One-step dual PCR method for detecting fire blight of pear
A technology of E. amylovora and its detection method, which is applied in biochemical equipment and methods, and the measurement/inspection of microorganisms. It can solve the problems that E. amylovora cannot be detected, meet the requirements of plant quarantine and disease monitoring, and have a simple process. , strong specific effect
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Embodiment 1
[0038] Implementation Example 1: Primer primer A / B and primer E / F use E. amylovora pus as a template.
[0039] 1) Preparation of E. amylovora pus template
[0040] 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.
[0041] 2) Synthesis of specific primers for detection of E. amylovora
[0042] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0043] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0044] and
[0045] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0046] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0047] 3) PCR amplification reaction
[0048]The primer A / B primer pair and the primer E / F primer pair are simultaneously carried out in the same reaction system fo...
Embodiment 2
[0055] Implementation Example 2: Primer primer A / B and primer primer E / F use the freshly cultured bacterial solution of E. amylovora as a template.
[0056] 1) Use freshly cultured bacteria as a template
[0057] 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.
[0058] 2) Synthesis of specific primers for detection of E. amylovora
[0059] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0060] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0061] and
[0062] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0063] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0064] 3) PCR amplification reaction
[0065] 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
[0072] Implementation Example 3: Primer primerA / B and primer primer E / F detect the sensitivity of E. amylovora and the determination of the minimum amount of detected bacteria
[0073] 1) Use the freshly cultivated bacterial solution as a template to pick bacterial pus from the NA medium, add it to the NA liquid medium, and culture it with shaking at 28°C for 1 day. Take the original bacterial solution and dilute it by 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.
[0074] 2) Synthesis of specific primers for detection of E. amylovora
[0075] Primer A: 5'-CGGTTTTTAACGCTGGG-3';
[0076] Primer B: 5'-GGGCAAATACTCGGATT-3'
[0077] and
[0078] Primer E: 5'-AATAAGGTGCCTGGTAATG-3'
[0079] Primer F: 5'-GGTGGAATGAGACTGGGTA-3' was synthesized by Shanghai Sangon Company.
[0080] 3) PCR amplification reaction
[0081] The primer A / B primer pair and the primer E / F primer pair are simultaneo...
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