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Method for rapidly detecting fusarium DNA

A fusarium and DNA molecule technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve problems such as sensitivity and selectivity cannot meet detection requirements, time-consuming and expensive sensitivity, low target DNA content, etc. Facilitates rapid detection and analysis, high sensitivity, and highly sensitive results

Inactive Publication Date: 2018-12-07
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The traditional fluorescence-labeled nucleic acid amplification detection technology requires fluorescent probe labeling, which is cumbersome, time-consuming and expensive, and its sensitivity is not high
Although the fluorescent sensor built on the basis of traditional technology has low background signal and strong stability, however, due to the complex composition of the actual sample and the extremely low content of target DNA, this requires the detection method to have extremely high sensitivity and specificity. Conventional fluorescently labeled nucleic acid amplification The sensitivity and selectivity of enhanced detection technology cannot meet its detection needs

Method used

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  • Method for rapidly detecting fusarium DNA
  • Method for rapidly detecting fusarium DNA
  • Method for rapidly detecting fusarium DNA

Examples

Experimental program
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Effect test

Embodiment 1

[0076] A method for detecting Fusarium DNA, the specific detection steps are as follows:

[0077] Step 1: Cut 100 mg of plant diseased leaf leaves, place the diseased leaf leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the plant tissue materials with viruses at room temperature until the juice fully flows out, and Centrifuge under a centrifuge to obtain plant tissue juice, heat at 93°C for 30s, and then heat at 93°C for 30s as the sample solution to be tested.

[0078] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 20nM,

[0079] Step 3: Take 4 μL of sample solution and 2 μL of DNA molecular machine solution, mix evenly by micro-oscillation to obtain a mixed solution,

[0080] Step 4: Add 10 μL buffer, 6 μL polymerase, 6 μL nickase, 6 μL Thioflavin T, 6 μL dNTP, 6 μL Mg 2+ Add to the mixed solution obtained in step 3 to obtain a reaction solution.

[0081] Wherein, the above dNTP concen...

Embodiment 2

[0100] A method for detecting Fusarium DNA, wherein the sequence of the DNA molecular machine is 5'-CCCCAAAACCCCAAAACCCCAAAACCCCTGCATTCGACTCGTCTTGTCTTCC-3'.

[0101] The specific detection steps are as follows:

[0102] Step 1: Cut 100 mg of plant diseased leaf leaves, place the diseased leaf leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the plant tissue materials with viruses at room temperature until the juice fully flows out, and The plant tissue juice was obtained by centrifuging in a centrifuge, and heated at 93° C. for 30 seconds as the sample solution to be tested.

[0103] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 60nM,

[0104] Step 3: Take 8 μL of sample solution and 4 μL of DNA molecular machine solution, mix evenly by micro-oscillation, and obtain a mixed solution;

[0105] Step 4: Add 40 μL buffer, 10 μL polymerase, 10 μL nickase, 10 μL Thioflavin T, 10 μL dNTP, 10 μL ...

Embodiment 3

[0115] A method for detecting Fusarium DNA, wherein the sequence of the DNA molecular machine is 5'-CCCCAAAACCCCAAAACCCCAAAACCCCTGCATTCGACTCGTCTTGTCTTCC-3'.

[0116] The specific detection steps are as follows:

[0117] Step 1: Cut 100 mg of plant diseased leaf leaves, place the diseased leaf leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the plant tissue materials with viruses at room temperature until the juice fully flows out, and The plant tissue juice was obtained by centrifuging in a centrifuge, and heated at 93° C. for 30 seconds as the sample solution to be tested.

[0118] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 100nM,

[0119] Step 3: Take 4 μL of the sample solution and 4 μL of the DNA molecular machine solution, and mix evenly by micro-oscillation to obtain a mixed solution.

[0120] Step 4: Add 20 μL buffer, 6 μL polymerase, 7 μL nickase, 10 μL Thioflavin T, 3 μL dNTP...

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Abstract

The invention relates to a method for rapidly detecting fusarium DNA. The method comprises the following steps: (S1) providing a DNA molecular machine for specifically recognizing fusarium DNA, wherein the DNA molecular machine is a nucleotide sequence containing a G-quadruplex complementary sequence; (S2) mutually mixing the DNA molecular machine with a to-be-tested sample; (S3) introducing DNA polymerase and nickase for the DNA molecular machine so as to generate DNA amplification reaction; and (S4) introducing thioflavine T for inducing and generating a G-quadruplex structure, and binding with the G-quadruplex structure to generate fluorescence; and detecting and recording the fluorescence. According to the method, a nucleic acid signal in the to-be-tested sample suspected to contain fusarium DNA is converted into G-quadruplex, and the continuous amplification of G-quadruplex can be realized at the constant room temperature, so that the DNA signal of the nucleic acid is greatly amplified; and a stable fluorescence signal is generated through the binding of thioflavine T and G-quadruplex, so that the high-sensitivity, rapid and qualitative analysis assay determination of nucleicacid is realized.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for rapidly detecting Fusarium DNA. Background technique [0002] Fusarium is a worldwide distribution of fungi, it can not only survive winter and summer in the soil, but also infect a variety of plants (food crops, economic crops, medicinal plants and ornamental plants), causing plant root rot, stem There are more than 100 species of host plants, infecting the vascular system of host plants, destroying the vascular bundles of plant transport tissues, and producing toxin hazards in the process of growth, development and metabolism Crops, causing crop wilting and death, affecting yield and quality, is one of the most difficult important diseases to control in production. [0003] At present, the isothermal detection of Fusarium can be regarded as detection, and most of them utilize loop-mediated isothermal amplification assays or PCR assays. [0004] Although constant temp...

Claims

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Application Information

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IPC IPC(8): C12Q1/6844C12Q1/04
CPCC12Q1/6844C12Q2521/101C12Q2531/119C12Q2563/107C12Q2525/173C12Q2521/301
Inventor 高飞张晓婷周琳许娟杜鹏强张艳丽谢源李洪连
Owner HENAN AGRICULTURAL UNIVERSITY
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