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Quantitative detection method of plasmodiophora brassicae in different kinds of samples

A quantitative detection method and technology for Phytophthora rhizogenes are applied in the field of quantitative detection of Phytophthora rhizogenes, which can solve the problems of inability to identify and diagnose bacteria, difficulty in disease prevention and control, and inability to cultivate Phytophthora rhizogenes.

Inactive Publication Date: 2019-09-17
SHENYANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In agricultural practice, once clubroot occurs, it will be very difficult to prevent and control the disease. In addition, clubroot cannot be cultivated on artificial medium. Traditional pathogen identification research methods cannot quickly and easily identify the pathogen. identification and diagnosis of

Method used

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  • Quantitative detection method of plasmodiophora brassicae in different kinds of samples
  • Quantitative detection method of plasmodiophora brassicae in different kinds of samples
  • Quantitative detection method of plasmodiophora brassicae in different kinds of samples

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] The establishment of the regression line quantitative detection equation of different samples of embodiment 1

[0052] Using Soil Genomic DNA Extraction Kit (Qiagen), Fungal Genomic DNA Extraction Kit (FastDNASpin Kit, CWBio), Plant Genomic DNA Extraction Kit (TIANGEN), Gel Recovery Kit (AXYGEN), Plasmid Extraction Kit (TIANGEN) to extract plasmid.

[0053] Construct a plasmid containing the target gene (Plustridiobacter ITS gene fragment), pure dormant spores, fungus-carrying soil, common representative cruciferous plants (rape, cabbage, mustard) with mycorrhizal tissues and fungus-carrying seeds.

[0054] 1. Plasmid

[0055] The quantitative concentration after extracting the total plasmid DNA was 472.6ng / μL, according to the formula: copies / μL=(6.02×1023copies / mol)(plasmid concentration g / μL) / (plasmid molecular weight g / mol)(Johnson et al, 2015) Calculate the plasmid copy number concentration, 472.6ng / μL is equivalent to 1010.91 copy number / μL, DNA is diluted 10 ti...

Embodiment 2

[0064] The establishment of embodiment 2 data fitting and general model

[0065] Comparing the mathematical models and regression equations established corresponding to the above-mentioned different types of samples, using the SAS statistical software linear regression analysis, fitting to establish a "general model" and equations applicable to various types of samples.

[0066] The results show that although the regression lines (y=ax+b) corresponding to different types of samples are slightly different, the parallelism between the regression lines is relatively high, that is, the slopes (b) of different lines are very close, but the intercepts (b) are different. , which may be related to the detection of background differences in different types of samples ( Image 6 ).

[0067] In addition, principal component analysis software was used to further analyze the regression equations corresponding to different types of samples, and it was found that the primers (slope) used as...

Embodiment 3

[0069] Application of Embodiment 3 General Model

[0070] Conventional quantitative detection methods need to accurately prepare a certain series of gradient-carrying samples according to different samples, and use the quantitative PCR method to establish a corresponding regression equation ( Figure 1-Figure 5 ), and then use the obtained regression equation to estimate the number of Plasmodium in the sample.

[0071] The method of the invention does not need to consider the type of samples, and at the same time, it does not need to prepare a series of gradient samples and establish a regression equation during the detection process. It is only necessary to use the qPCR method to obtain the Ct value of the test sample, use the "general model", refer to Table 1 to obtain the corresponding sample type equation or use the "general model" of the corrected intercept, to realize the evaluation of the number of bacteria carried by different samples. That is, y=ax+b+△c, where x repr...

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Abstract

The invention provides a quantitative detection method of plasmodiophora brassicae in different kinds of samples. The method comprises the steps of extracting DNA including plasmids, resting spores, germ carrying soil, crucifer germ carrying root tissue and seeds. ITS region gene fragments of the plasmodiophora brassicae in different samples are detected through a fluorescent quantitation PCR technique, and a regression equation between the number of the plasmodiophora brassicae in the samples and Ct value is established. The regression equations of different samples are fitted through statistics software, a universal model and an equation suitable for different samples are obtained, and quantitative detection for assessing the plasmodiophora brassicae in different samples through the universal model is realized. According to the quantitative detection method disclosed by the invention, a molecular biology technique is used, a mathematics and statistics analysis method is combined for use, the universal models for quantitative detection are fitted, the purpose of accurately assessing the quantity of the plasmodiophora brassicae in different samples is achieved, the quantitative detection method has the characteristics of saving test cost, shortening detection time and the like, and technical means and theory reference are provided for the respects of early diagnosis, epidemic early warning, prevention and control and the like of cruciferae club roots.

Description

technical field [0001] The invention belongs to the technical field of plant pathogenic fungus monitoring and crop disease diagnosis and prevention, and in particular relates to a quantitative detection method of clubroot bacteria carried in different types of samples. Background technique [0002] Plasmodiophora brassicae is an obligate parasitic plant pathogen that infects Brassicaceae and other crops and causes clubroot. With the expansion of the planting area of ​​main host crops such as Cruciferae, the occurrence and damage of clubroot are becoming more and more serious worldwide. Especially in my country, clubroot has occurred and spread continuously in Sichuan, Beijing, Shandong, Tibet, Jiangsu, Hunan, Jiangxi, Anhui, Liaoning, Jilin and Heilongjiang provinces and cities. Clubroot can cause severe decline in yield and quality of cruciferous crops, or even yield failure. The main transmission routes of clubroot are various, among which the spread of bacteria-carrying...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6851C12Q1/06C12R1/645
CPCC12Q1/6895C12Q1/6851C12Q2531/113C12Q2563/107C12Q2537/165
Inventor 梁月邢曼竹关格格朴钟云
Owner SHENYANG AGRI UNIV
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