A kind of pcr detection primer group of blast blight and its application
A technology for detecting primers and primer sets, applied in the biological field, can solve problems such as short incubation period of diseases, difficult to control, and difficult to control diseases, and achieve obvious control effects, good application prospects, and good specificity
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Embodiment 1
[0019] In this example, the specificity of the PCR primer sets shown in SEQ ID No.4 and SEQ ID No.5 is detected, and the steps are as follows:
[0020] (1) Extraction of DNA from common fungi on millet, including grain blast fungus and other 6 common phytopathogenic fungi on millet, namely Magnaporthe oryzae, Sclerospora graminicola, and Rhizoctonia solani), Ustilago crameri, Bipolarissetariae, Curvularia lunata, Uromyces setariae-italicae, the detailed steps are as follows:
[0021] a. After the solid PDA has been activated, pick the bacterial plate and inoculate it into the liquid PDA medium, collect the mycelium after culturing for seven days, and use a sterile The bacterial filter paper was pressed dry and used to extract genomic DNA. The rust and smut fungus directly use spore powder to extract DNA, while the white fungus uses its oospores to extract DNA.
[0022] b. The pathogenic bacteria on the millet take CTAB to extract genomic DNA. The specific steps are: ① Take ...
Embodiment 2
[0025] In this example, the sensitivity of the PCR primer sets shown in SEQ ID No.4 and SEQ ID No.5 is analyzed, and the steps are as follows:
[0026] 1. DNA concentration dilution
[0027] Using NanoDrop 1000 spectrophotometer to detect the concentration of the extracted genomic DNA of the valley blast strains, the DNA concentration was adjusted to the initial concentration of 10 μg / μL, and the 10-fold concentration was sequentially diluted to 10 μg / μL, 1 μg / μL, 100 ng / μL , 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL 7 concentration gradients.
[0028] 2. PCR amplification sensitivity detection
[0029] (1) The PCR amplification system is 25 μL, including 2.0 μL of DNA template, 0.5 μL of upstream primer Gw-1, 20.5 μL of downstream primer Gw-1, 12.5 μL of 2×Es Taq MasterMix, ddH ...2... O 9.5 μL. The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 30s, a total of 35 cycles,...
Embodiment 3
[0032] Detection of Cereal Blast in Inoculated Plants
[0033] (1) Sample treatment: Take healthy millet leaves with the same growth period and inoculate conidia (1×10 5 pcs / ml), placed in a 26°C constant temperature incubator for 1 day in the dark and moisturizing. After moisturizing cultivation, put the millet plants in the incubator for normal light cultivation, and spray in real time to maintain the humidity. Sampling was carried out 1 day, 2 days, and 3 days after culturing. In addition, leaves were also collected from the control group inoculated with millet with clear water. 5 leaves were collected for each sample, and genomic DNA was extracted after mixing.
[0034] (2) Millet leaf DNA extraction: The improved CTAB method was used to extract the genomic DNA of the millet leaf samples. The specific steps are as follows:
[0035] ① Take 0.2 g of millet leaves and grind them into powder with liquid nitrogen, transfer to a 1.5 mL centrifuge tube, add 650 μL of CTAB extra...
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