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Detection and identification kit of composite PCR of wheat stripe rust fungus and leaf rust fungus

A wheat stripe rust and kit technology, which is applied in the direction of microbial determination/inspection, biochemical equipment and methods, etc., can solve the problems of PCR and other problems in detecting and distinguishing between wheat stripe rust and leaf rust, and achieves strong specificity, The experimental operation is simple and the operation is simple and fast.

Inactive Publication Date: 2010-06-23
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

[0042] Example 1 Composite PCR detection of susceptible wheat leaves and urediospores of rust fungus and its specificity analysis.

[0043] ①Mingxian 169 wheat seedlings were inoculated with wheat leaf rust 07-24-17 and stripe rust S23 alone or mixed in the artificial climate chamber, and the diseased leaves were collected 7 days later to extract genomic DNA; cultivate and collect 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.

[0044] ② Establish PCR reaction system

[0045] 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 template DNA (20ng / μl), and cover the PCR thin-walled reaction t...

Embodiment 2

[0050] The mensuration of embodiment 2 wheat rust composite PCR detection sensitivity

[0051] ① Gradually dilute the ureterospore DNA of leaf rust 07-24-3 and stripe rust S23 to the final concentration of 1.00pg / uL, 5.00pg / uL, 50.00pg / uL, 1.00ng / uL, 20.00ng / uL 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 final concentrations of 5.00pg / uL, 50.00pg / uL, 1.00ng / uL, 10.00ng / uL, 20.00ng / uL and 50.00ng / uL.

[0052] ② Establish PCR reaction system

[0053] 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.

[0054] ③PCR amplification

[0055] 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 ann...

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Abstract

The invention relates to a detection and identification kit of a composite PCR of wheat stripe rust fungus and leaf rust fungus, which belongs to the technical fields of biotechnology, crop disease control and plant quarantine. The kit detects the specific genome DNA sequences of the wheat stripe rust fungus 171 bp and the leaf rust fungus 226 bp simultaneously in a primary PCR reaction by three primers from a fungal beta-tubulin gene conserved sequence and wheat stripe rust fungus and leaf rust fungus specific sequences. The invention provides supplement and improvement on the wheat stripe rust fungus and leaf rust fungus specific primer detecting method, and is suitable for the diagnostic detection of the stripe rust fungus and the leaf rust fungus in an infected wheat leaf before disease symptoms appear. Composite PCR technology is adopted in the kit, and in a PCR-SMART reaction solution, the three primers are amplified simultaneously, so the detection accuracy is increased, the detection time is saved, the operation is simple, and the kit can be applied and popularized at plant protection and plant quarantine departments in China.

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 Applications(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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