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Detection primer of Thecaphana schwarzmania, detection method and application thereof

A detection method and technology for detection primers, which are applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as aggravating environmental pollution, time-consuming, and delaying prevention and control opportunities, and achieve high detection sensitivity. , strong specificity, high precision effect

Inactive Publication Date: 2020-06-12
湖北省农业科学院中药材研究所
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  • Claims
  • Application Information

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Problems solved by technology

Due to the lack of guidance of professionals in plant protection of traditional Chinese medicinal materials, various chemical agents are used indiscriminately in major production areas of rhubarb, which not only fails to effectively prevent and control diseases, but also increases the resistance of pathogenic bacteria and aggravates environmental pollution. It is very inconsistent with the development concept of green medicinal materials, and at the same time seriously threatens the further development and growth of the rhubarb industry
The identification of traditional pathogenic bacteria can only be carried out after the plants are infected by pathogenic bacteria and show symptoms. It requires isolation of pathogenic bacteria, purification and culture, verification of Koch's law, morphological observation, physiological and biochemical detection, etc. for identification. If it goes well, the whole process can be completed It takes about 20-30 days, but once other bacteria contaminate the medium, it will affect the identification speed and take longer. At the same time, long-term identification delays the timing of prevention and control, which is not conducive to the timeliness of prevention and control
Traditional identification has the shortcomings of inaccurate identification, time-consuming, and laborious, which can not meet the requirements of accurate and rapid prevention and control of plant protection work.

Method used

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  • Detection primer of Thecaphana schwarzmania, detection method and application thereof
  • Detection primer of Thecaphana schwarzmania, detection method and application thereof
  • Detection primer of Thecaphana schwarzmania, detection method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] The establishment of embodiment 1 rhubarb smut pathogen-specific PCR detection method

[0032] 1. Extraction of pathogenic genomic DNA of rhubarb smut

[0033] The rhubarb smut pathogen Thecaphora schwarzmaniana was preserved by the Plant Protection Laboratory of the Institute of Chinese Materia Medica, Hubei Academy of Agricultural Sciences. After one week of culture on plates containing PSA, total genomic DNA of pathogenic bacteria was extracted with a bacterial DNA extraction kit.

[0034] 2. Design of primers for specific molecular detection

[0035]PCR amplification and sequencing was performed on the obtained total genomic DNA of the pathogenic bacteria, according to the genus Thecaphoraschwarzmaniana and the same genus and the genus Thecaphora oxytropis, Thecaphora thlaspeos, Thecaphora amaranthi, Pseudozyma flocculosa, Thecaphora solani, Thecaphoraseminis-convolvuli, Thecaphora hennenii 、Thecaphora thlaspeos、Thecaphoraaffinis、Thecaphora frezii、Thecaphora lepti...

Embodiment 2

[0057] Example 2 Specific detection of genomic DNA of Thecaphora schwarzmaniana

[0058] Extraction of Thecaphora schwarzmaniana, Thecaphoraoxytropis, Thecaphora thlaspeos, Thecaphora amaranthi, Pseudozyma flocculosa, Thecaphora solani, Thecaphora seminis-convolvuli, Thecaphora hennenii, Thecaphora thlaspeos, Thecaphora affinis, Thecaphora frezii, Thecaphora, Thecaphora spilan, Thecaphora spilan The genomic DNAs of melandrii, Thecaphora pustulata, Thecaphora alsinearum, Ustilago maydis, S. chinensis, Fusarium, Rhizoctonia, Pseudomonas culm, Botrytis cinerea, etc. were set as negative controls, and 1 μL of the DNA of the above pathogens was used as a template for PCR.

[0059] The reaction system was as follows: 12.5 μL of 2×Taq Master Mix, 1 μL of 10 μM upstream and downstream primers of one of the 4 pairs of specific primers designed in step 2 of Example 1, and 9.5 μL of double distilled water. The amplification reaction program was: pre-denaturation at 94°C for 5 minutes, fo...

Embodiment 3

[0063] Example 3 Detection of Thecaphora schwarzmaniana genomic DNA by conventional PCR

[0064] The Thecaphora schwarzmaniana genomic DNA was sequentially diluted into 9 concentration gradients of 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, and then PCR was performed.

[0065]The total volume of the PCR reaction system was 25 μL, and the reaction system was: 12.5 μL of 2×Taq Master Mix, 1 μL of 10 μM upstream and downstream primers BXTF / BXTR (4 pairs of specific primers designed in Example 1), and 9.5 μL of double distilled water. The amplification reaction program was: pre-denaturation at 94°C for 5 minutes, followed by 25 cycles of 94°C for 30 seconds, 54°C for 30 seconds, 72°C for 1 minute and 30 seconds, and finally extension at 72°C for 3 minutes.

[0066] Take 7 μL of amplification product, use 1% (mass / volume) agarose gel, add 100 μL (mass to volume) of Goldview type I nucleic acid stain to 100 mL agarose gel, electrophoresis at 12...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to a detection primer of Thecaphana schwarzmania, a detection method and application thereof. According to the invention, a specific primer is designed according to the composition difference of basic groups in ITS regions of pathogenic bacteria Thecaphana schwarzmania of the rhubarb smut disease and ustilago fungi;the invention also provides the detection primer for the Thecaphana schwarzmania, the detection method thereof and an application of Thecaphana schwarzmania. The method has the characteristics of rapidness, accuracy and sensitivity, and the convenience, timeliness and accuracy of detecting the disease caused by Thecaphana schwarzmania are effectively improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer, a detection method and applications of the smut pathogenic bacterium Schwarzmania solani. Background technique [0002] Chinese medicinal materials rhubarb include Rheum palmatum L., Rheumtanguticum Maxim.ex Balf. and Rheum officinale Baill. of the Polygonaceae plant. Fire, cooling blood, removing blood stasis, detoxification and other effects, it is the raw material of various Chinese patent medicines, widely distributed in Shaanxi, southeastern Gansu, Hubei, Qinghai, western Sichuan, Yunnan and eastern Tibet, and also in Europe and Japan. [0003] With the development of traditional Chinese medicine, the amount of Chinese herbal medicine rhubarb is increasing year by year, and the planting area is expanding year by year. The main producing areas are widespread, and the disease mainly damages leaves, shoots and petioles. After the leaves are damaged...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6848C12Q1/6895C12Q2531/113
Inventor 游景茂唐涛王帆帆郭杰段媛媛郭晓亮
Owner 湖北省农业科学院中药材研究所
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