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Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application

A cruciferous vegetable and fluorescence quantitative technology, which is applied in the field of fluorescence quantitative PCR detection, can solve the problems that the detection method is not fast enough and cannot quantify Phytophthora rhizogenes, and achieves the effects of good practicability, rapid detection and high sensitivity

Inactive Publication Date: 2014-08-06
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

These detection methods are not fast enough and cannot quantify Plasmodium in plant, water and soil samples. And the development of multiplex PCR, on the other hand, the development from qualitative PCR to real-time fluorescence quantitative PCR (RTi-PCR), which provides us with a strong foundation for the rapid detection of Plasmodium in plants, soil and water

Method used

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  • Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application
  • Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application
  • Fluorescent quantitative PCR detection technology of brassicaceous vegetable plasmodiophoromycetes, and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Embodiment 1: the specific primer detection of clubroot

[0040] Using common soil-borne pathogenic bacteria in the rhizosphere microorganisms of cruciferous vegetable crops as materials, the primer pairs PBF2 / PBR2 and PBF3 / PBR3 of clubroot were specifically detected. The swollen roots of cabbage were stored in a -20°C refrigerator. Other soil-borne pathogenic fungal strains were cultured with PDA or PD liquid medium. All the strains were first activated on solid medium, and then transferred to liquid medium for culture. One week later, the hyphae or cells were collected to extract DNA. DNA was extracted using a modified CTAB extraction method.

[0041] Using the genomic DNA of Plasmodium brassicae and other pathogenic fungi of soil-borne diseases as templates, the primers PBF2 / PBR2 were used for ordinary PCR amplification to test the specificity of the primers. The general PCR reaction system is as follows: total reaction volume 25 μL, 10×PCR buffer 2.5 μL, dNTP (10...

Embodiment 2

[0046] Embodiment 2: Detection of root clubroot inside the cabbage growth period

[0047] In early September 2012, samples were taken at the experimental base of Qingdao Academy of Agricultural Sciences during the growth period of cabbage. The samples were the roots of cabbage 15 days after planting in the field with clubroot disease. In addition, the roots of plants that did not show symptoms of disease were taken, and the samples were washed repeatedly to ensure that there were no dormant spores of Plasmodium spp. on the surface of the samples. The DNA was extracted and purified using (MP Biomedicals, USA, FastDNA SPIN Kit for Soil). The DNA of each root was dissolved in 30 μl TE buffer, and 2 μl of the DNA solution was taken as a template, and the primer pair GZ10 / GZ11 was used for PCR detection of each sample, and the amplified product was detected by 1.5% agarose electrophoresis. PCR detection.

[0048] The products amplified by common PCR show no bands on the agarose ge...

Embodiment 3

[0050] Example 3: Soil sample detection of natural disease in the field

[0051] In areas where clubroot of cruciferous vegetables in our country has serious incidence, during the field disease investigation from July 2011 to July 2012, the soil with natural disease in the fields of Hubei, Yunnan, Guizhou, Qingdao, Sichuan, Chongqing and other places was collected Samples, a total of 60 soil samples were collected, 32 of the 60 soil samples were seriously diseased fields, and these fields had been planting cruciferous vegetable crops before 2011, and 20 were lightly diseased fields, and these fields were planted with cruciferous vegetables The vegetable crops in the family were not more than 3 years old, and 8 of them were fields without disease symptoms. The previous crop was Chinese cabbage. Most of the crop varieties cultivated in these areas were susceptible varieties. The soil samples taken from the field were immediately stored in a -20°C refrigerator, and freeze-dried d...

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Abstract

The invention relates to a fluorescent quantitative PCR detection technology of plasmodiophoromycetes, and its application. The technology is used for rapidly and accurately quantitatively detecting brassicaceous vegetable plasmodiophoromycetes. The technology comprises the following steps: extracting DNA from a sample, designing a specific primer, carrying out real-time fluorescent quantitative PCR detection on the ITS region gene fragment of the plasmodiophoromycetes by applying an SYBRGreen I based dye process fluorescent quantitative PCR technique, constructing a standard curve, and calculating the copy number of the ITS region gene fragment in the sample and the concentration of the plasmodiophoromycetes. The detection technology has the advantages of simple and rapid operation, strong specificity and high sensitivity, realizes a highest detection sensitivity reaching 24 copy numbers per reaction, and can also realize the analysis of large batch samples. The technology provides a good basis for the early-stage prediction forecasting of diseases, so the technology is adapted to be widely applied in the plant disease diagnosis detection field.

Description

technical field [0001] The invention relates to a fluorescence quantitative PCR detection technology and application of cruciferous vegetable clubroot, which is specially used for detecting cruciferous vegetable clubroot, and belongs to the technical field of crop disease diagnosis and prevention. Background technique [0002] Plasmodium brassicae ( Plasmodiophora brassicae ) causes clubroot of cruciferous vegetables, mainly parasitic on the roots of many Brassica plants. It is distributed all over the world. In recent years, its damage range has expanded from cruciferous vegetables to some flower plants, infecting more than 100 species (variants) of cultivated and wild plants. Clubroot causes worldwide losses of 10-15% per year. After the infected plant is infected by Plasmodium, clubroot tissue is formed, and the plant is short and prone to wilting (Voorips, 1995). The pathogen is released from decaying clubroot tissue to form dormant spores that can survive for many ye...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
CPCC12Q1/6851
Inventor 谢学文李金萍李宝聚石延霞柴阿丽
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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