Method for detecting plant pathogenic bacteria sensitivity by improving PCR technology

A technology for the detection of plant pathogenic bacteria and technology, which is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of complex operation, low sensitivity, and low detection sensitivity, so as to achieve accurate detection results and overcome low sensitivity. , the effect of improving the sensitivity

Inactive Publication Date: 2003-11-19
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

However, PCR technology also has its undeniable limitations, making its sensitivity often not high
Especially for the detection of pathogenic bacteria on asymptomatic plant materials, due to the low bacterial content and the presence of a large number of PCR reaction inhibitors in the sample liquid, the actual detection sensitivity is often relatively low, which limits its practical application in the detection of plant pathogens
The reason is: 1) direct

Method used

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  • Method for detecting plant pathogenic bacteria sensitivity by improving PCR technology
  • Method for detecting plant pathogenic bacteria sensitivity by improving PCR technology
  • Method for detecting plant pathogenic bacteria sensitivity by improving PCR technology

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Experimental program
Comparison scheme
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Embodiment 1

[0031] 1) Preparation of pathogenic bacteria-specific antiserum

[0032] Using traditional antiserum preparation methods, use glutaraldehyde-fixed whole bacterial cells or extracted and purified bacterial lipopolysaccharides to immunize rabbits to prepare specific anti-bacterial antibodies against plant pathogenic bacterial whole cells or lipopolysaccharide (LPS). For serum (technical method is mature, please refer to relevant literature), agar double diffusion method (ODD) is used to determine the titer, and the antiserum with titer reaching or exceeding 1:32 is selected;

[0033] 2) Coating of PCR reaction tube

[0034] Prepare coating buffer: Na 2 CO 3 1.59g; NaHCO 3 2.93g, add distilled water to 1000ml, pH 9.6 Select a thin-walled PCR reaction tube (ordinary PCR tube) that has not been silanized, dilute the antiserum 500 times with coating buffer, add 50 μl diluted antiserum to each tube, and store at 37°C Incubate for 2 hours or overnight at 4°C;

[0035] 3) Washing ...

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Abstract

A process for increasing the sensitivity of RCR technique in detecting the pathogenic bacteria of plant features that the fire blight bacterium of pear is used as target, and includes such steps as preparing the antiserum specific to pathogenic bacterium, coating PCR reaction tube, washing the PCR reaction tube, adsorbing the target bacteria, and PCR amplifying. It can increase the sensitivity by 1000 times.

Description

(1) Technical field [0001] The invention "a method for improving the sensitivity of PCR technology for detecting plant pathogenic bacteria" belongs to the field of biotechnology. It is specially used to detect pathogenic bacteria and improve the sensitivity of traditional PCR technology to detect pathogenic bacteria. (2) Background technology [0002] With the development of science and technology, the detection technology of plant pathogenic bacteria has been greatly improved. The risk of pest introduction has a lot to do with whether there are accurate and sensitive detection methods. With advanced and reliable detection technology, the risk of introduction of alien pests can be reduced. At present, the detection technology of quarantine plant pathogenic bacteria in my country is relatively weak, and many ports have not yet carried out the detection of quarantine pathogenic bacteria, and there is an urgent need for simple, accurate and sensitive detection methods. Curre...

Claims

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Application Information

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IPC IPC(8): C12Q1/04C12Q1/68
Inventor 胡白石许志刚卢玲刘凤权
Owner NANJING AGRICULTURAL UNIVERSITY
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