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Primer for molecularly detecting phytophthora capsici leonian and application thereof

A technology for molecular detection of Phytophthora capsici and molecular detection, which is applied in the field of primers for molecular detection of Phytophthora capsicum, can solve the problems of poor specificity and low sensitivity, and achieve the effects of high sensitivity, strong specificity, and strong practicability

Inactive Publication Date: 2009-06-03
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of capsici pathogen molecular detection primer and its application, for the biological detection method of capsici pathogen in the prior art required cycle is long, current capsici pathogen molecular detection method specificity is poor, the sensitivity is low The problem is to provide a specific molecular detection primer for Phytophthora capsici. The method has reliable results, is easy to operate, has strong specificity, and high sensitivity. It can be used for high-sensitivity and rapid molecular detection of plant tissues and soils with bacteria, and can be used for the detection of pepper blight in the field. Early diagnosis and monitoring and identification of pathogens, this technology is of great significance for early monitoring before Phytophthora capsici causes disease symptoms and to determine the best period for disease control

Method used

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  • Primer for molecularly detecting phytophthora capsici leonian and application thereof
  • Primer for molecularly detecting phytophthora capsici leonian and application thereof
  • Primer for molecularly detecting phytophthora capsici leonian and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Primer specific amplification to Phytophthora capsici

[0035] 1. Specific detection of Phytophthora capsici

[0036] PCR reaction system 25μl, including 2.5μl 10×PCR reaction buffer, 2.0mmol / L Mg 2+ , 50μmol / L dNTPs, 1.25U Taq DNA polymerase, 0.5μmol / L each of primers PCA1F and PCA2R and 10ng template DNA, d.d.H 2 Make up 25 μl of O, and the PCR reaction conditions are: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 1 min, annealing at 60°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10 min.

[0037] 2. Test results

[0038] Specificity of detection: In addition to the specific amplification of 364bp products from the DNA of Phytophthora capsici from Fujian, Anhui, Jiangsu and other provinces in my country, other 13 species of Phytophthora, Downy mildew, Pythium and other oomycetes were detected. The fungal and bacterial strain DNA failed to amplify any products, which has strong specificity.

Embodiment 2

[0039] Embodiment 2: Sensitivity detection of primers to Phytophthora capsici

[0040] 1. Dilution of DNA concentration: the extracted genomic DNA of Phytophthora capsici is diluted with serial concentrations after the concentration is measured by a spectrophotometer.

[0041] 2. Sensitivity detection of Phytophthora capsici

[0042] PCR reaction system 25μl, including 2.5μl 10×PCR reaction buffer, 2.0mmol / L Mg 2+ , 50μmol / L dNTPs, 1.25U Taq DNA polymerase, 0.5μmol / L primers PCA1F / PCA2R and 10ng template DNA, d.d.H 2 Make up 25 μl of O, and the PCR reaction conditions are: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 1 min, annealing at 60°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10 min.

[0043] 3. Detection results: In a 25 μl reaction system, 1 pg of Phytophthora capsici genomic DNA can obtain obvious amplified bands, and the detection sensitivity can reach 1 pg.

Embodiment 3

[0044] Example 3: Detection of Phytophthora capsici in diseased plant tissues.

[0045] 1. Sample collection: Plant tissue samples were collected from the vegetable base of Nantong Town, Fuzhou City, Fujian Province

[0046] 2. DNA extraction and detection

[0047] The diseased plant tissue was extracted with the CTAB method, and PCR amplification was carried out according to the method implemented in the above kit. The PCR reaction system was 25 μl, including 2.5 μl 10×PCR reaction buffer, 2.0 mmol / L Mg 2+ , 50μmol / L dNTPs, 1.25U TaqDNA polymerase, 0.5μmol / L each of primers PCA1F and PCA2R and 10ng template DNA, d.d.H 2 Make up 25 μl of O, and the PCR reaction conditions are: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 1 min, annealing at 60°C for 30 sec, extension at 72°C for 1 min, a total of 35 cycles; extension at 72°C for 10 min. Amplified products were detected by electrophoresis.

[0048] 3. Test results

[0049] see results image 3 , a clear spe...

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Abstract

The invention provides a primer for molecularly detecting phytophthora capsici leonian and the application thereof. The peculiar primer comprises an upstream primer PCA1F: 5'-GTATAGCAGAGGTTTAGTGAA-3' and a downstream primer PCA2R: 5'-ACTGAAGTTCTGCGTGCGTT-3'; a peculiar augmentation product with a segment length of 364bp is peculiarly augmented on phytophthora capsici leonian pure DNA and bacteria-bearing pathogenetic tissues and soil through PCR augmentation and agarose gel electrophoresis so as to rapidly detect the phytophthora capsici leonian. The peculiar molecular detection primer and the application method thereof can be used for rapidly, sensitively and peculiarly detecting plant tissues infected by the phytophthora capsici leonian and the phytophthora capsici leonian in soil in productive practice and simultaneously used for early diagnosis of field plant diseases and monitoring and indentifying harmful bacteria, thereby providing a reliable technical and theoretical foundation for the prevention and the cure of plant diseases caused by the phytophthora capsici leonian.

Description

technical field [0001] The invention belongs to the field of crop disease detection, identification and prevention technology, and more specifically relates to a molecular detection primer for Phytophthora capsici and its application. Background technique [0002] Pepper blight is a devastating disease caused by the infection of Phytophthora capsici, which was first discovered in New Mexico, USA in 1918, and has now become a major disease of pepper production in countries all over the world (Ristaine et al., 1999 ). Phytophthora capsici has a wide range of hosts. In addition to peppers, it can also infect more than 50 crops such as tomato, eggplant, watermelon and zucchini in the family Solanaceae and Cucurbitaceae (Tian et al., 2004; Hausbeck, et al., 2004), It occurs in a wide area and causes serious damage. Under suitable disease conditions, a pandemic can cause host crop failure (Gevens et al., 2007; Jiang Yanquan et al., 2008; Liu Xuemin et al., 2007). At the same tim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 陈庆河翁启勇兰成忠李本金赵健
Owner INST OF PLANT PROTECTION FAAS
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