Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof

A cucumber Phytophthora, detection method technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problems of poor specificity, long cycle and low sensitivity of detection methods, and achieve specificity Strong, reliable results and high sensitivity results

Inactive Publication Date: 2013-01-30
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The object of the present invention is to provide a kind of LAMP detection primer of cucumber Phytophthora and relia

Method used

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  • Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof
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  • Cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and rapid detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Specific amplification of LAMP primers to Phytophthora cucumber

[0044] 1. Specific Detection of Phytophthora Cucumber LAMP

[0045] ①LAMP reaction system 25 ul: containing 0.2uM each of F3 and B3, 1.6uM each of FIP and BIP, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction condition is to incubate at 63°C for 60 minutes, then incubate at 80°C for 10 minutes.

[0046] ② Add 1 ul of a chromogenic agent to the final amplification product of the LAMP reaction, the chromogenic agent is SYBR green I, and the green fluorescence is judged to be positive while the orange is judged to be negative. Or take 2ul of the amplified product and use 2% agarose gel electrophoresis to detect it. If the characteristic trapezoidal band of LAMP appears, it is judged as positive, a...

Embodiment 2

[0049] Embodiment 2: Sensitivity detection of LAMP primers to Phytophthora cucumber

[0050] 1. LAMP Sensitivity Detection of Phytophthora Cucumber

[0051] The extracted Phytophthora cucumber DNA was diluted into 10 different concentration gradients of 1000ng, 100ng, 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg by 10-fold concentration serial dilution method.

[0052] ①LAMP reaction system 25ul: containing 0.2uM each of F3 and B3, 1.6uM each of FIP and BIP, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction condition is to incubate at 63°C for 60 minutes, then incubate at 80°C for 10 minutes.

[0053] ② Add 1 ul of a chromogenic agent to the final amplification product of the LAMP reaction, the chromogenic agent is SYBR green I, and the green fluorescence is judged t...

Embodiment 3

[0055] Example 3: Detection of Phytophthora cucumber in diseased tissue or soil.

[0056]1. Sample collection: Plant tissue samples were collected from cucumber production base in Fuzhou City, Fujian Province; soil samples were collected from cucumber production base in Longhai City, Fujian Province.

[0057] 2. DNA extraction and detection

[0058] The DNA of Phytophthora cucumber was extracted by NaOH rapid cracking method from the diseased plant tissue, and the DNA of Phytophthora cucumber was extracted by soil DNA extraction method from the diseased soil.

[0059] Perform LAMP detection as follows:

[0060] ①LAMP reaction system 25 ul: containing 0.2uM each of F3 and B3, 1.6uM each of FIP and BIP, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 10mM KCl, 8mM MgSO 4 , 0.1% Tween-20, 0.8M Betaine, 1.4 mM dNTPs, 8U Bst Large fragment of DNA polymerase, 25ng of DNA template, make up the deficiency with sterile double distilled water; the LAMP reaction condition is to incubate ...

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Abstract

The invention discloses cucumber phytophthora LAMP (Loop-mediated Isothermal Amplification) primer and a rapid detection method thereof, which are specifically used for the specific detection of cucumber phytophthora. The rapid detection method is characterized in that a cucumber phytophthora LAMP primer is mainly adopted and designed, detailed information can be seen in SEQ NO.1-SEQ NO.4. Green fluorescent light or an LAMP characterized ladder-shaped pattern can be observed through the LAMP and the color developing by adding an SYBR green I color agent for developing color or the agarose gel electrophoresis detection. The LAMP primer and the rapid detection method, which are disclosed by the invention, can be used for quickly, sensitively and accurately detecting the cucumber phytophthora in plants and soil which are affected by the cucumber phytophthora in production practice and can be simultaneously used for the early diagnosis of diseases in filed and the monitoring and the identification of germs, and reliable technical and theoretical basis is provided for preventing the disease caused by the cucumber phytophthora.

Description

technical field [0001] The invention relates to a LAMP primer for Phytophthora cucumber and a rapid detection method thereof, which is specially used for high-sensitivity and rapid molecular detection of Phytophthora cucumber, and can be used for early diagnosis of cucumber blight in the field and monitoring and identification of pathogens, belonging to the detection, identification and identification of crop diseases. Prevention technology field. Background technique [0002] Cucumber Phytophthora ( Phytophthora melonis Katsura) was first isolated from a diseased cucumber by Katsura in 1968, and identified as a new species. Subsequently, China, Japan, Egypt, Turkey, South Korea, India and other countries also reported this pathogen one after another. Cucumber blight caused by this fungus is one of the most serious diseases in China's cucumber production areas. It mainly damages the stems, leaves and fruits of plants, and can occur from the seedling stage to the adult pla...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 李本金陈庆河翁启勇兰成忠刘裴清
Owner INST OF PLANT PROTECTION FAAS
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