PCR detection primers and detection method of Phytophthora cowpea
A detection method and technology for detection primers, which are applied in the fields of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. problem, to achieve the effect of strong practicability, high sensitivity and high accuracy
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Embodiment 1
[0028] Example 1: Design of primer sequences for PCR detection and specific amplification of primers to Phytophthora cowpea
[0029] 1. Design and synthesis of primers
[0030] According to GenBank Phyophthora of different species Ypt1 Differences between gene sequences (aligned in BioEdit), Phytophthora cowpea designed with Primer Primer5 software Phytophthora vignae Specific primers, the primer sequence is: upstream primer PVF: 5'-CTCAGAATGGCCAAGGTTTC -3', downstream primer PVR: 5'-CGTTCCGCGAGACTTT TCTA -3', the primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.
[0031] 2. Extraction of Genomic DNA of Tested Strains
[0032] The genomic DNA of the tested strain was extracted by the CTAB method. The specific method is as follows: take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 μL of 2% CTAB (hexadecyl trimethylammonium bromide) extract (2% CTAB...
Embodiment 2
[0037] Embodiment 2: Sensitivity detection of primers to Phytophthora cowpea genomic DNA
[0038] 1. Conventional PCR amplification
[0039] The genomic DNA of Phytophthora cowpea was diluted with sterile ultrapure water, and prepared into a series concentration of 10-fold order of magnitude for future use. Use the primer PVF / PVR described in the present invention to carry out PCR amplification to the genomic DNA of different series concentrations, evaluate the sensitivity of this primer to Phytophthora cowpea genomic DNA detection, amplification reaction system and reaction procedure are as follows: PCR reaction system 25 μ L, Contains 2.5 µL 10×PCR buffer (Mg 2+ free), 2.0 mmol / L MgCl 2 , 0.2 mmol / L dNTP, 1.0 U Taq DNA polymerase (Takara Dalian Bao Biological Engineering Co., Ltd.), primers PVF / PVR 0.4 μmol / L, 25 ng DNA template, and the insufficient part was made up with sterile ultrapure water. The amplification reaction program was as follows: pre-denaturation at 94...
Embodiment 3
[0043] Embodiment 3: the detection of Phytophthora cowpea in diseased plant tissue
[0044] Extraction of Phytophthora cowpea DNA from diseased plant tissue: DNA was extracted by NaOH rapid cleavage method, and the specific process was as follows: 0.5 mol / L NaOH 10 µL was added to 1.0 mg diseased plant tissue (washed diseased cowpea root or stem), and the The tissue was fully ground into a paste, then transferred to a 1.5 mL centrifuge tube, centrifuged at 12,000 rpm for 6 min, 5 µl of the supernatant was added to 495 µL of 0.1mol / L Tris-HCl (pH=8.0) and mixed evenly, and 1.0 µL was used as a PCR template Amplify. PCR amplification detection: use the primer PVF / PVR of the present invention to perform PCR amplification. PCR reaction system 25 µL, including 2.5 µL 10×PCRbuffer (Mg 2+ free), 2.0 mmol / L MgCl 2 , 0.2 mmol / L dNTP, 1.0 U Taq DNA polymerase (Takara Dalian Bao Biological Engineering Co., Ltd.), primers PVF / PVR 0.4 μmol / L each, 25 ng DNA template, the insufficient...
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