One-step dual PCR method for detecting fire blight of pear
A technology of E. amylovora and its usage, which is applied in the direction of biochemical equipment and methods, and the measurement/inspection of microorganisms. It can solve the problems that E. amylovora cannot be detected and meet the requirements of plant quarantine, disease monitoring and detection accuracy. High, simple process effect
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Embodiment 1
[0040] Implementation Example 1: Primer A / B and Primer E / F use E. amylovora pus as a template.
[0041] 1) Preparation of E. amylovora pus template
[0042] To activate the E. amylovora stored at -80°C, first pick the bacterial liquid and put it on the NA medium (3 grams of beef extract, 0.5 grams of yeast extract powder, 5 grams of peptone, 10 grams of glucose, 1000 ml of distilled water, pH 7.0-7.2 ) at 28°C, cultivate for 1-2 days, pick out the pus, and add it directly to the PCR reaction system.
[0043] 2) Synthesis of specific primers for detection of E. amylovora
[0044] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0045] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0046] and
[0047] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0048] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0049] 3) PCR amplification reaction
[0050] The primer A / B primer pair and the primer E / F primer pair are simultaneously carried out in the same reaction system for double...
Embodiment 2
[0057] Implementation Example 2: Primer primer A / B and primer primer E / F use the freshly cultured bacterial solution of E. amylovora as a template.
[0058] 1) Use freshly cultured bacteria as a template
[0059] Pick the bacterial pus from the NA medium, add it to the NA liquid medium, and incubate with shaking at 28°C for 1 day, take 1 μl of the bacterial liquid as a template for PCR amplification, and directly add it to the PCR reaction system.
[0060] 2) Synthesis of specific primers for detection of E. amylovora
[0061] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0062] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0063] and
[0064] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0065] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0066] 3) PCR amplification reaction
[0067] The primer A / B primer pair and the primer E / F primer pair are simultaneously carried out in the same reaction system for double PCR reaction, in which 10×PCR buffer 5μl, 15mM MgCl...
Embodiment 3
[0074] Implementation Example 3: Primer primer A / B and primer primer E / F detect the sensitivity of Phytophthora amylovora and the determination of the minimum amount of detected bacteria
[0075] 1) Use freshly cultured bacteria as a template
[0076] Bacterial pus was picked from NA medium, added to NA liquid medium, cultured with shaking at 28°C for 1 day, and the original bacterial solution was taken and diluted 10 1 -10 5 1 μl of bacterial solution was used as a template for PCR amplification, and was directly added to the PCR reaction system.
[0077] 2) Synthesis of specific primers for detection of E. amylovora
[0078] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0079] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0080] and
[0081] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0082] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0083] 3) PCR amplification reaction
[0084] The primer A / B primer pair and the primer E / F primer pair are simultaneousl...
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