Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian

A detection kit, the technology of Phytophthora capsicum, is applied in the directions of fluorescence/phosphorescence, determination/inspection of microorganisms, biochemical equipment and methods, etc., to achieve the effects of simple and quick operation, high detection sensitivity and high accuracy

Inactive Publication Date: 2011-02-16
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This technology not only realizes the quantification of DNA templates, but also has the characteristics of high sensitivity, stronger specificity and reliability, multiple reactions, high degree of automation, no pollution, real-time and accuracy, etc., and has b

Method used

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  • Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian
  • Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian
  • Fluorescence quantitative polymerase chain reaction (PCR) detection method and detection kit for phytophthora capsici leonian

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Experimental program
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Effect test

Embodiment 1

[0044] Cloning and sequencing of the Phytophthora capsici ITS region of embodiment 1

[0045] 1) Phytophthora capsici DNA preparation:

[0046] Phytophthora capsici was cultured on a plate, placed in an incubator at 30°C, and grown for about a week. The mycelium was scraped with a sterilized dissecting needle, collected in an EP tube, and stored at -20°C for later use. The mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted by conventional CTAB method.

[0047] 2) Amplification of the ITS region of Phytophthora capsici

[0048] Universal primers ITS1 / ITS4 for fungal rDNA ITS amplification (White, T.J., Bruns, T., Lee, S., et al., Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenies. Innis MA, Glefand D H, Sninsky JJ, et al. a Guide to Methods and Applications. San Diego: Academic Press, New York, 1990, 315-322.) Amplification of the ITS region of Phytophthora capsici.

[0049] The PCR reaction system is shown in Ta...

Embodiment 2

[0064] The specific primer detection of embodiment 2 Phytophthora capsici

[0065] Materials: 1. Phytophthora capsici 2. Phytophthora infestans 3. Phytophthora boehmeriae 4. Phytophthora parasitica 5. Phytophthora cryptogea 6. Melons Pythium aphanidermatum

[0066] The microorganisms used in the present invention have all been recorded in existing documents, and are also preserved in this laboratory, which can be distributed to the public for verification tests.

[0067] 1) DNA preparation of each strain:

[0068] All Phytophthora strains were grown on rye medium and all strains except Phytophthora were grown on PDA medium.

[0069] Cultivate strains on a plate, place them in an incubator at a suitable temperature, and grow for about a week. Scrape mycelia with a sterilized dissecting needle, collect them in EP tubes, and store them at -20°C for later use. The mycelium was ground with liquid nitrogen, and the DNA of each strain was extracted by conventional CTAB method.

...

Embodiment 3

[0082] Sensitivity detection of embodiment 3 Phytophthora capsici specific primer

[0083] 1) The concentration of Phytophthora capsici DNA template was serially diluted from high to low:

[0084] The Phytophthora capsici DNA template concentration was serially diluted 10× from high to low, and serially diluted from high concentration 2 μg / mL to 0.2 ng / mL.

[0085] 2) Specific primers for detecting Phytophthora capsici:

[0086] Specific primer ljym1: 5'-TTGTTTTAAACCCATTTCACAATT-3',

[0087] Specific primer ljym2: 5'-CCACAGCAGGAAAAGCATT-3'.

[0088] Synthesized by Shanghai Shenggong Company.

[0089] 3) PCR reaction system for detecting Phytophthora capsici:

[0090] PCR reaction system, in which 10×PCR buffer 2μL, 15mM MgCl 2 0.5 μL, 1.6 μL of 2.5 mM dNTPs, 0.5 μL of each 5 μM primer, 0.2 μL of Taq enzyme (5U / μL), 1 μL of DNA template, 13.7 μL of sterile double-distilled water, and the final volume was 20 μL.

[0091] 4) PCR amplification program for detecting Phytophth...

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Abstract

The invention relates to a fluorescence quantitative polymerase chain reaction (PCR) detection method and a detection kit for phytophthora capsici leonian. In the method, a primer pair ljym1/ljym2 is designed according to an ITS area of the phytophthora capsici leonian rDNA; the reaction liquid contains the pair of specific primers and fluorescent dye; a phytophthora capsici leonian DNA template is added into the reaction liquid; and the fluorescence intensity of the reaction system is detected in real time along with the PCR reaction, and a Ct value is calculated according to analysis software so as to calculate the content of phytophthora capsici leonian in a sample. The kit designed according to the method has simple and fast operation and good specificity and high sensitivity, is free from the culture conditions based on the nucleotide detection, can actually reflect the infection conditions of phytophthora capsici leonian into plants by quantitative detection, realizes high-flux sample detection at the same time, conducts fast quantitative detection on the phytophthora capsici leonian and replaces the conventional identification method of isolated culture on a selective medium, and is suitable for wide popularization and application in the field of plant disease diagnosis and detection.

Description

technical field [0001] The invention discloses a method for detecting Phytophthora capsici by fluorescent quantitative PCR, which is specially used for detecting Phytophthora capsici, belongs to the technical field of diagnosis and control of crop diseases, and also includes a detection kit. Background technique [0002] Phytophthora capsici has a wide host range and can infect aboveground and underground parts of plants, causing diseases of Solanaceae and melons. Phytophthora survives in soil as chlamydospores or oospores. During the planting and cultivation of crops in protected areas, the soil-borne diseases on peppers caused by Phytophthora capsici are becoming more and more serious. The severely infected pepper seedlings first form dark green water-soaked lesions at the base of the stem, and quickly brown rot and constrict And cataplexy. Some stem bases are dark brown, and the seedlings wither and die. The leaves are susceptible, and the lesion is round or nearly rou...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/06G01N21/64
Inventor 李园曹坳程郭美霞崔瑞吴篆芳赵海滨
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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