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Visual detection kit for wheat pathogenic fungi

A technology for detecting kits and pathogenic fungi, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, etc., and can solve problems such as different pathogens and medication errors

Pending Publication Date: 2021-06-08
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnosis of wheat diseases mainly depends on the observation of symptoms by professional agricultural technicians, and the pathogens of the same symptoms are different, which will lead to medication errors

Method used

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  • Visual detection kit for wheat pathogenic fungi
  • Visual detection kit for wheat pathogenic fungi
  • Visual detection kit for wheat pathogenic fungi

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1 prepares double-stranded DNA probe

[0044] In this example, a DNA duplex formed by complementary base pairing between the mispaired strand and the substrate strand embedded with silver ions was prepared, and the double-stranded DNA played a role in molecular recognition in the process of detecting the target wheat RNA.

[0045] The specific method of synthesis of double-stranded DNA (molecular recognition reagent) is as follows:

[0046] 5 μL of Ag at a concentration of 3 μM +, 5 μL of 1.5 μM DNA substrate strand, 5 μL of 1.5 μM DNA mismatch strand and 15 μL buffer solution (1M NaNO3, pH=6.9) were added to a PCR tube, placed at 90°C for 5 min , followed by incubation at room temperature for 30 min. With the participation of mercury ions, the cytosine pairs, the mismatched strand and the substrate strand complement each other to form a double-stranded structure, and a double-stranded DNA solution is obtained.

Embodiment 2

[0047] Example 2 RNA Extraction of Wheat Pathogenic Bacteria

[0048] In the present embodiment, the specific operation of heating and extracting wheat pathogenic bacteria RNA is as follows:

[0049] Take wheat leaves infected with stripe rust, stem rust, leaf rust, Gibberella, and Fusarium graminearum, put them in a centrifuge tube, mash them with a grinding rod, add 50 μL of molecular water, heat at 70°C for 1-10 min, draw the supernatant and store it for later use.

Embodiment 3

[0050] The selectivity test of embodiment 3 wheat pathogens

[0051] In the present embodiment, investigate the selection specificity of the inventive method to the stripe rust RNA, the steps are as follows:

[0052] (1) Take 5 μL of the RNA stock solution in Example 2

[0053] (2) Detection of wheat pathogenic bacteria sample solution

[0054] ①Take 15 μL of the double-stranded DNA solution prepared in Example 1, which is formed by combining the mismatched strand m-probe and the substrate strand s-probe, mix well, and incubate at room temperature for 35 min, so that the mercury Ions and mispaired strands are replaced. Then add 2 μL of ultrapure water and 3 μL of 90 nM urease, mix well, and place at room temperature for 35 min

[0055] ②Take 5 μL each of 1.5 mM phenol red solution and 2.5 M urea aqueous solution, mix with 15 μL ultrapure water

[0056] ③ After mixing the solutions of ① and (1), place it at room temperature for 30 minutes, and measure the ultraviolet absorp...

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Abstract

The invention discloses a visual detection kit for wheat pathogenic fungi, and belongs to the field of molecular detection. The kit comprises a sample pretreatment part, a probe identification part, a detection result visualization part and a data management part; a heating extraction method is adopted for the sample pretreatment part, and the time is within 5 min; DNA double strands embedded with silver ions are adopted as a detection probe; The visual result is composed of a test paper reaction tank, a smart phone signal acquisition and a monitoring data management cloud webpage. The detection principle is based on replacement of mismatched chains by pathogenic bacteria RNA, release of silver ions, inactivation of urease and prevention of urea decomposition, and the paper-based chip is yellow under the action of phenol red , otherwise the color is red. The kit can be used for simultaneously screening a plurality of common wheat pathogenic bacteria diseases including stripe rust, root rot, fusarium graminearum and the like. The amount of RNA of puccinia striiformis can be calculated through an image management webpage according to a test result, the number of viable bacteria of pathogenic bacteria is estimated, the wheat disease infection condition is accurately judged, and the accuracy, stability and convenience of wheat disease detection are further improved.

Description

technical field [0001] The invention belongs to the field of molecular detection. Background technique [0002] Wheat is the main grain supply of our country, and maintaining a high yield of wheat every year is crucial to maintaining our country's food security. In 2019, my country's wheat output was 133 million tons, the consumption was 128 million tons, the import volume was 3.205 million tons, and the export volume was 1.1 million tons. Referring to the wheat production trend chart from 2008 to 2019, it can be seen that my country's wheat market is large, and the overall and unit yield per mu both show a steady upward trend. [0003] The improvement of crop yield mainly relies on breeding and plant protection. Wheat is very difficult to breed due to its complex genome (hexaploid), so plant protection measures have become more important to ensure wheat output. However, there are many diseases and insect pests of wheat, which are difficult to control. Major diseases suc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6834C12Q1/6806
CPCC12Q1/6834C12Q1/6806C12Q2565/525C12Q2537/165C12Q2527/101
Inventor 邓锐杰张婷王雅敏
Owner SICHUAN UNIV
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