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Loop-mediated isothermal amplification primers for detecting Solanum lycopersicum L Alternaria solani, and applications thereof

A tomato early blight bacteria, ring-mediated isothermal technology, applied to the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of long time-consuming methods, long detection time, low accuracy, etc. , to achieve reliable results, low cost and high sensitivity

Inactive Publication Date: 2018-07-27
INST OF PLANT PROTECTION FAAS
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0006] The object of the present invention is to provide a loop-mediated isothermal amplification primer for detecting tomato early blight and its application. In the prior art, the detection and identification of tomato early blight is mainly based on morphological characteristics, and the method is time-consuming, cumbersome and cumbersome. Strong experience, low accuracy, it is difficult to timely monitor the occurrence of diseases and control the spread and prevalence of pathogenic bacteria, and the existing PCR molecular detection needs to rely on expensive instruments such as amplifiers, and the detection time is long, etc. Provides a new molecular detection method for tomato early blight pathogen, LAMP detection of tomato early blight pathogen, short detection cycle, high accuracy, high sensitivity, and the detection results can be observed with naked eyes

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  • Loop-mediated isothermal amplification primers for detecting Solanum lycopersicum L Alternaria solani, and applications thereof
  • Loop-mediated isothermal amplification primers for detecting Solanum lycopersicum L Alternaria solani, and applications thereof
  • Loop-mediated isothermal amplification primers for detecting Solanum lycopersicum L Alternaria solani, and applications thereof

Examples

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Effect test

Embodiment 1

[0029] Example 1: Design of specific primers for detection of tomato early blight pathogen loop-mediated isothermal amplification (LAMP) and verification of primer specificity

[0030] 1. Extraction of genomic DNA of the tested strains

[0031] 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 (volume ra...

Embodiment 2

[0044] Example 2: Detection Sensitivity of Loop-Mediated Isothermal Amplification (LAMP) for Tomato Early Blight

[0045] 1. Preparation of genomic DNA at different concentrations

[0046] Dilute the genomic DNA of the tomato early blight bacteria with sterile ultrapure water, and prepare a series concentration of 10 times order of magnitude for later use;

[0047] 2. Sensitivity determination and result observation of LAMP detection method

[0048] Using different concentrations of the tomato early blight genomic DNA as a template, F3, B3, FIP and BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 1.0 μL each of 5 μM primers F3 and B3, and 40 μM primers FIP and 1.0 μL for each BIP, 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 mM dNTPs, 0.2wt.% Trion X-100] 12.5 μL, 8 U Bst Polymerase 1.0 μL, DNA template 1.0 μL, make up to 25 μL with sterilized ultrapu...

Embodiment 3

[0052] Example 3: LAMP detection of tomato early blight bacteria in diseased tissues

[0053] Sample collection: Collect tomato early blight symptoms and healthy leaves from Zhangzhou, Sanming, Fuzhou, and Xiapu in Fujian and bring them back to the laboratory for later use;

[0054] Plant tissue DNA extraction: DNA was extracted by NaOH rapid cleavage method, the specific process is as follows: add 10µL 0.5 mol / L NaOH to each mg of plant tissue, fully grind the tissue into a paste in a mortar and transfer it to a 1.5mL centrifuge tube centrifuge at 12,000 rpm for 6 min, 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.

[0055] Amplification detection and observation: Using the above-mentioned extracted DNA as a template, use F3, B3, FIP and BIP for LAMP amplification. The LAMP detection reaction system is 25 μL, including 5 μM primers F3 and 1.0 μL each of B3, and 40 μM primer FIP and ...

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Abstract

The invention provides loop-mediated isothermal amplification primers for detecting Solanum lycopersicum L Alternaria solani, and applications thereof, wherein the primers comprise four specific primers F3, B3, FIP and BIP4, and the nucleotide sequences represented by SEQ ID NO.1-4. According to the present invention, the problems of long period, time consumption, labor consumption, complexity, poor specificity, and requirement of thermal cycle instrument and other expensive equipment by PCR detection technology in the detection method for Solanum lycopersicum L Alternaria solani in the priorart can be solved with the primers; and the detection system of the present invention can rapidly, efficiently, highly-specific and highly-sensitive detect Solanum lycopersicum L Alternaria solani under the LAMP amplification condition, is suitable for the on-site detection of pathogenic bacteria and diseases, is suitable for large-range promotion and application, and can provide the reliable technical and theoretical basis for the prevention and treatment of Solanum lycopersicum L early blight.

Description

technical field [0001] The invention belongs to the technical field of detection, identification and prevention of crop diseases, and in particular relates to a loop-mediated isothermal amplification (LAMP) detection primer for tomato early blight and its application, which can be used for rapid, sensitive and specific molecular detection of tomato early blight, At the same time, it can be used for early diagnosis of tomato early blight and monitoring and identification of pathogens. Background technique [0002] tomato( Solanum lycopersicum L.) is a solanaceous vegetable with rich nutrition. It is a kind of vegetable that people generally like. It has high edible value and strong adaptability. It is widely cultivated all over the country. It is popular with consumers and has remarkable economic benefits. With the development of protected planting and facility agriculture technology, tomato planting has achieved annual supply production, but there are also some problems in...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6848C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 兰成忠游泳阮宏椿吴玮姚锦爱
Owner INST OF PLANT PROTECTION FAAS
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