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Specific PCR primers for Plasmodiophora brassicae Woron and detection method

A kind of swollen bacteria, specific technology, applied in the field of detection of clubroot bacteria, can solve the problems of inability to identify and diagnose the bacteria, difficulty in disease control, etc., and achieve the effect of simple operation, high sensitivity and good specificity

Pending Publication Date: 2018-04-20
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In agricultural practice, the field soil and crops before sowing are infected but not symptomatic, and the infection and disease diagnosis of clubroot bacteria cannot be avoided. As a result, once the cruciferous clubroot occurs, it is very difficult to prevent and control the disease.
In addition, clubroot bacteria cannot be cultured on artificial medium, and traditional pathogen identification research methods cannot quickly and easily identify and diagnose the bacteria.

Method used

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  • Specific PCR primers for Plasmodiophora brassicae Woron and detection method
  • Specific PCR primers for Plasmodiophora brassicae Woron and detection method
  • Specific PCR primers for Plasmodiophora brassicae Woron and detection method

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Effect test

Embodiment 1

[0033] Example 1 Design and synthesis of specific primers for clubroot

[0034] According to the conserved internal transcribed spacer (ITS) sequence (NCBI accession number KX011135.1) in the whole genome sequence of Plasmodiophora brassicae, it is closely related to other fungi Spongospora in the phylum Plasmodiophora Subterranea), Polymyxa graminis (Polymyxa graminis), Maullinia ectocarpii (Maullinia ectocarpii), Woronina pythii (Woronina pythii) partial ITS sequence similarity alignment ( figure 1 ) to find specific regions of clubroot. The specific primers Pb4871F / Pb4871R (Table 1) designed by software and manual correction were used, and the size of PCR amplification product was 244bp.

[0035] Table 1 Sequences of primers for rapid detection of P. rhizogenes

[0036]

Embodiment 2

[0037] Embodiment 2 The specific PCR detection method of Brassica rhizogenes

[0038] 1. Template DNA extraction

[0039]The template DNA was extracted by different methods. The DNAsecure Plant Kit (TianGen) was used for dormant spores and plant and seed samples of clubroot, the modified CTAB method was used for fungi, the Bacteria Genomic DNAKit (CWBio) was used for bacteria, and the DNeasy PowerSoil Kit (Qiagen) was used for soil samples. . DNA templates extracted by different methods were quantitatively analyzed by ultra-micro spectrophotometer (Table 2).

[0040] Table 2 Concentrations of different biological DNA templates

[0041]

[0042] 2. PCR reaction system of clubroot

[0043] The template DNA (50ng) was based on the recommended annealing temperature (62°C) of the designed primers, and the PCR reaction system was optimized (Table 3). The results showed that different PCR reaction systems were able to successfully amplify the target fragment of P. figure 2 )...

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Abstract

The present invention provides specific PCR primers for Plasmodiophora brassicae Woron and a detection method. The specific PCR primers Pb4871F and Pb4871R (SEQ ID NO: 1-2) are designed based on conserved internal transcribed spacer sequences in the whole genomic sequence of the Plasmodiophora brassicae. The pair of primers can be used for molecular identification and disease epidemic dynamic monitoring of the Plasmodiophora brassicae. A rapid, accurate, simple and effective method for the early diagnosis and bacteria detection of clubroot of crucifers is provided, and an important tool and technical support are provided for effective monitoring of development and progression of the Plasmodiophora brassicae in a field and prediction and forecast of the clubroot of crucifers.

Description

technical field [0001] The present invention relates to the detection of clubroot, in particular to specific PCR primers and a detection method of brassica clubroot. Background technique [0002] Plasmodiophora brassicae is an obligate parasitic phytopathogen that infects cruciferous crops and causes clubroot disease. With the expansion of the planting area of ​​cruciferous and other crops, the occurrence and damage of clubroot disease is becoming more and more serious in the world. Fujian, Guangxi, Yunnan, Tibet, Sichuan, Guizhou, Guangdong and other provinces, municipalities and autonomous regions have occurred. The annual economic loss caused by clubroot disease can reach 10-15% worldwide. Cruciferous clubroot mainly damages the roots and forms tumors of different sizes. Plants infected by clubroot grow slowly, and are easily mixed with saprophytic fungi to cause rot, accompanied by a fishy odor. The main transmission routes of clubroot include bacteria-carrying soil, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12N15/11
CPCC12Q1/6895
Inventor 梁月关格格朴钟云孙慧颖陈彩霞庞文星李晓楠
Owner SHENYANG AGRI UNIV
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