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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

Inactive Publication Date: 2005-11-30
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the detection of E. amylovora which does not contain the pEA29 plasmid cannot be carried out

Method used

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  • One-step dual PCR method for detecting fire blight of pear
  • One-step dual PCR method for detecting fire blight of pear

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a one-step dual PCR method for detection of erwinia amylovora. It belongs to the technique sphere of agricultural pest control and plant quarantine inspection. It designs two-pair specific primer according to pEA29 plasmid and genome ams gene order, simultaneously detecting erwinia amylovora in the same reaction system, augmentation product being 1.0kb and 1.5kb, and detection sensibility of the two-pair primer reaching three bacterial cells. It is a supplementary and improvement to the method for detection of specific primer of erwinia amylovora pEA29 plasmid. And it also can detect the strain of erwinia amylovora containing no pEA29 plasmid. Using dual PCR technique, augmentation of two-pair primer in the same reaction system, it improves the detection accuracy, saves the testing time, and it can largely spread as a form of reagent case.

Description

(1) Technical field [0001] The "one-step double PCR method for detection of Phytophthora amylovora" of the present invention is specially used for detection of Phytophthora amylovora and belongs to the technical field of crop disease control and plant quarantine. (2) Background technology [0002] Erwina amylovora is a devastating bacterial disease on pears, apples and other Rosaceae plants. It was first discovered in 1780 and is now mainly distributed in Central and North America, Oceania, Europe, West Asia, Egypt, Japan, Korea and more than 40 countries or regions. During the decade from 1951 to 1960, the United States lost an average of $4 million per year. my country has not found the harm of the disease so far, and it is listed as a first-class foreign quarantine object in my country. [0003] Pear fire blight mainly harms flowers, leaves, young shoots, fruits, etc., generally reducing production by 25%. [0004] The cultivation area of ​​fruit trees in my country is...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 冯洁许景升徐进何礼远张争张杨
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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