Molecule detection technique of cayenne pepper phytophthora capsici pectin lyase (Pcpel) 1 genes

A technology of pectin lyase and Phytophthora capsici, which is applied in the field of molecular biology to achieve comprehensive prevention and control and efficient treatment, predict the development trend of the epidemic, and reduce the cost of disease prevention and control

Inactive Publication Date: 2010-02-10
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Before the application of the present invention, except for the molecular detection of the pathogen using Phytophthora capsici rDNA / ITS sequence-specific primers, there was no molecular analysis of the growth and decline dynamics of Phytophthora capsici using target gene-specific primers of important pathogenic enzymes. Data and information on detection and disease warning

Method used

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  • Molecule detection technique of cayenne pepper phytophthora capsici pectin lyase (Pcpel) 1 genes
  • Molecule detection technique of cayenne pepper phytophthora capsici pectin lyase (Pcpel) 1 genes
  • Molecule detection technique of cayenne pepper phytophthora capsici pectin lyase (Pcpel) 1 genes

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Implementation 1: Design and synthesis of specific primers for the target Pcpel 1 gene

[0057] (1) Specific primer design: Use DNAMAN to detect known oomycetes such as Phytophthora capsici, Phytophthora soybean, Phytophthora rubberum, fungi such as Aspergillus and Alternaria, various plants such as tomato, peach, lemon, etc. All the found pectin lyase genes were compared, and specific primers for Pcpel 1 were designed, Pcpel 1-F, Pcpel 1-R, and the length of the extended fragment was 574bp. Pcpel 1-F: 5'-TCGTGCAGAACATCACATCAC-3'22bp, Pcpel 1-R: 5'-CGTCCCAGTTGAGCCTTGC-3'19bp.

[0058] (2) Primer synthesis: Send the designed and synthesized primers to Shanghai Boshang Biological Co., Ltd. or Shanghai Dingan Biological Co., Ltd., and they will be synthesized and sent back by the biological company for direct application.

Embodiment 2

[0059] Implementation 2: Technical steps and methods for verification of specific primers

[0060] 1) Test strains and their cultivation

[0061] For the test, Phytophthora capsici (P.capsici) (Shandong), Phytophthora soybean (P.sojae) (Jilin), Phytophthora parasitic (P.parasitic) (Yunnan), Phytophthora infestans (P.infestans), Camphor P. cinnamomi (provided by CBS), P. drechsleri (provided by CBS), P. palmivora (provided by CBS), P. tropicalis (provided by CBS) , Phytophthora ramie (P.boehmeriae) (Fujian), Phytophthora cactorum (P.cactorum) (provided by CBS), Phytophthora alfalfa (Shaanxi), Phytophthora colocasiae (P.colocasiae) (Guangdong), Phytophthora cryptogae ( P.cryptogea) (CBS), Pea Phytophthora (P.erythroseptica) (Yunnan), Strawberry Phytophthora (P.fragariae) (Shandong), Nobita Phytophthora (P.megasperma) (ATCC), Bean Phytophthora (P. phaseoli) (Shandong), P.porri, P.hibemalis (CBS119904), Pythiumaphanidermatum (Shandong), and Saprolegnia sp. (Shandong), respective...

Embodiment 3

[0069] Implementation 3: Specific primer sensitivity verification technical method

[0070] 1) DNA extraction of zoospores of Phytophthora capsici

[0071] Add 350 zoospores (directly recorded under a microscope) to 50 μL of sterile water, add 0.05 g of quartz sand, shake vigorously on a vortex for 3 minutes, and centrifuge slightly. The supernatant is the crude DNA extract of zoospores. Take 0.5-10 μL of the supernatant directly as a template for PCR amplification.

[0072] 2) DNA extraction and purification of soil oospores

[0073] Refer to the method reported by Zhu et al. (Applied and Environmental Microbiology, 1996, 62: 316-322) (slightly improved). Take soil containing 20, 10, 5.0, 2.0, 1.0, 0.05, 0.01 oospores / 1g and put them into 5mL sterilized centrifuge tubes, add appropriate amount of quartz sand, and shake vigorously for 1min. Then add 2mLNa 3 PO 4Buffer solution (0.12mol / L, pH8.0), shake at 30°C for 15min (160rpm / min), centrifuge at 10000rpm / min for 10min, ...

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Abstract

The invention relates to a molecule detection technique of cayenne pepper phytophthora capsici pectin lyase (Pcpel) 1 genes, belonging to the field of molecular biology. A pair of specific primers aredesigned and synthesized by the cayenne (sweet) pepper phytophthora capsici pectin lyase (Pcpel) 1 genes, the specificity of a plurality of tested bacteria species is verified by PCR, and then the sensitivity of the specific primers is verified by carrying out quantitative molecule detection for a soil cayenne pepper phytophthora oospore and a zoospore; and on the basis, the qualitative and quantitative molecule detection and the early warning of cayenne pepper phytophthora capsici contained in cayenne pepper diseased field soil, diseased stems, diseased leaves and diseased fruits are carriedout. The invention can detect and early warn the cayenne pepper phytophthora capsici in different periods and pathopoiesia characters thereof, thereby being convenient to establish a comprehensive prevention and control technology strategy, determining and selecting an optimal prevention and control period to carry out comprehensive prevention and control and high-efficiency management for the cayenne pepper phytophthora capsici, really controlling the infection and the harm of pathogenic bacteria from a source and enhancing the comprehensive prevention and control effect of the cayenne (sweet) pepper phytophthora capsici.

Description

technical field [0001] The invention belongs to the field of molecular biology. Specifically, the technical invention provides a pair of specific primers designed and synthesized from the pectin lyase Pcpel 1 gene of Phytophthora capsici, and provides evidence for detecting the specificity and sensitivity of Phytophthora capsici. In addition, the present invention mainly relates to qualitative and quantitative molecular detection of Phytophthora capsici in the soil, diseased stems, leaves, and fruits of pepper diseased fields using this technology, as well as early warning of epidemic trends, comprehensive prevention and control of pepper disease in due course and Efficient governance. Background technique [0002] Phytopathogenic oomycetes are an important class of plant pathogens, which can infect and harm a variety of plants and cause devastating diseases of a variety of plants, such as Phytophthora infestans, P. capsici, soybean blight P. sojae, P. parasitic, P. cinnamo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/645
Inventor 张修国
Owner SHANDONG AGRICULTURAL UNIVERSITY
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