Molecular detection primer for banana cladosporium cucumerinum and detection method
A molecular detection and banana technology, applied in the field of crop disease detection and biology, can solve the problems of low accuracy, long consumption, and difficult separation, and achieve the effects of good practicability, high sensitivity and high accuracy
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Embodiment 1
[0029] Example 1 Molecular Detection Primer Design and Establishment of a Specific Molecular Detection Method for Banana Scab
[0030] 1. Extraction of Banana Scab Genomic DNA:
[0031] The CTAB method was used to extract the genomic DNA of 6 strains of Banana scab bacteria 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 (the volume ratio of each 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;
[0035] (4) Take 350 μL of th...
Embodiment 2
[0052] Example 2 Banana scab specific amplification
[0053] 1. Using the CTAB method to extract 2 strains of banana scab, banana anthracnose, Curvularia banana leaf spot, banana black spot, banana gerbilia leaf spot, banana dark bisporus leaf spot, banana Fusarium wilt and Genomic DNA of Phalomyces asparagus.
[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 BMF and BMR, 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, 56°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. Specific amplification results
[0056] Such as f...
Embodiment 3
[0057] Embodiment 3 The sensitivity detection of primers of the present invention to banana scab
[0058] 1. Using the CTAB method to extract the genomic DNA of the banana scab fungus;
[0059] 2. After the genomic DNA of the extracted banana scab bacterium is measured by a spectrophotometer, it is diluted with sterile ultrapure water to prepare a series of concentrations for subsequent use;
[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 Taq with a concentration of 5 U / μL Enzyme, 0.5 μL each of 10 μmol / L BMF and BMR, 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, 56°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 ...
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