Chickpea ascochyta blight fungal molecule detection primer and applications thereof
The invention relates to a technology for molecular detection of Ascoliaceae, which is applied in the field of molecular detection primers for Chickpea Ascotolius blight, and can solve the problems of long cycle and low accuracy.
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Embodiment 1
[0137] Example 1: Primers for specific amplification of Ascochyta rabeie:
[0138] Specific Detection of Ascochyta rabeie, the Pathogen of Chickpea Dispora Leaf Blight
[0139] PCR amplification reaction system 25 μL, including 10 × buffer 2.5 μL, deoxynucleic acid triphosphate substrate (dNTPs) (2.5 mMeach) 2.5 μL, primer 7-5f / 7-5r (10 μM) each 0.5 μL, Taq DNA polymerase (Takara ) 0.5 μL, DNA template 2 μL, sterile deionized water to make up to 25 μL; PCR reaction conditions: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 50 s, annealing at 57.5 °C for 50 s, extension at 72 °C for 2 min, a total of 30 cycles, and finally The temperature is 72°C for 10 minutes, and the temperature is 4°C for storage;
[0140] Test results:
[0141] Specificity of detection: In addition to the DNA of Ascochyta rabeie, the pathogenic bacterium Ascochyta rabeie from Mulei County, Xinjiang, my country, which can specifically amplify a 440bp product, other Rhizoctonia solani and A...
Embodiment 2
[0142] Embodiment 2: the sensitivity amplification of primers to the chickpea shell bisporus leaf blight pathogen Ascochyta rabeie:
[0143] Dilution of DNA concentration: the extracted genomic DNA of Ascochyta rabeie, the pathogenic bacteria of chickpea husk bisporus leaf blight, was diluted by 10 times after the concentration was measured by a spectrophotometer;
[0144] Sensitivity detection of Ascochyta rabeie, the pathogen of chickpea dispora leaf blight:
[0145] PCR amplification reaction system 25 μL, including 10 × buffer 2.5 μL, deoxynucleic acid triphosphate substrate (dNTPs) (2.5 mM each) 2.5 μL, primer 7-5f / 7-5r (10 μM) each 0.5 μL, Taq DNA polymerase ( Takara) 0.5 μL, DNA template 2 μL, and sterile deionized water to make up to 25 μL. PCR reaction conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 57.5°C for 50 s, extension at 72°C for 2 min, 30 cycles in total, final temperature at 72°C for 10 min, storage at 4°C;
[01...
Embodiment 3
[0148] Embodiment 3: the specific detection of chickpea shell dispora leaf blight diseased body, soil with bacteria, and seeds with bacteria:
[0149] DNA extraction and detection:
[0150] The residue of chickpea husk dispora leaf blight, fungus-carrying soil, and fungus-carrying seeds were all extracted by CTAB method, and PCR amplification was carried out according to the above method. The PCR amplification reaction system was 25 μL, including 10×buffer2.5 μL, and Triphosphate substrate (dNTPs) (2.5mM each) 2.5μL, primer 7-5f / 7-5r (10μM) 0.5μL each, Taq DNA polymerase (Takara) 0.5μL, DNA template 2μL, sterile deionized water to make up to 25 μL; PCR reaction conditions: Pre-denaturation at 94°C for 5 min, denaturation at 94°C for 50 s, annealing at 57.5°C for 50 s, extension at 72°C for 2 min, 30 cycles in total, final temperature at 72°C for 10 min, storage at 4°C, electrophoresis detection of amplified products ;
[0151] Test results:
[0152] The test results showed ...
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