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Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof

A technology for Phytophthora capsicum and a detection method is applied in the field of Phytophthora capsicum LAMP primers and rapid detection thereof, which can solve the problems of long cycle, low sensitivity, poor specificity of detection methods and the like, and achieve reliable results, high sensitivity and strong specificity. Effect

Inactive Publication Date: 2014-01-22
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a kind of LAMP detection primer of Phytophthora capsici and reliable result, easy to operate, aim at the biological detection method of Phytophthora capsici in the prior art that required period is long, detection method specificity is poor, sensitivity is low. Rapid detection method of Phytophthora capsici with strong specificity and high sensitivity

Method used

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  • Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof
  • Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof
  • Phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and rapid detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1: Specific amplification of LAMP primers to Phytophthora capsici and verification of primer sequences

[0043] The key technology of the present invention is the primer sequence and the amplification method for efficient and specific amplification of Phytophthora capsici. In order to verify the specific primer sequence of Phytophthora capsici, the present invention uses Phytophthora capsici, 11 kinds of Phytophthora and Pythium oomycetes and 22 different fungi as test materials, and adopts CTAB method to extract the DNA of Phytophthora capsici in tissues. The specific method is as follows: take 50 mg of freeze-dried mycelium powder in a 1.5 ml centrifuge tube, add 900 μl of 2% CTAB (cetyltrimethylammonium bromide) extract (the formula of the extract is: 2% CTAB; 100 mmol / L Tris-HCl (trishydroxymethylaminomethane hydrochloride), pH 8.0; 20mmol / L EDTA (disodium ethylenediamine tetraacetate), pH8.0; 1.4 mol / L NaCl) and 90μl 10 % SDS (sodium dodecylbenzene sulfo...

Embodiment 2

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

[0050] 1. LAMP Sensitivity Detection of Phytophthora capsici

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

[0052] ①LAMP reaction system 25μl: containing 0.25uM 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 DNA template, make up the deficiency with sterile double-distilled water; LAMP reaction conditions are incubation at 64°C for 60 minutes, and incubation at 80°C for 10 minutes.

[0053] ② Add 1 μl of chromogen to the final amplification product of the LAMP reaction, the chromogen is 50 μM Calcein-500 μM MnCl 2 , Green fluorescence was observed as a positive result of col...

Embodiment 3

[0055] Embodiment 3: detection of Phytophthora capsici in pathogenic tissue or soil

[0056] 1. Sample collection: plant tissue samples were collected from pepper production bases in Fuzhou, Fujian, Shaoguan, Guangdong, and Nanchang, Jiangxi; soil samples were collected from pepper production bases in Ningde, Fujian Province.

[0057] 2. DNA extraction and detection

[0058] Phytophthora capsici DNA was extracted from diseased plant tissue by NaOH rapid lysis method. The specific process is as follows: (1) Wash and dry the diseased leaves or stems of peppers, and cut off the diseased parts; (2) Add 10 μl (0.5mol / L NaOH, 0.5%PVP) to 1mg of diseased leaves, and fully Grind into a paste and centrifuge in a 12,000g centrifuge for 5 minutes; (3) Take 20 μl of the supernatant and mix it with an equal volume of 0.1 mol / L Tris-HCl (pH8.0); (4) Dilute 10 times, 100 times, 1000-fold solution, 1 μl stock solution, 10-fold, 100-fold, and 1000-fold solution were used as PCR templates...

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Abstract

The invention discloses a phytophthora capsici LAMP (Loop-mediated isothermal Amplification) primer and a rapid detection method thereof, which are specially used for phytophthora capsici specific detection. The invention mainly designs a phytophthora capsici LAMP primer (F3, B3, FIP and BIP); the primer is subjected to isothermal amplification and a 50micromole / L Calcein-500micromole / L MnCl2 developing agent is added to develop or agarose gel electrophoresis detection is carried out, so as to observe green fluorescent light or a ladder belt with an LAMP characteristic. The phytophthora capsici LAMP primer and the rapid detection method thereof can be used for rapid, sensitive and accurate detection of plants infected by phytophthora capsici and the phytophthora capsici in the soil in production practice and can also be used for early diagnosis of field diseases and the monitoring and identifying of bacteria, so as to provide reliable technological and theoretical foundations for preventing and treating the diseases caused by the phytophthora capsici.

Description

Technical field [0001] The present invention involves a LAMP primer and its rapid detection method of pepper -epidemic mold. It is used for high -sensitivity fast molecular detection of pepper and moldy mildew.Prevention and control technology. Background technique [0002] Pepper -epidemic is a destructive fungal disease that occurs around the world. It first discovered in 1918 in New Mexico, USA. Phytophone Capsici Leonian was named by Leon Leonian in 1922, and it belongs to plant pathogenic bacteria.Pepper -of -the -peppercorities with oval or thick spores in the soil disease can survive in the soil disease. They can survive for several months or even longer. The host range is wide. It mainly invades pepper, tomato, eggplant, cucumber, pumpkin and other important vegetable crops.Both pepper epidemic diseases and protection areas can occur. The epidemic of the disease causes serious losses of pepper. The general disease plant rate is 15–30 %, and it is more than 80 % in severe ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
CPCC12Q1/04C12Q1/6844C12Q2531/119
Inventor 刘裴清陈庆河李本金董中美尹容美翁启勇
Owner INST OF PLANT PROTECTION FAAS
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