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
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
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...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap