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Rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and application of rapid PCR molecular detection method

A technology for molecular detection and D. destructor, which is applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem that there is no specific PCR detection method that can simultaneously detect type A and type B potato nematodes

Inactive Publication Date: 2014-04-23
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

[0006] There have been research reports on the ITS region of D. destructor ribosomal DNA at home and abroad, but most of the primers developed so far can only detect a certain type of D. Specific PCR detection method for D. destructor

Method used

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  • Rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and application of rapid PCR molecular detection method
  • Rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and application of rapid PCR molecular detection method
  • Rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and application of rapid PCR molecular detection method

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: the establishment of D. destructor specific PCR molecular detection method of potato

[0036] 1.1 Extraction of Nematode DNA

[0037] Put the single-headed nematodes of the D. hand elegans population into a centrifuge tube containing 7 μl of 10× PCR buffer, 3 μl of proteinase K solution (600 μg / ml) and 10 μl of sterilized double-distilled water, and freeze in a -20°C refrigerator for 1 hour . Glass rods sterilized with 75% alcohol were rolled until the ice melted and then frozen at -20°C for at least 2 h. After 2 hours, the centrifuge tube was taken out of the refrigerator, incubated at 65°C for 1.5h, then at 95°C for 10 minutes, and finally centrifuged at 10,000 rpm for 1 minute, and the supernatant was stored at -20°C for later use.

[0038] 1.2 Design and screening of specific primers for D. destructor

[0039] Using general primers TW81 (5'-TTGATTACGTCCCTGCCCTTT-3') and AB28 (5'TTTCACTCGCCGTTACTAAGG-3'), the ITS sequence of D. destructor was amplifi...

Embodiment 2

[0046] Example 2: Specificity and sensitivity detection of D. destructor PCR molecular detection method.

[0047] 2.1 Specific detection of potato D. destructor PCR molecular detection method.

[0048] The specific primer DdF1 / DdR1 and universal primer D2A / D3B designed by the present invention are synthesized by Shanghai Biological Engineering Co., Ltd., PCR reaction system: 2.5 μl 10×PCR Buffer (containing Mg 2+ ), 2μl 10mM dNTP (2.5mM), 1.5μl primer pair DdF1 / DdR1 (20μM), 0.2μl Taq DNA polymerase (5U / μl), 2μl template DNA, sterilized ddH2O to make up to 25μl. Negative worm DNA template was used as negative control. Amplification was performed on an Eppendorf PCR cycler. The PCR reaction conditions were as follows: 95°C, 8min, 95°C, 45s, 55°C, 30s, 72°C, 1min, 35 cycles; 72°C, 10min, 4°C storage. Take 5 μl of amplified product and 1 μl of loading buffer, electrophoresis on 1.0% agarose gel, use 0.5×TAE as electrophoresis buffer, 120V / 40min, stain with EB, observe and take ...

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Abstract

The invention discloses a rapid PCR (Polymerase Chain Reaction) molecular detection method for ditylenchus destructor thorne and the application of the rapid PCR molecular detection method, and belongs to the technical field of molecular detection of plant nematodes. A PCR system is involved in the rapid PCR molecular detection method for the ditylenchus destructor thorne. The method is characterized in that the PCR system comprises a group of specific primers, wherein the specific primers are DdF1 and DdR1, and can specifically amplify fragments with lengths of 495bp from A-type and B-type ditylenchus destructor thorne, thereby realizing the rapid molecular detection of the ditylenchus destructor thorne. The method is high in sensitivity and specificity, rapid and accurate, and has high actual application value in the aspects of early diagnosis of ditylenchus destructor thorne, field monitoring and early warning and the like.

Description

technical field [0001] The invention belongs to the technical field of molecular detection of plant nematodes, and relates to a fast PCR molecular detection method for D. destructor potato and its application. Background technique [0002] Potato rot nematode (Ditylenchus destructor Thorne) is one of the main pathogens causing potato and sweet potato. Sweet potato stem nematode disease was reported in my country as early as 1937, and it was once thought to be caused by D. dipsaci (Liu Xianbao, Ge Jianjun, Tan Zhiqiong, Cao Aixin. The first report of potato rot nematode harming potatoes in China. Plant Protection, 2006, 32 (6):157-158), until 1979, Zhang Xiangrong believed that the pathogen was D. Systematic identification was carried out, and it was confirmed that the causative agent of the stem nematode on sweet potatoes in Shandong and Jiangsu was D. destructor (Ding Zaifu, Lin Maosong. Identification of stem nematodes on sweet potatoes and mint. Plant Protection, 1982, 9(3...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2531/113
Inventor 彭德良彭焕贺文婷
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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