Phytophthora sojae molecule detecting primer and detection reagent kit thereof
A technology for root rot fungus and Phytophthora sojae, applied in microorganisms, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity and poor specificity, and achieve simple and fast operation, good practicability, fast and reliable. The effect of detection and identification
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Embodiment 1
[0043] Embodiment 1: Primer is to the specific amplification of soybean Phytophthora root rot bacterium
[0044] 1. Specific detection of soybean Phytophthora root rot
[0045] PCR reaction system 25μl, including 2.5μl 10×PCR reaction buffer, 5mM MgCl 2 2.5μl, 10mM dNTPs2.0μl, 1.0U Taq DNA polymerase, 20pmol / μl primer PSD11 / PSD12 each 0.2μl, weight concentration 99.5% DMSO0.5μl and 10ng template DNA,, d.d.H 2 O to make up 25 μl. The PCR amplification program was: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 68°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min.
[0046] 2. Test results
[0047] Specificity of detection: In addition to the 382bp product that can be specifically amplified from the DNA of Phytophthora root rot of soybean from Fujian, Heilongjiang and other provinces in my country and abroad, other 13 species of Phytophthora, downy mildew, and rot of oomycetes were detected. Mold and o...
Embodiment 2
[0048] Example 2: Detection of Phytophthora sojae root rot in diseased plant tissues.
[0049] 1. Sample collection: Plant tissue samples were collected from the soybean vegetable base in Bangshan Town, Longhai City, Fujian Province
[0050] 2. DNA extraction and detection
[0051] The diseased plant tissue was extracted with the CTAB method, and PCR amplification was carried out according to the method implemented in the above kit. The PCR reaction system was 25 μl, including 2.5 μl 10×PCR reaction buffer, 5 mM MgCl 2 2.5μl, 10mM dNTPs 2.0μl, 1.0UTaq DNA polymerase, 20pmol / μl primer PSD11 / PSD12 0.2μl each, weight concentration 99.5% DMSO 0.5μl and 10ng template DNA, d.d.H2O make up 25μl. The PCR amplification program was: denaturation at 94°C for 5 min; denaturation at 94°C for 1 min, annealing at 68°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally extension at 72°C for 10 min. Amplified products were detected by electrophoresis.
[0052] 3. Test results ...
Embodiment 3
[0054] Example 3: Detection of Phytophthora soybean root rot in soil samples.
[0055] 1. Sample collection: Soil samples were collected from soybean vegetable base in Fugong Town, Longhai City, Fujian Province
[0056] 2. DNA extraction and detection
[0057] (1) Extract DNA from diseased soil samples:
[0058] Take the sieved soil, freeze and dry it for 24-48 hours, add a small amount of quartz sand, pour liquid nitrogen into it and grind it thoroughly, divide the ground soil fine powder into 1.5ml centrifuge tubes, add 500μl weight concentration of 0.4% skimmed milk powder to each tube solution, vortexed to mix. Centrifuge at 12000rpm for 15min. Take the supernatant and add an equal volume of proteinase K buffer to a final concentration of 10 μg / ml proteinase K, and bathe in water at 55°C for 1-3h. After the water bath, add 1 / 2 volume of 7.5M NH 4 AC solution, mix up and down. Centrifuge at 12000rpm for 15min. Aspirate the supernatant and add 2 times the volume of ab...
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