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A kind of molecular detection primer and rapid detection method of maize leaf spot bacterium

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

Active Publication Date: 2019-03-19
INST OF PLANT PROTECTION FAAS
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Problems solved by technology

[0005] Aiming at the detection and identification of Pseudomonas spp. in the prior art, which 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 Pseudomonas spp. Provided are a molecular detection primer and a detection method for maize leaf spot bacterium

Method used

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  • A kind of molecular detection primer and rapid detection method of maize leaf spot bacterium
  • A kind of molecular detection primer and rapid detection method of maize leaf spot bacterium
  • A kind of molecular detection primer and rapid detection method of maize leaf spot bacterium

Examples

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Embodiment 1

[0028] Example 1 Molecular Detection Primer Design and Establishment of Specific Molecular Detection Method for Septoria maize

[0029] 1. Extraction of Genomic DNA of Pseudomonas maize:

[0030] The CTAB method was used to extract the genomic DNA of 6 strains of P. maize spp. preserved in our laboratory, and the specific steps were as follows:

[0031] (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;

[0032] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (the volume ratio of each is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 minutes at room temperature;

[0033] (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;

[0034](4) Take 350 μ...

Embodiment 2

[0051] Example 2 Specific Amplification of Pseudomonas maize

[0052] 1. Using the CTAB method to extract 2 strains of different sources of maize leaf spot, maize gray spot, maize round spot, maize small spot, maize southern rust, maize sheath blight, rice blast fungus and peanut brown spot Genomic DNA of bacteria.

[0053] 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 ESF and ESR, 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, 55°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.

[0054] 3. Specific amplification results

[0055...

Embodiment 3

[0056] Example 3 Sensitivity detection of primers of the present invention to Pseudomonas maize

[0057] 1. Using the CTAB method to extract the genomic DNA of P. maize;

[0058] 2. After measuring the concentration of the extracted genomic DNA of Leptosphaeria maize with a spectrophotometer, dilute it with sterile ultrapure water to prepare a series of concentrations for subsequent use;

[0059] 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 ESF and ESR, 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, 55°C annealing for 45 sec, 72°C extension for 45 sec, a total of 35 cycles; 72°C extension for 10 min; electrophore...

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Abstract

The invention relates to Exserohilum turcicum 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 ESF: 5'-GCCGGCTGCCAATTGTTTTA-3' and a reverse primer ESR: 5'-CCCATGTCTTTTGCGCACTT-3'. The primers can be subjected to PCR (polymerase chain reaction) amplification and agarose gel electrophoresis, and used for specific amplification in Exserohilum turcicum pure DNA bacteria-bearing corn leaves or sheaths to obtain the specific amplification product with the segment length of 356bp. 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 Exserohilum turcicum, can effectively distinguish grey speck disease, round spot and leaf blight on corn, and has important meanings for prewarning and controlling Exserohilum turcicum and controlling dispersion and propagation of diseases.

Description

technical field [0001] The invention relates to a molecular detection primer and a detection method for the large spot disease of corn, which is specially used for the rapid molecular detection of the large spot disease of the corn, and at the same time can realize the early diagnosis of the large spot disease of the corn and the monitoring and identification of the pathogen, and belongs to the detection and identification of crop diseases. field of biotechnology. Background technique [0002] Corn is widely planted in many countries in the world due to its advantages of wide application, high yield and low requirements on planting conditions. In my country, corn is an important food and economic crop with the largest planting area and the second largest output. In addition to being used as food, the modern corn industry has also developed multiple uses such as energy, feed, fruits and vegetables. The healthy development of the corn industry plays a very positive and import...

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 石妞妞杜宜新阮宏椿陈福如杨秀娟甘林
Owner INST OF PLANT PROTECTION FAAS
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