Method for high sensitivity detection of Phytophthora hibernalis Carne
A high-sensitivity technology for Phytophthora winteris, which is applied in the field of detection of Phytophthora winteris brown rot pathogen of citrus, can solve the problem that the amplified bands of citrus samples with bacteria are not particularly obvious, and achieve high sensitivity, good repeatability, and easy operation. easy effect
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Embodiment 1
[0027] The total volume of the RCA reaction system in this example is 10 μl. The specific detection method is as follows: (1) Add 5.9 μl of sterilized deionized water, 1 μl of 10×phi29 DNA polymerase buffer, 0.5 μl of sense primer PHIB1 with a concentration of 10 μM, and reverse reaction primer with a concentration of 10 μM in a sterile centrifuge tube. The volume of sense primer PHIB2 is 0.5 μl and the volume of target DNA with a concentration of 200 ng / μl is 1 μl, and then they are mixed evenly and centrifuged, and the reaction solution is centrifuged to the bottom of the tube;
[0028] (2) Heat the centrifuge tube at 95°C for 3 minutes with a PCR instrument, and then quickly ice-bath for 15 minutes;
[0029] (3) Add 0.5 μl of dNTPs mixture with a concentration of 2.5 mM each, 0.1 μl of BSA with a concentration of 1%, and 0.5 μl of phi29 DNA polymerase with a concentration of 10 U / μl in the centrifuge tube, mix well, and centrifuge. The reaction solution was centrifuged to ...
Embodiment 2
[0035] The total volume of the RCA reaction system in this example is 10 μl. The specific detection method is as follows: (1) Add 5.9 μl of sterilized deionized water, 1 μl of 10×phi29 DNA polymerase buffer, 0.5 μl of sense primer PHIB1 with a concentration of 10 μM, and reverse reaction primer with a concentration of 10 μM in a sterile centrifuge tube. The volume of sense primer PHIB2 is 0.5 μl and the volume of target DNA with a concentration of 110 ng / μl is 1 μl, and then they are mixed evenly and then centrifuged, and the reaction solution is centrifuged to the bottom of the tube;
[0036] (2) Heat the centrifuge tube with a PCR instrument at 95°C for 5 minutes, and then quickly ice-bath for 18 minutes;
[0037] (3) Add 0.5 μl of dNTPs mixture with a concentration of 2.5 mM each, 0.1 μl of BSA with a concentration of 1%, and 0.5 μl of phi29 DNA polymerase with a concentration of 10 U / μl in the centrifuge tube, mix well, and centrifuge. The reaction solution was centrifuge...
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