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Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum

A real-time fluorescent quantitative technology for Pythium melons and fruits, which is applied in the field of crop disease diagnosis and control, can solve the problems of few applications, achieve high detection accuracy, good specificity, and save detection time

Inactive Publication Date: 2016-02-10
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 been widely used at present. In the fields of molecular biology research and medical research, but it is rarely used in the qualitative and quantitative detection research of agricultural plant pathogenic fungi, especially in the qualitative and quantitative research of Pythium melon and fruit

Method used

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  • Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum
  • Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum
  • Fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum

Examples

Experimental program
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Effect test

Embodiment 1

[0039] The specific primer detection of embodiment 1 Pythium melon and fruit

[0040] 1) DNA preparation of each strain:

[0041] Culture the strains on a plate, place them in an incubator at a suitable temperature, and grow for about a week. Scrape the 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 with Takara DNA extraction kit.

[0042] 2) Specific primers for detecting Pythium melon and fruit:

[0043] Specific primer (GF1F): 5'-GCAACCTCTATTGGCGGTATG-3',

[0044] Specific primer (GF1R): 5'-ATATCAGGTCCAATTAGAAAGTTGTTC-3',

[0045] Synthesized by Shanghai Shenggong Company.

[0046] 3) PCR reaction system for detecting Pythium melon and fruit:

[0047] PCR reaction system, in which l0×PCRbuffer 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), 2 μL of DNA template, ...

Embodiment 2

[0054] Sensitivity detection of embodiment 2 Pythium melon and fruit specific primer

[0055] 1) Serial dilution of Pythium melon and fruit DNA template concentration from high to low:

[0056] The Pythium melon and fruit DNA template concentration was serially diluted from high to low by 10×, from a high concentration of 10 ng / mL to 0.001 ng / mL.

[0057] 2) Specific primers for detecting Pythium melon and fruit:

[0058] Specific primer (GF1F): 5'-GCAACCTCTATTGGCGGTATG-3',

[0059] Specific primer (GF1R): 5'-ATATCAGGTCCAATTAGAAAGTTGTTC-3',

[0060] Synthesized by Shanghai Shenggong Company.

[0061] 3) PCR reaction system for detecting Pythium melon and fruit:

[0062] PCR reaction system, in which l0×PCRbuffer 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), 2 μL of DNA template, 12.7 μL of sterile double-distilled water, and the final volume is 20 μL.

[0063] 4) PCR amplification program for detecting Pythium ...

Embodiment 3

[0069] Example 3 Fluorescence quantitative PCR method detects the quantity of Pythium melon and fruit in the leaves and fruits of diseased tomato plants

[0070] 1) Sample DNA preparation:

[0071] The diseased leaves and fruits of different parts of tomato plants inoculated in the greenhouse were collected in EP tubes and stored at -20°C for later use. Each sample was ground with liquid nitrogen, and the DNA of the sample was extracted with the Takara DNA extraction kit.

[0072] 2) Specific primers for detecting Pythium melon and fruit:

[0073] Specific primer (GF1F): 5'-GCAACCTCTATTGGCGGTATG-3'

[0074] Specific primer (GF1R): 5'-ATATCAGGTCCAATTAGAAAGTTGTTC-3'

[0075] Synthesized by Shanghai Shenggong Company.

[0076] 3) Fluorescence quantitative PCR reaction system for detecting Pythium melon and fruit:

[0077] The total reaction volume is 20 μL, SYBRGreenSupermix 10 μL, specific primer GF1F (10 μM / L) 1 μL, specific primer GF1R (10 μM / L) 1 μL, DNA template 2 μL, a...

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Abstract

The invention relates to a fluorescent quantitative PCR detection kit for quickly detecting pythium aphanidermatum. Pythium aphanidermatum DNA is taken as a template for carrying out PCR reaction, and a specific primer GF1F / GF1R designed in the ITS region of pythium aphanidermatum rDNA is amplified and can be specifically amplified to obtain a product of 282bp. The sequence of the specific primer is respectively as follows: the specific primer GF1F: 5'-GCAACCTCTATTGGCGGTATG-3'; and the specific primer GF1R: 5'-ATATCAGGTCCAATTAGAAAGTTGTTC-3'. The method provided by the invention has high detection sensitivity, can be used for detecting botrytis cinerea DNA with concentration of 0.01ng / mL, and can accurately detect 62 spores for botrytis cinerea.

Description

technical field [0001] The invention relates to a kit for detecting Pythium melon and fruit by fluorescent quantitative PCR method, which is specially used for detecting Pythium melon and fruit, and belongs to the technical field of diagnosis and control of crop diseases. Background technique [0002] Pythium aphanidermatum (Pythiumaphanidermatum), a fungus belonging to the flagellate subphylum. The mycelium grows luxuriantly and is in the form of white cotton wool. Mycelium is colorless, without septum, 2.3-7.1μm in diameter. There is no obvious difference between hyphae and cystineria. Sporangia filamentous or branched, or irregularly enlarged, with a size of 63-725×4.9-14.8 (μm). The vesicles are spherical and contain 6-26 zoospores. The ovum is spherical, with a diameter of 14.9-34.8μm. The oospore is spherical, smooth, not full, with a diameter of 14.0-22.0μm. The fungus occurs more frequently in places with high annual average temperature. The fungus survives th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6895C12Q1/686C12Q2563/107C12Q2545/114C12Q2531/113
Inventor 李园任宗杰曹坳程颜冬冬郭美霞王秋霞欧阳灿彬刘秋梅
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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