Method for detecting DNA of wheat stunt virus

A technology of wheat dwarf virus and DNA molecules, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve the problems of high price, high operator cost, unfavorable site, rapid analysis, etc., and achieve simple steps , strong sensitivity, easy to quickly detect and analyze the effect

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

AI Technical Summary

Problems solved by technology

However, this type of method often has high requirements for operators and requires professional equipment, such as expensive precision temperature control equipment (for nucleic acid amplification and bulky), complex fluorescence detection system (for product detection), not conducive to on-site, rapid analysis

Method used

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  • Method for detecting DNA of wheat stunt virus
  • Method for detecting DNA of wheat stunt virus
  • Method for detecting DNA of wheat stunt virus

Examples

Experimental program
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Embodiment 1

[0074] A method for detecting wheat dwarf virus DNA, the specific detection steps are as follows:

[0075] Step 1: Cut 100 mg of wheat diseased leaves, place the diseased leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the virus-carrying plant tissue materials 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.

[0076] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 20nM,

[0077] 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,

[0078] 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.

[0079] Wherein, the above-mentioned dNTP co...

Embodiment 2

[0098] A method for detecting wheat dwarf virus DNA, wherein the DNA molecular machine sequence is 5'-CCCCAAAACCCCAAAACCCCAAAACCCCTGCATCGACTCGAAGCACGAAGCTTG-3'.

[0099] The specific detection steps are as follows:

[0100] Step 1: Cut 100 mg of wheat diseased leaves, place the diseased leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the virus-carrying plant tissue materials 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.

[0101] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 60nm,

[0102] 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;

[0103] Step 4: Add 40 μL buffer, 10 μL polymerase, 10 μL nickase, 10 μL Thioflavin T, 10 μL dNTP, 10 μL Mg ...

Embodiment 3

[0113] A method for detecting wheat dwarf virus DNA, wherein the sequence of the DNA molecular machine is

[0114] 5'-CCCCAAAAACCCCAAAACCCCAAAACCCCTGCATCGACTCGAAGCACGAAGCTTG-3'.

[0115] The specific detection steps are as follows:

[0116] Step 1: Cut 100 mg of wheat diseased leaves, place the diseased leaves in a liquid mortar, add liquid nitrogen and grind them into powder; grind the virus-carrying plant tissue materials 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.

[0117] Step 2: Synthesize the DNA molecular machine and configure it into a solution with a concentration of 100nM,

[0118] 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.

[0119] Step 4: Add 20 μL buffer, 6 μL polymerase, 7 μL nickase, 10 μL Thioflavi...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a method for detecting DNA of a wheat stunt virus. The method comprises the following steps: providing a DNA molecular machine for specifically recognizing DNA of the to-be-tested wheat stunt virus, wherein the DNA molecular machine is a nucleotide sequence containing a G-quadruplex synthesis template; mixing the DNA molecular machine with a to-be-tested sample; providing DNA polymerase and nickase for the DNA molecular machine so as to generate a series of reactions of generating DNA new chain synthesis, forming a single-chain notch and continuously striping to generate G-rich gene segments; providing thioflavine ThT 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 DNA signal of the wheat stunt virus in the sample is converted into G-quadruplex, and the continuous application of G-quadruplex can be realized at the room temperature, so that the DNA signal of the wheat stunt virus is greatly amplified, and the high-sensitivity rapid qualitative assay determination of nucleic acid is realized. The method is strong in specificity, expensive or huge-volume precise temperature control equipment does not need to be used, and the rapid detection and analysis are facilitated.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for detecting wheat dwarf virus DNA. Background technique [0002] Wheat dwarf virus DNA (Wheatdwarfvirus, WDV) is a member of the genus Zeavirus of the family Synzymeviridae and belongs to the subgroup I Synzymevirus. At present, the detection of wheat dwarf virus DNA is detected by PCR method. Polymerase Chain Reaction (PCR) based on nucleic acid amplification has become the most commonly used nucleic acid molecular detection method at home and abroad, with high specificity. However, this type of method often has high requirements for operators and requires professional equipment, such as expensive precision temperature control equipment (for nucleic acid amplification and bulky), complex fluorescence detection system (for detection of increased products), which is not conducive to on-site and rapid analysis. [0003] G-quadruplex (G-quadruplex) is a higher-order struct...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/682
CPCC12Q1/682C12Q1/701C12Q2563/107C12Q2563/137
Inventor 张晓婷谢源张艳丽孙柄剑周琳李洪连
Owner HENAN AGRICULTURAL UNIVERSITY
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