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
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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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