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Compound pcr detection and identification kit for wheat stripe rust and leaf rust

A technology of wheat stripe rust and kit, which is applied in the field of biotechnology, crop disease control and plant quarantine, can solve the problem of no detection and differentiation of wheat stripe rust and leaf rust by PCR, etc., and achieves strong specificity and simple experimental operation , Easy and fast operation

Inactive Publication Date: 2012-02-01
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0006] At present, there is no special PCR kit for the detection and distinction of wheat stripe rust and leaf rust at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Multiplex PCR detection of susceptible wheat leaves and urediospores of rust fungus and its specificity analysis.

[0046] ①In the artificial climate chamber, wheat leaf rust 07-24-17 and stripe rust S23 were used to inoculate Mingxian 169 wheat seedlings alone or mixed, and the diseased leaves were collected 7 days later to extract genomic DNA; culturing and collecting wheat leaf rust (07- 24-17, 07-18-7, 07-24-3), stripe rust (S23, 07-6-3-11), uredospores of stem rust, and gibberella, powdery mildew, sheath blight, leaf blight Genomic DNA was extracted from the mycelia of , root rot, light fishy, ​​and dwarf bacterium, and prepared as a 20ng / μl amplification reaction template.

[0047] ② Establish PCR reaction system

[0048] Take out the PCR-SMART reaction solution, melt at room temperature, mix well, add 7.5 μl to each PCR thin-walled reaction tube; then add 0.5 μl Taq enzyme, 16 μl ddH 2O. 1 μl template DNA (20ng / μl), and cover the PCR thin-walled react...

Embodiment 2

[0053] Example 2 Determination of Sensitivity of Detection of Wheat Rust Composite PCR

[0054] ① Gradually dilute the ureterospore DNA of Phyllostachys 07-24-3 and P. uL and 50.00ng / uL; Equal amounts of urediospore DNA of leaf rust 07-24-3 and stripe rust S23 were mixed and serially diluted to the final concentrations of 5.00pg / uL, 50.00pg / uL, 1.00ng / uL, 10.00ng / uL, 20.00ng / uL and 50.00ng / uL.

[0055] ② Establish PCR reaction system

[0056] Take out the PCR-SMART reaction solution, melt at room temperature, mix well, add 7.5 μl to each PCR thin-walled reaction tube; then add 0.5 μl Taq enzyme, 16 μl ddH 2 O. 1 μl of template DNA, and cover the PCR thin-walled reaction tube.

[0057] ③PCR amplification

[0058] Place the PCR thin-walled reaction tube in the PCR machine, and set the PCR reaction program: 94°C pre-denaturation for 5 minutes, 1 cycle; 94°C denaturation for 30S, 55°C annealing for 30S, 72°C extension for 40S, 35 cycles; 72°C extension 5min 1 cycle.

[0059]...

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PUM

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Abstract

The invention discloses a composite PCR detection and identification kit for wheat stripe rust and leaf rust, belonging to the technical fields of biotechnology, crop disease prevention and control, and plant quarantine. The kit uses three primers derived from the conserved sequence of the fungal β-tubulin gene and the specific sequences of wheat stripe rust and leaf rust to simultaneously detect the specificity of wheat stripe rust 171bp and leaf rust 226bp in one PCR reaction. Genomic DNA sequence. It is a supplement and improvement to the specific primer detection method of wheat stripe rust and leaf rust, and is also suitable for the diagnostic detection of stripe rust and leaf rust in infected wheat leaves before disease symptoms appear. This kit adopts compound PCR technology. In the PCR-SMART reaction solution, three primers are simultaneously amplified, which improves the accuracy of detection, saves detection time, and is easy to operate. It can be popularized and applied by plant protection and plant inspection departments in my country.

Description

technical field [0001] The invention belongs to the fields of biotechnology, crop disease prevention and plant quarantine, and specifically relates to a compound PCR detection and identification kit for wheat stripe rust and leaf rust. Background technique [0002] Wheat rust is a worldwide cereal disease, with a wide range of occurrence, a high epidemic disaster rate, and serious damage. It is an important biological disaster that affects the safe production of wheat. It can reduce wheat production by about 30% in a pandemic year, and 50% in a particularly severe epidemic year. % to 60%, or even no harvest. As my country's second largest food crop, wheat production is directly related to my country's food security issues. However, in different wheat planting areas in my country, the source of rust fungus exchanges and spreads on a large scale, and the epidemic of rust goes back and forth. [0003] The common symptom of wheat stripe rust and leaf rust is the appearance of ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
Inventor 曹丽华孟颢光陈万权徐世昌刘太国蔺瑞明王振跃
Owner HENAN AGRICULTURAL UNIVERSITY
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