Molecular detection method for rapid detection of new puccnia striiformis West f.sp.tritici strain
A technology for wheat stripe rust and bacterial strains, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, which can solve the problems of heavy workload, difficulty in identifying a large number of standard samples, and long cycle time
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Embodiment 1
[0029] 1. Sample collection
[0030] On October 5, 2012, the plant disease experimental station of Northwest Agriculture and Forestry University collected wheat leaf samples inoculated with stripe rust bacteria T4, Su11-4, V26, CYR29, CYR31, CYR32, and CYR33, and washed the wheat leaves with deionized water. Store at -80°C for later use.
[0031] 2. Detection of new toxic strain V26 of wheat stripe rust
[0032] (1) Extract the genomic DNA of uredospores of wheat leaf stripe rust;
[0033] (2) PCR amplification: 15 μL reaction system was placed in PCR tubes in turn: 10×Buffer H 1.5 μL, 5U / μL TaqDNA polymerase 0.15 μL, 25 mmol / L MgCl 2 0.9 μL, 0.3 μL of 10 mmol / L dNTPs, 0.75 μL of 10 ng / μL upstream primer and 0.75 μL of downstream primer, 1.5 μL of 50 ng / μL DNA, and finally make up to 15 μL with sterile deionized water. Centrifuge for 10 sec, put the PCR thin-walled tube into the PCR instrument for amplification. The reaction was carried out on a PTC-100 thermal cycler (MJ...
Embodiment 2
[0038] 1. Sample collection
[0039] On November 7, 2012, in Yangling, Shaanxi, 200 asymptomatic wheat leaves were randomly collected; the wheat leaves were rinsed with deionized water and stored at -80°C for later use.
[0040] 2. Detection of new toxic strain V26 of wheat stripe rust
[0041] (1) Extract the genomic DNA of uredospores of wheat leaf stripe rust;
[0042] (2) PCR amplification: 15 μL reaction system was placed in PCR tubes in turn: 10×Buffer H 1.5 μL, 5U / μL TaqDNA polymerase 0.15 μL, 25 mmol / L MgCl 2 0.9 μL, 0.3 μL of 10 mmol / L dNTPs, 0.75 μL of 10 ng / μL upstream primer and 0.75 μL of downstream primer, 1.5 μL of 50 ng / μL DNA, and finally make up to 15 μL with sterile deionized water. Centrifuge for 10 sec, put the PCR thin-walled tube into the PCR instrument for amplification. The reaction was carried out on a PTC-100 thermal cycler (MJ RESEARCH, Inc.), and the amplification program was as follows: the first cycle was 94 °C for 5 min, then 94 °C for 1 min...
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