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 bacterial content, so as to achieve accurate detection results and overcome low sensitivity. , the effect of strong signal strength
Inactive Publication Date: 2005-01-26
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) directly amplifying the sample, because the plant tissue contains a large amount of phenolic substances, carbohydrates and other PCR reaction inhibitors, which seriously affects the sensitivity of the PCR reaction
2) The sample volume added to the PCR reaction system is very small, usually only 1-5ul, if expressed in cell / ml, the number will be very large
However, this method requires certain equipment, the operation is more complicated, and the cost is higher.
Method used
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Embodiment 1
[0035] 1) Preparation of pathogenic bacteria specific antiserum
[0036] Using traditional antiserum preparation methods, glutaraldehyde-fixed whole bacterial cells or extracted and purified bacterial lipopolysaccharides are used to immunize rabbits to prepare specific antisera against plant pathogenic bacteria whole cells or lipopolysaccharide (LPS) Serum (the technical method is mature, please refer to relevant literature), the agar double diffusion method (ODD) measures the titer, and select the antiserum with a titer of 1:32 or more;
[0037] 2) Coating of PCR reaction tube
[0038] Preparation of coating buffer: Na 2 CO 3 1.59g; NaHCO 3 2.93g, add distilled water to 1000ml, pH 9.6
[0039]Choose a thin-walled PCR reaction tube (common PCR tube) that has not been silanized, dilute the antiserum 500 times with coating buffer, add 50μl of diluted antiserum to each tube, and incubate at 37°C for 2 hours or 4°C overnight;
[0040] 3) Washing of PCR reaction tube
[0041] Prepare 0...
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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 to detect 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 introduction risk of harmful organisms has a lot to do with whether there are accurate and sensitive detection methods. The detection technology is advanced and reliable, and the risk of the introduction of foreign pests can be reduced. my country's current detection technology for quarantine plant pathogenic bacteria 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, easy, accurate and sensitive detection met...
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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