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Detection method for phytopathogen content in soil

A technology for plant pathogenic bacteria and detection methods, applied in the direction of microbial-based methods, biochemical equipment and methods, microbial measurement/testing, etc., can solve the problems of poor biological representation and unsuitability for the detection of a large number of soil samples

Active Publication Date: 2015-06-10
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, kit methods are mostly used for the preparation of soil sample DNA, and the processing amount of soil samples is 0.25g-1g, which has poor biological significance and is not suitable for the detection of a large number of soil samples.

Method used

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  • Detection method for phytopathogen content in soil
  • Detection method for phytopathogen content in soil
  • Detection method for phytopathogen content in soil

Examples

Experimental program
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Effect test

Embodiment 1

[0079] Embodiment 1, utilize the detection method of plant pathogen content in soil to detect the content of cruciferous clubroot in soil

[0080] The complete set of reagents for identifying Cruciferous Clubroot consists of PCR primer pairs and probes for identifying Cruciferous Clubroot; the primer pair for identifying Cruciferous Clubroot consists of a single sequence shown in SEQ ID No.1 Strand DNA and the single-stranded DNA composition shown in SEQ ID No.2; The probe is the single-stranded DNA shown in SEQ ID No.3 in the sequence listing, and the 5' end of the probe is marked with FAM, and the The 3' end of the probe is labeled with TAMARA.

[0081] The specific steps of the method for detecting the content of cruciferous clubroot bacteria in soil are as follows:

[0082] 1. Preparation of standard curve

[0083] 1.1 Construction of standard plasmids

[0084] The DNA molecule shown in the DNA sequence of the cruciferous clubroot bacterium whose gene accession number i...

Embodiment 2

[0108] Embodiment 2, the sensitivity experiment of the detection method of cruciferous clubroot content in soil

[0109] 1. Sensitivity experiment of a complete set of reagents for identification of Cruciferous clubroot

[0110] The concentration in Example 1 is 1×10 9 The copy / μl linear carrier T solution was serially diluted with sterile water to obtain linear carrier T solutions with concentrations of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 copies / μl, respectively.

[0111] Using the DNA in the linear carrier T solution with a concentration of 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 copies / μl respectively as a template, carry out fluorescent quantitative PCR with the complete set of reagents for identifying Brassicaceae clubroot in step 1.3 of Example 1 Reaction, the reaction system is 1 copy / 25 μl reaction system, 2 copies / 25 μl reaction system, 3 copies / 25 μl reaction system, 4 copies / 25 μl reaction system, 5 copies / 25 μl reaction system and 6 copies / 25 μl reaction system.

[0112] The...

Embodiment 3

[0118] Embodiment 3, the specificity experiment of the detection method of cruciferous clubroot content in soil

[0119] According to the method of 2.1 of embodiment 1 step 2, clubroot pathogen of cruciferae is replaced by Java different water mold (Allomyces javanicus), tobacco red spot fungus (Alternaria alternata), cabbage black spot fungus (Alternaria brassicae), white radish black Alternaria raphani, Aspergillus niger, Bipolaris sp., Botrytis sp., Colletotrichum dematium, Fusarium graminearum ), Fusarium oxysporum (Fusarium oxysporum), Penicillium sp. (Penicillium sp.), Rhizoctonia solani (Rhizoctonia solani) and Verticillium dahliae (Verticillium dahliae), other steps are all unchanged, respectively to get Java different water Standard soil microbial DNA for mould, standard soil microbial DNA for Alternaria Alternaria in tobacco, standard soil microbial DNA for cabbage black spot fungus, standard soil microbial DNA for white radish black spot fungus, standard soil microb...

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Abstract

The invention discloses a detection method for phytopathogen content in soil. The detection method for the phytopathogen content in the soil includes that S1) using a soil microorganism DNA extracting method to extract the microorganism DNA of the soil to be detected to obtain the microorganism DNA of the soil to be detected; S2) using the microorganism DNA of the soil to be detected as a template, using reagent set for identifying the phytopathogen to perform fluorescent quantitative PCR, and confirming the content of the phytopathogen in the soil to be detected through the fluorescence intensity of the PCR product. The soil microorganism DNA extracting method includes that S21) adding extracting buffer solution to the soil to obtain cell suspension; S22) adding SDS to the cell suspension to obtain cell lysis solution, performing cell lysis to obtain lysate, wherein the mass percent concentration of the SDS in the cell lysis solution is 4%; removing impurities from the lysate to obtain the microorganism DNA of the soil to be detected.

Description

technical field [0001] The invention relates to a method for detecting the content of plant pathogenic bacteria in soil in the field of biotechnology. Background technique [0002] Soilborne diseases are serious damage to plants. Its pathogenic microorganisms exist in the soil, the environment is stable, and the pathogens have strong resistance to stress, so it is difficult to carry out chemical control. For example: Cruciferous clubroot (Clubroot) is a worldwide soil-borne disease caused by obligate parasitic Plasmodiophora brassicae Woronin. The disease harms many cultivars such as Brassicaceae cabbage, rapeseed, cabbage, and sage, causing root swelling, poor growth, wilting, dwarfing, and impaired yield and quality, which seriously threatens the production of Brassicaceae crops. The main determinant of the occurrence of plant soil-borne diseases is the content of pathogenic bacteria in the soil before sowing, and the quantitative detection of plant pathogenic bacteria i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12N15/10C12R1/645
CPCC12N15/10C12Q1/06C12Q1/6844C12Q1/689C12Q1/6895C12Q2561/101C12Q2545/114C12Q2531/113
Inventor 张力群郭松张俊威陈伟
Owner CHINA AGRI UNIV
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