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Specific pcr detection method of clubroot pathogen in soil

A detection method and specific technology, which is applied in the field of specific PCR detection of clubroot in soil, can solve the problems of low sensitivity and interference, and achieve the effects of wide applicability, rapid detection and sensitive method

Active Publication Date: 2011-12-28
SHANGHAI ACAD OF AGRI SCI
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  • Description
  • Claims
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AI Technical Summary

Benefits of technology

This patented technique allows researches on how plants grow without harmful chemicals like pesticides or fertilizers that may be used during their growth process. By testing different types of root-associated gene sequences found within these crops, we have developed an improved way to monitor them quickly and accurately. Additionally, this approach provides us with valuable insights into what causes disease called Cronobrassy grass rot caused by BSCCO strains isolated through laboratory experiments conducted at home and abroad.

Problems solved by technology

This patents discuss various methods aimed towards prevention of diseases called Cobbler' s Disease (CWD), particularly those affecting Brussels sprouts grown mainly around China. Current treatments involve pesticides applied directly onto these germplasm parts without any consideration given how they may cause damage if taken carelessly. There currently exist effective ways to identify and isolate damaged members of plants based upon their ability to release phytophilic microsporidia when exposed during growth. However, current techniques require significant amounts of laborious work and have limitations in terms of effectiveness over different environmental conditions.

Method used

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  • Specific pcr detection method of clubroot pathogen in soil
  • Specific pcr detection method of clubroot pathogen in soil
  • Specific pcr detection method of clubroot pathogen in soil

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Determination of Molecular Characteristic Sequence of Clubroot Phytophthora in Shanghai Area

[0059] 1) Root group DNA extraction of clubroot

[0060] Grind the deformed and swollen diseased roots of the cleaned vegetables with liquid nitrogen, take 50 mg of ground diseased root samples and add 1 mL of extract (50 mmol / L Tris-HCl, 150 mmol / L NaCl, 100 mmol / L EDTA) to 1.5 mL In an Eppendorf centrifuge tube, shake and mix, then add 0.1mL 20% SDS to mix, then place the centrifuge tube in a shaker at 37°C and shake slowly for 1h, add 0.15mL 5mol / L NaCl solution to each tube, mix slowly, add 0.13mL CTAB / NaCl solution (10% CTAB in 0.7mol / L NaCl), mix well, put in water bath at 65°C for 20min, shake once every few minutes, cool to room temperature, add an equal volume of chloroform / isoamyl alcohol, Shake vigorously for 5 minutes, centrifuge at 10,000r / min for 12min, centrifuge the supernatant at 12,000r / min for 5min, transfer the supernatant to a new tube, add 0.6 ...

Embodiment 2

[0068] Example 2 Comparison of the Molecular Characteristic Sequences of Clubroot of Cabbage in Shanghai Area with Different Types of Pathogens in the Class Plasmodium and Other Different Types of Soil Pathogens

[0069] Compare the molecular signature sequences of the above-mentioned obtained Plasmodium cabbage strains with different types of pathogenic bacteria of the Placidiomycetes class, different types of pathogenic bacteria within the Oomycetes class, and different types of Fusarium bacteria in the soil, respectively, to find the strains of Plasmodium cabbage pathogens Specific regions of DNA.

[0070] 1) Sequence similarity comparison of the molecular characteristics of Plasmodium cabbage and different types of pathogenic bacteria of Plasmodium (wherein, the bases with higher identical frequency at the same position are marked in gray):

[0071] Wherein A: the sequence (SEQ ID No 10) of Pb-Shanghai of Shanghai cabbage clubroot

[0072] B: Plasmodiophora brasiccae sequ...

Embodiment 3

[0105] Example 3 Specific PCR Amplification of Brassicola brassica in Roots of Cabbage Clubroot

[0106] By comparing the sequence with other species of Clubroot, Oomycetes pathogenic species in soil and Fusarium species, 6 primer pairs were designed, and according to the Tm value of the primer pairs, the annealing temperature of PCR amplification was determined, and then the The root clubroot of bok choy was amplified by PCR, and the amplification reaction system was the same as in Example 1.

[0107] The amplification reaction conditions are as follows: 95°C for 5min for 1 cycle; 95°C for 30sec, 54°C for 30sec, 72°C for 45sec, 35 cycles; 72°C for 10min for 1 cycle.

[0108] The above-mentioned PCR amplification products were subjected to 1% Agrose gel electrophoresis, and the results showed that: the primer pairs 1, 2, 4, 5, and 6 of the 6 primers designed in the experiment amplified the target pathogen--Pluzophylla brassicae root, respectively. The only specific fragments ...

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Abstract

The invention discloses a specific PCR detection method for plasmodiophora brassicae in soil. The invention is characterized in that primers are designed as shown in SEQ ID No 1,2,3,5 according to 1-1028 sequences in a SEQ ID No 10 to form primer pairs 2,4,5 for performing the specific PCR detection. The method of the invention has the advantages of rapid detection, sensitive method, good specificity, wide application and simple operation, and can be taken as an effective technology means for detecting brassica plasmodiophora brassicae in soil and plant organization which has high practical value.

Description

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Claims

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Application Information

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Owner SHANGHAI ACAD OF AGRI SCI
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