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Wheat stripe rust bacteria molecalar detecting method

A technology for molecular detection of wheat stripe rust, applied in biochemical equipment and methods, analytical materials, measuring devices, etc., can solve the problems that the molecular detection of wheat stripe rust has not been reported.

Inactive Publication Date: 2007-01-03
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the detection of pathogenic bacteria by PCR technology, but the molecular detection of wheat stripe rust has not been reported at home and abroad

Method used

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  • Wheat stripe rust bacteria molecalar detecting method
  • Wheat stripe rust bacteria molecalar detecting method
  • Wheat stripe rust bacteria molecalar detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1. Sample collection

[0026] On December 22, 2003, in Changwu, Shaanxi, 300 asymptomatic wheat leaf samples were randomly collected. The wheat leaves were rinsed with deionized water and stored at -80°C for later use.

[0027] 2. Detection of wheat stripe rust

[0028] (1) extracting wheat leaf genomic DNA;

[0029] (2) PCR amplification, 25μL reaction system was placed in PCR thin-walled tubes: 10×Buffer 2.5μL, 25mmol / LMgCL2 2.0μL, 2.5mmol / LdNTP 1.5μL, upstream primer and downstream primer 20ng each, wheat leaf Genomic DNA 10ng, Taq DNA polymerase 1.0U, and finally make up to 25μL with sterile deionized water. Centrifuge for 10 sec, put the PCR thin-walled tube into the PCR instrument for amplification. Amplification conditions were as follows: the first cycle of pre-denaturation at 94°C for 3 minutes; followed by denaturation at 94°C for 50 sec, annealing at 54°C for 90 sec, and extension at 72°C for 2 min, a total of 35 cycles; the last cycle, extension at 72°C ...

Embodiment 2

[0034] 1. Sample collection

[0035] On February 19, 2004, 410 asymptomatic wheat leaf samples were randomly collected in Peng Zuyuan, Baoji, Shaanxi. The wheat leaves were rinsed with deionized water and stored at -80°C for later use.

[0036] 2. Detection of wheat stripe rust

[0037] (1) extracting wheat leaf genomic DNA;

[0038] (2) PCR amplification, 25μL reaction system was placed in PCR thin-walled tubes: 10×Buffer 2.5μL, 25mmol / LMgCL2 2.0μL, 2.5mmol / LdNTP 1.5μL, upstream primer and downstream primer 30ng each, wheat leaf Genomic DNA 30ng, Taq DNA polymerase 1.0U, and finally make up to 25μL with sterile deionized water. Centrifuge for 10 sec, put the PCR thin-walled tube into the PCR instrument for amplification. Amplification conditions were as follows: the first cycle of pre-denaturation at 94°C for 3 minutes; followed by denaturation at 94°C for 50 sec, annealing at 54°C for 90 sec, and extension at 72°C for 2 min, a total of 35 cycles; the last cycle, extensio...

Embodiment 3

[0043] 1. Sample collection

[0044] On March 24, 2004, 391 asymptomatic wheat leaf samples were randomly collected in Gong Town, Baoji County, Shaanxi Province. The wheat leaves were rinsed with deionized water and stored at -80°C for later use.

[0045] 2. Detection of wheat stripe rust

[0046] (1) extracting wheat leaf genomic DNA;

[0047](2) PCR amplification, 25μL reaction system was placed in PCR thin-walled tubes: 10×Buffer 2.5μL, 25mmol / LMgCL2 2.0μL, 2.5mmol / LdNTP 1.5μL, upstream primer and downstream primer 40ng each, wheat leaf Genomic DNA 40ng, Taq DNA polymerase 1.0U, and finally make up to 25μL with sterile deionized water. Centrifuge for 10 sec, put the PCR thin-walled tube into the PCR instrument for amplification. Amplification conditions were as follows: the first cycle of pre-denaturation at 94°C for 3 minutes; followed by denaturation at 94°C for 50 sec, annealing at 54°C for 90 sec, and extension at 72°C for 2 min, a total of 35 cycles; the last cycle...

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Abstract

A wheat stripe rust germina molecule testing method, that is: the differential probe (the upsteam primers: 5'-TCTGTAAGATGTTAGATGC and the downsteam primers 5' -ATGCTGGCAGTGTGGTTG), expands by PCR (the expending parameter 94 centigrade pre-denaturalization 3min; then 94 centigrade denaturalization 50sec, 54 centigrade annealing 90sec 72 centigrade extends 2min, altogether 35 circulations; 72 centigrade extends 10min), the gelose gelatin electrophoresis, tests the disease-carrying situation of the wheat by the expanding production. The invention is used for forepart inspection of wheat stripe rust disease situation during winter and spring, provides the reliable technical support and the theory basis for the wheat stripe rust disease preventing and controlling decision-making.

Description

technical field [0001] The invention relates to PCR (Polymerase Chain Reaction) technology in biotechnology, in particular to a method for detecting wheat stripe rust by designing PCR special primers and using PCR technology. Background technique [0002] Wheat stripe rust is an important worldwide disease caused by wheat stripe rust (Puccinia striiformis West.f.sp.tritici). When the disease is prevalent, it can cause large-scale epidemic hazards in wheat producing areas in my country. In epidemic years, wheat production can be reduced by 20%-30%. [0003] Wheat stripe rust is an endemic disease in large regions, that is, a long-distance transmission disease. The pathogenic bacteria can reach thousands of meters with the updraft, and then spread to areas thousands of kilometers away with the high air. The fungus likes cool and is afraid of heat, and can survive winter but not summer in warm winter plains, and can survive summer but often cannot survive winter in cool alpine...

Claims

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Application Information

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IPC IPC(8): G01N27/447C12Q1/68
Inventor 康振生王小杰郑文明黄丽丽赵杰韩青梅魏国荣高小宁
Owner NORTHWEST A & F UNIV
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