Method used

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  • Primer for detecting aphelenchoides besseyi in rice seeds and seedlings and application thereof
  • Primer for detecting aphelenchoides besseyi in rice seeds and seedlings and application thereof
  • Primer for detecting aphelenchoides besseyi in rice seeds and seedlings and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The design of embodiment 1 rice stem sharp nematode specific primer

[0030] Reference ITS sequence for primer design: 6 other species (Aphelenchoides besseyi, KJ009342, EU186069, JF826517, JF826518, JF826519, JF932290, KP757371, KP757372, KP757373) of the same genus (Aphelenchoides besseyi, KJ009342, KP757373) and other 6 species (Aphelenchoides 1, Aphelenia 9choides 1, 1 菊花滑刃线虫(Aphelen choides ritzemabosi,EU186068),花生滑刃线虫(Aphelenchoides arachidis,KC771278),Aphelenchoides paradalianensis(HQ454505),Aphelenchoidesbicaudatus(GU984233),Aphelenchoides subtenuis(EF213109)的线虫和同科的松材线虫(Bursaphelenchus xylophilus, KP644770).

[0031] The software used in this experiment is the Primer designing tool of NCBI, and the DNA sequence used is the KJ009342 sequence of the rice stem nematode, which consists of part of the 18S rDNA sequence, ITS1, 5.8S rDNA sequence, ITS2, and part of the 28S rDNA sequence. .

[0032] Both the forward and reverse primers of the present invention can comp...

Embodiment 2

[0033] The detection of the dry sharp nematode of embodiment 2 mass culture

[0034] Extraction of a large amount of nematode DNA:

[0035] F8 (Liaoning japonica rice), Ningjing48 (Ningxia japonica rice), cy44 (Jiangsu japonica rice), Mianzhu (Sichuan indica rice) were selected as nematode strains, with 3 replicates for each line.

[0036] 1) Collect 100 μL of mass-cultured A. sativa in a 2 mL centrifuge tube;

[0037] 2) After quick-freezing in liquid nitrogen, add 700 μL of DNA extraction buffer, mix well, add 5 μL of 20 mg / mL proteinase K, and mix well;

[0038] 3) In a water bath at 55°C for 30 minutes, add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1) extract and mix well;

[0039] 4) Centrifuge at 12000rpm for 10min at room temperature, and take the supernatant into a new 1.5mL centrifuge tube;

[0040] 5) Add 0.8 times the volume of frozen isopropanol and 0.1 times the volume of 3M sodium acetate (pH5.2), and mix well;

[0041] 6) After standing at ...

Embodiment 3

[0052] The detection of embodiment 3 single stem sharp nematodes

[0053] Extraction of single nematode DNA:

[0054] F8 (Liaoning japonica), Ningjing48 (Ningxia japonica), cy44 (Jiangsu japonica) and Mianzhu (Sichuan indica) were selected as nematode strains, with 5 replicates for each line. The method refers to the single nematode DNA extraction method of Wang Jiangling et al. (2011).

[0055] a. Pick a single nematode and add 8μL ddH 2 O and 1 μL of 10×PCR Buffer (Mg2+free) in a PCR tube;

[0056] b. Quickly freeze in liquid nitrogen for 1 minute, then heat at 85°C for 2 minutes;

[0057] c. Add 1 μL 20mg / mL proteinase K to the PCR tube;

[0058] d. Heat at 56°C for 15 minutes and at 95°C for 10 minutes; the obtained DNA extract can be directly used for PCR amplification.

[0059] PCR amplification of D. elegans DNA:

[0060] PCR system: same as PCR to amplify single-headed nematode DNA. PCR amplification program: 94°C, 4min; 94°C for 30s, 55-60°C for 30s, 72°C for 4...

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Abstract

The invention discloses a primer for detecting aphelenchoides besseyi in rice seeds and seedlings and application thereof. Alkaline base sequences of the primer are shown in SEQ ID NO:1 and SEQ ID NO:2. The primer has the advantages that the primer can be used for detecting the aphelenchoides besseyi in the rice seeds and seedlings, and a kit for detecting the aphelenchoides besseyi in the rice seeds and seedlings can be prepared; compared with the prior art, the detection speed is quick, and the specificity is good; the popularization and application value is realized.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a primer for detecting the nematode nematode in rice seeds and seedlings and an application thereof. Background technique [0002] Rice apical nematode is one of the most widely distributed plant parasitic nematodes in the world. It can occur in upland rice, irrigated rice and deep-water rice. Generally, the yield is reduced by 10% to 20%, and in serious cases it can reach more than 30%. The rice stem nematode cannot survive in water and soil for a long time, so the irrigation water and soil spread less, and the infected rice seeds are the primary source of infection. Although A. sativa has been detected in seeds in many places now, A. sativa is still a quarantine nematode, because A. sativa is mainly spread through seeds with insects. [0003] The morphological identification characteristics of D. oryzae sativa need to be observed with a high-power microscope, and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 杨芳姬红丽谢家廉
Owner INST OF PLANT PROTECTION SICHUAN ACAD OF AGRI SCI
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