Phytophthora colocasiae LAMP (loop-mediated isothermal amplification) detection primer and detection method

A loop-mediated isothermal and detection method technology is applied in the fields of identification and control, and crop disease detection, which can solve the problems of long detection time, cumbersome procedures and low accuracy, and achieve reliable results, high sensitivity and good practicability. Effect

Active Publication Date: 2017-02-22
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide Phytophthora taro ring-mediated isothermal amplification detection primers and detection methods, aiming at the detection and identification of Phytophthora taro in the prior art mainly based on morphological characteristics, the method is time-consuming, cumbersome, empirical, and The accuracy is low, it is difficult to monitor the occurrence of disea...

Method used

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  • Phytophthora colocasiae LAMP (loop-mediated isothermal amplification) detection primer and detection method
  • Phytophthora colocasiae LAMP (loop-mediated isothermal amplification) detection primer and detection method
  • Phytophthora colocasiae LAMP (loop-mediated isothermal amplification) detection primer and detection method

Examples

Experimental program
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Embodiment 1

[0035] Example 1: Design of specific primers for the detection of Phytophthora colocatus Loop-Mediated Isothermal Amplification (LAMP) and the specificity verification of the primers

[0036] 1. Extraction of genomic DNA of the tested strains

[0037] The genomic DNA of the tested strain (Table 1) was extracted by the CTAB method. The specific method was as follows: Take a small amount of mycelium powder in a 1.5 mL centrifuge tube (it is better that the mycelium powder just covers the bottom of the semicircle), add 900 µL of 2% CTAB ( cetyltrimethylammonium bromide) extract (2% CTAB; 100 mmol / L Tris-HCl, pH 8.0; 20 mmol / L EDTA, pH 8.0; 1.4 mol / LNaCl) and 90 µL SDS (ten Sodium dialkylbenzene sulfonate) [Note: CTAB, SDS needs to be preheated at 60°C], use a shaker to shake and mix, 60°C water bath for 1h (DNA is released into the buffer), 12000 r.min -1 Centrifuge for 15 min; take 700 µL of the supernatant, add an equal volume of phenol, chloroform, isoamyl alcohol mixture (vo...

Embodiment 2

[0053] Example 2: Determination of Sensitivity of Phytophthora taro Loop-Mediated Isothermal Amplification (LAMP) Detection

[0054] 1. Preparation of genomic DNA at different concentrations

[0055] Phytophthora taro genome DNA is diluted with sterile ultrapure water, and prepared into a series concentration of 10 times order of magnitude for subsequent use;

[0056] 2. Sensitivity determination and result observation of LAMP detection method

[0057] Using different concentrations of Phytophthora colocasia genomic DNA as a template, the outer primers PcoF3 / PcoB3 and inner primers PcoFIP / PcoBIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 5 μM outer primers PcoF3 and 1.0 μL each of PcoB3, 40 μM inner primer PcoFIP and PcoBIP each 1.0 μL, LAMP reaction mixture [40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20 mM KCl, 16 mM MgSO 4 , 1.6 mol / L Betaine (Betaine), 2.0 mMdNTPs, 0.2% Trion X-100] 12.5 μL, 8 U Bst 1.0 μL of polymerase, 1.0 μ...

Embodiment 3

[0060] Example 3: LAMP detection of Phytophthora taro in diseased leaves of taro

[0061] Sample collection: Collect leaves with typical symptoms of taro blight and healthy leaves from Fuzhou, Xiapu, and Fuding in Fujian and bring them back to the laboratory for later use;

[0062] Extraction of Phytophthora taro DNA from diseased leaves: DNA was extracted by NaOH rapid cleavage method, the specific process was as follows: Add 10 µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar, and then transfer to Centrifuge at 12,000 rpm for 6 min in a 1.5 mL centrifuge tube, take 5 µl of the supernatant and add 495 µL of 0.1 mol / L Tris-HCl (pH=8.0) to mix well, and take 1.0 µL as a PCR template for amplification.

[0063] LAMP amplification detection and observation: Using the above-mentioned extracted DNA as a template, use the outer primer PcoF3 / PcoB3 and inner primer PcoFIP / PcoBIP for LAMP amplification. The LAMP detection reaction system is ...

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Abstract

The invention discloses a phytophthora colocasiae LAMP (loop-mediated isothermal amplification) detection primer and it rapid detection method, which can be used for the specific detection of the phytophthora colocasiae. With the phytophthora colocasiae LAMP detection primer, via isothermal amplification, green fluorescence light or trapezoidal bands with LAMP characteristics can be observed by color developing of SYBR-green-I color developing agent or electrophoretic detection of agarose gel. The LAMP primer and the detection method of the invention have high accuracy, specificity, convenience in operation and good practicability, and realize the constant temperature amplification, which provides a new technology platform for the detection of phytophthora colocasiae, can be used for quickly, flexibly and accurately detecting of infected plants of phytophthora colocasiae, and phytophthora colocasiae of the initial infection stage in the production practice and for the early diagnosis, monitoring and identification of the crop diseases, and can provide reliable technical and theoretical basis for the prevention and control of the diseases.

Description

technical field [0001] The invention belongs to the technical field of crop disease detection, identification and prevention, and in particular relates to a loop-mediated isothermal amplification (LAMP) detection primer and a rapid detection method of Phytophthora taro, which can be used for rapid, sensitive and specific molecular analysis of Phytophthora taro It can also be used for early diagnosis of taro blight and monitoring and identification of pathogens. Background technique [0002] Phytophthora taro ( Phytophthora colocasiae ) belongs to Oomycota ( Oomycota ), Oomycetes ( Oomycetes ), Peronomyces ( Peronosporals ), Pythium ( Pythiaceae ), Phytophthora ( Phytophthora ). Phytophthora taro is a very serious pathogen in taro production. Taro blight caused by Phytophthora taro infection is one of the main diseases in taro cultivation, and it is widely distributed and occurs in taro production areas. The initial infection of the disease mainly comes from the ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
Inventor 兰成忠姚锦爱阮宏椿吴玮
Owner INST OF PLANT PROTECTION FAAS
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