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

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

Inactive Publication Date: 2015-06-17
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

Examples

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Effect test

Embodiment 1

[0038] Example 1: Specific amplification of LAMP primers to Phytophthora capsici and verification of primer sequences

[0039] The key technology of the present invention is a primer sequence for efficient and specific amplification of Phytophthora capsicum and an amplification method thereof. In order to verify the specific primer sequences of Phytophthora capsicum, the present invention uses Phytophthora capsicum, 11 species of Phytophthora and Pythium oomycetes and 22 different fungi as test materials, and uses CTAB method to extract the DNA of Phytophthora capsicum from tissues. The specific method is as follows: take 50mg of freeze-dried mycelium powder in a 1.5ml centrifuge tube, add 900μl of 2% CTAB (hexadecyltrimethylammonium bromide) extract (the formula of the extract is: 2% CTAB; 100 mmol / L Tris-HCl (tris-hydroxymethylaminomethane hydrochloride), pH 8.0; 20 mmol / L EDTA (disodium EDTA), pH8.0; 1.4 mol / L NaCl) and 90 μl 10 % SDS (sodium dodecyl benzene sulfonate) a...

Embodiment 2

[0044] Example 2: Sensitivity detection of LAMP primers to Phytophthora capsici

[0045] 1. LAMP Sensitive Detection of Phytophthora capsicum

[0046] The extracted Phytophthora capsicum DNA was diluted into 10 different concentration gradients of 10ng, 1ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg by 10-fold concentration serial dilution method.

[0047] ①LAMP reaction system 25μl: contains 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 DNA polymerase large fragment, 25ng DNA template, and the insufficient part was supplemented with sterile double-distilled water; the LAMP reaction conditions were incubated at 64°C for 60 min, and incubated at 80°C for 10 min.

[0048] ②Add 1 μl of chromogenic reagent to the final amplification product of the LAMP reaction, the chromogenic reagent is 50 μM Calcein-500 μM MnCl 2 , the green fluorescence is observed ...

Embodiment 3

[0050] Example 3: Detection of Phytophthora capsicum in diseased tissue or soil

[0051] 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.

[0052] 2. DNA extraction and testing

[0053] Phytophthora capsicum 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 pepper, and cut off the diseased parts; (2) Add 10 μl (0.5mol / L NaOH, 0.5% PVP) according to 1 mg of diseased leaves, and the tissue is fully Grind into a paste and centrifuge in a 12,000g centrifuge for 5 min; (3) Take 20 μl of supernatant and mix with an equal volume of 0.1 mol / L Tris-HCl (pH 8.0); (4) Dilute 10 times, 100 times, 1000-fold solution, 1 μl stock solution, 10-fold, 100-fold, and 1,000-fold solution were taken as P...

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Abstract

The invention discloses a Phytophthora capsici LAMP primer and a rapid detection method thereof, which are specially used for the specific detection of Phytophthora capsici. Mainly designed a LAMP primer (F3, B3, FIP, BIP) of Phytophthora capsici, using this primer for constant temperature amplification and adding 50μM Calcein-500μM MnCl2 color reagent or agarose gel electrophoresis detection, it can be observed to green fluorescence or a trapezoidal band characteristic of LAMP. The present invention can be used for rapid, sensitive and accurate detection of Phytophthora capsici in plants and soil infected by Phytophthora capsici in production practice, and can be used for early diagnosis of field diseases and monitoring and identification of pathogens. Provide reliable technical and theoretical basis for disease control.

Description

technical field [0001] The invention relates to a LAMP primer for Phytophthora capsici and its rapid detection method, which is specially used for high-sensitivity and rapid molecular detection of Phytophthora capsicum, and can be used for early diagnosis of pepper blight in the field and monitoring and identification of pathogens, and belongs to the field of detection and identification of crop diseases. Prevention technology field. Background technique [0002] Pepper blight is a devastating fungal disease that occurs worldwide. It was first discovered in New Mexico, USA in 1918. Phytophthora capsici caused by pepper blight Phytophthora capsici Leonian was named by Leon Leonian in 1922, and it belongs to plant pathogenic oomycetes. Phytophthora capsici overwinters in diseased soil as oospores or chlamydospores, and can survive for several months or even longer. It has a wide range of hosts and mainly infects many important vegetable crops such as peppers, tomatoes, eggp...

Claims

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Application Information

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Patent Type & Authority Patents(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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