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A kind of molecular detection primer and rapid detection method of corn leaf spot fungus

A technology for the detection of small spot bacteria and molecules of corn, which is applied in the field of crop disease detection and biology, can solve the problems of high identification experience requirements, long consumption, low accuracy, etc., and achieve wide applicability, high sensitivity, and good practicability Effect

Active Publication Date: 2019-03-19
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The detection and identification of corn spot disease in the prior art is mainly based on the morphological characteristics of the pathogen, the procedures are cumbersome, time-consuming, require high identification experience, and the accuracy is low, and it is difficult to meet the actual needs of the diagnosis of corn spot disease. Provided are a molecular detection primer and a detection method for corn leaf spot fungus

Method used

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  • A kind of molecular detection primer and rapid detection method of corn leaf spot fungus
  • A kind of molecular detection primer and rapid detection method of corn leaf spot fungus
  • A kind of molecular detection primer and rapid detection method of corn leaf spot fungus

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Example 1 Design of primers for molecular detection and establishment of a specific molecular detection method for P. maize spot

[0030] 1. Extraction of Genomic DNA of Pseudomonas maize:

[0031] The CTAB method was used to extract the genomic DNA of 22 strains of P. maize from different sources preserved in our laboratory. The specific steps are as follows:

[0032] (1) Take 0.1 g of mycelium powder in a 1.5 mL centrifuge tube, add 900 μL of 2% CTAB extract, shake and mix with a shaker, bathe in 60°C water for 60 min, and centrifuge at 12,000 r / min for 15 min at room temperature;

[0033] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (each volume ratio is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 minutes at room temperature;

[0034] (3) Take 500 μL of the supernatant, add an equal volume of chloroform and extract again, and centrifuge at 8000 r / min for 10 min at room temperature;

[003...

Embodiment 2

[0052] Example 2 Specific Amplification of Corn Leaf Spot Bacteria

[0053] 1. Using the CTAB method to extract 2 strains of different sources of corn spot disease, corn gray spot fungus, corn round spot fungus, Curvularia maize leaf spot fungus, maize southern rust fungus, corn sheath blight fungus, maize spot fungus and Genomic DNA of P. arachis.

[0054] 2. Use the DNA extracted from the test bacteria as a template for PCR amplification: 25 μL of PCR reaction system, including 2.5 μL of 10×PCR buffer, 2.0 μL of dNTP Mixture with a concentration of 2.5 mmol / L, and 0.15 μL of Taq with a concentration of 5 U / μL Enzyme, 0.5 μL each of 10 μmol / L primers BSF and BSR, 1 μL DNA template, add ddH 2 O to a total volume of 25 μL; PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 sec, 54°C annealing for 45 sec, 72°C extension for 45 sec, a total of 35 cycles; 72°C extension for 10 min; electrophoresis detection of amplified products.

[0055] 3. Speci...

Embodiment 3

[0057] Example 3 Sensitivity detection of primers of the present invention to corn leaf spot bacterium

[0058] 1. Using the CTAB method to extract the genomic DNA of the corn leaf spot fungus;

[0059] 2. After measuring the concentration of the extracted genomic DNA of corn spot bacterium with a spectrophotometer, dilute it with sterile ultrapure water, prepare a series of concentrations, and set aside;

[0060] 3. Carry out routine PCR amplification using the prepared series of DNA as a template: 25 μL of PCR reaction system, including 2.5 μL of 10×PCR buffer, 2.0 μL of dNTP Mixture with a concentration of 2.5 mmol / L, and 0.15 μL of dNTP Mixture with a concentration of 5 U / μL Taq enzyme, 0.5 μL each of 10 μmol / L primers BSF and BSR, 1 μL DNA template, add ddH 2 O to a total volume of 25 μL; PCR reaction conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 45 sec, 54°C annealing for 45 sec, 72°C extension for 45 sec, a total of 35 cycles; 72°C extension for 10...

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Abstract

The invention relates to Helminthosporium maydis molecular detection primers and a detection method thereof, belonging to the technical fields of crop disease detection and biology. The specific primers comprise a forward primer BSF: 5'-TGCAAAATATGTGCTGCGCT-3' and a reverse primer BSR: 5'-CCCATGTCTTTTGCGCACTT-3'. The primers can be subjected to PCR (polymerase chain reaction) amplification and agarose gel electrophoresis, and used for specific amplification in Helminthosporium maydis pure DNA bacteria-bearing corn leaves or sheaths to obtain the specific amplification product with the segment length of 392bp. The detection primers have the advantages of high specificity and high sensitivity. The detection method has the advantage of high practicality, and is simple and quick to operate. The method can implement early detection of Helminthosporium maydis, can effectively distinguish grey speck disease, round spot and leaf blight on corn, and has important meanings for prewarning and controlling Helminthosporium maydis and controlling dispersion and propagation of diseases.

Description

technical field [0001] The invention relates to a molecular detection primer and a detection method for corn spot disease, which is specially used for rapid molecular detection of corn spot disease, and can realize early diagnosis of corn spot disease and monitoring and identification of the pathogen at the same time, and belongs to the field of crop disease detection and field of biotechnology. Background technique [0002] Corn is an important food crop and economic crop in my country. It also has multiple uses such as energy, feed, fruits and vegetables. It is the crop that ranks first in planting area and second in output, and is in great demand. The healthy development of the corn industry plays a very positive and important role in improving the structure of my country's agricultural industry and ensuring food security. [0003] Corn spot disease is caused by the umbilicalspora zea Bipolaris maydis ) Infection caused by leaf fungal diseases, the suitable temperature ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895
Inventor 杜宜新陈福如杨秀娟石妞妞代玉立阮宏椿甘林
Owner INST OF PLANT PROTECTION FAAS
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