Method for detecting three potato pathogenic bacteria by multiple PCR (polymerase chain reaction) techniques

A technical detection and pathogenic bacteria technology, which is applied in the field of detecting three potato pathogenic bacteria using multiplex PCR technology, can solve the problems of not meeting the detection requirements, increasing the detection complexity, and inaccurate detection results, achieving fast detection speed, no potential safety hazards, The effect of high sensitivity

Inactive Publication Date: 2019-08-16
FUJIAN AGRI & FORESTRY UNIV
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  • Description
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Problems solved by technology

[0005] Traditional pathogenic bacteria detection methods have the disadvantages of cumbersome steps, long time-consuming and inaccurate detection results due to the need for microscopes, isolation cultures, bioassays, serology and other techniques.
In addition, in the field, diseases are often caused by the mixed infection of multiple pathogenic bacteria, which increases the complexity of detection. At this time, conventional isolation and identification methods cannot meet the detection requirements.

Method used

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  • Method for detecting three potato pathogenic bacteria by multiple PCR (polymerase chain reaction) techniques
  • Method for detecting three potato pathogenic bacteria by multiple PCR (polymerase chain reaction) techniques
  • Method for detecting three potato pathogenic bacteria by multiple PCR (polymerase chain reaction) techniques

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Experimental program
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Effect test

Embodiment 1

[0045] Embodiment 1 The establishment of detection method

[0046] (1) Genomic DNA extraction: HP Fungal DNA kit was used to extract the genomic DNA of Phytophthora infestans, Alternaria solani and Rhizoctonia solani (AG3 fusion group) respectively.

[0047] (2) Primer design: According to the specific gene INF1 gene of Phytophthora infestans (GenBank: AY830094.1), the specific gene histone H3 gene of Alternaria solani (GenBank: KF308960.1) and Rhizoctonia solani (AG3 The primers were designed for the gene sequence of the specific gene RSENDO1 gene (GenBank: KM522918.1), which were Pi-F / R, As-F / R and Rs-F / R in sequence. The nucleotide sequences of the primers used are shown in Table 1:

[0048] Table 1 Nucleotide sequence list of primers

[0049]

[0050] (3) Multiplex PCR reaction:

[0051] The 25μL amplification system is shown in Table 2:

[0052] Table 2 The concentration of each component in the multiplex PCR system

[0053]

[0054] Reaction program: react at ...

Embodiment 2

[0057] Example 2 Pathogen Detection Sensitivity Experiment

[0058] The template DNA concentrations of the three pathogenic bacteria were diluted to five concentration gradients of 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL and 0.1 pg / μL, and obtained according to the method established in Example 1 For test results, see figure 2 As shown (1: 1ng / μL; 2: 100 pg / μL; 3: 10 pg / μL; 4: 1 pg / μL; 5: 0.1 pg / μL; C: blank control), by figure 2 It is known that when the template concentration is 10 pg / μL, all three pathogens can be detected; when the template concentration is 1 pg / μL, except Phytophthora infestans, the other two pathogens can be detected. When the template concentration was 0.1 pg / μL, none of the three pathogenic bacteria could be detected. Therefore, the detection limits of Phytophthora infestans, Alternaria solani and Rhizoctonia solani (AG3 fusion group) are successively 10 pg / μL, 1 pg / μL and 1 pg / μL, so it can be judged that the established The multiplex PCR detection...

Embodiment 3

[0059] Example 3 Pathogen Detection Specificity Experiment

[0060] According to the method established in Example 1, three kinds of potato pathogenic bacterium genomic DNAs are used as the positive control of the template, and sterile deionized water is used as the negative control of the template. Alternaria solani, Rhizoctonia solani (AG3 fusion group), Phytophthora sojae, Phytophthora capsici, Alternaria, Alternaria adenocarp, Rhizoctonia solani (AG1 fusion group) and Phytophthora solani Genomic DNA of Rhizoctonia sp. (AG4 fusion group) was amplified by PCR. See the test results image 3 , when adding one, two or three DNA templates of the bacteria to be detected in the present invention, the electrophoresis results of the PCR product correspondingly only appear the target bands (1-7 swimming lanes) of the added bacteria, while adding the DNA of other bacteria The electrophoresis results of the multiple PCR products of the template have no bands (lane 8-13), thus indicat...

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Abstract

The invention discloses a method for detecting three potato pathogenic bacteria by multiple PCR (polymerase chain reaction) techniques, and belongs to the technical field of biological detection. According to the method, special genes of three pathogenic bacteria including phytophthora infestans, alternaria solani and rhizoctonia solani AG3 anastomosis groups are simultaneously amplified by multiple PCR once, and a PCR amplifying product is detected through agarose gel electrophoresis. The method includes the steps: (1) extraction of genome DNA (deoxyribonucleic acid) of the three potato pathogenic bacteria; (2) design of three pairs of amplification primers; (3) implementation of multiple PCR; (4) electrophoresis detection. According to the method, the three pathogenic bacteria are simultaneously detected by the multiple PCR techniques, and the method has the advantages that efficiency is high, speed is high, time is saved, and cost is reduced.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for detecting three potato pathogenic bacteria by using multiplex PCR technology. Background technique [0002] potato( Solanum tuberosum L.) is a dual-purpose crop for grain, vegetables, feed, and processing. It has the characteristics of short production cycle, wide adaptability, high yield and rich nutrition, so it is widely planted in the world. my country is the largest potato producer in the world, and the main planting areas are distributed in North China, Northeast China, Southwest China and South China. Potato tubers are rich in starch, protein, antioxidants and vitamins. They have the characteristics of food, vegetables and feed. They can be processed in a variety of ways. They play an important role in increasing food nutrition sources, ensuring food security and promoting economic development. effect. Under the background of over-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12N15/11C12R1/645
CPCC12Q1/686C12Q1/6895C12Q2537/143
Inventor 詹家绥詹芳芳王甜汤一凡
Owner FUJIAN AGRI & FORESTRY UNIV
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