Detection primers, detection kits and application of tomato cinerea Lamp

A technology of tomato botrytis cinerea and a detection kit, which is applied in the direction of microorganisms, recombinant DNA technology, and methods based on microorganisms, can solve the problems of difficult monitoring and control of pathogenic bacteria, long detection time, and low accuracy. Reliance on expensive equipment, high sensitivity, and good practicality

Active Publication Date: 2019-08-27
INST OF PLANT PROTECTION FAAS
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  • Abstract
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  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to detect and identify Botrytis cinerea in the prior art mainly based on morphological characteristics, the method takes a long time, the procedure is cumbersome, the experience is strong, the accuracy is low, and it is difficult to monitor and control the occurrence of the disease in time 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. A new molecular detection method for Botrytis cinerea is provided, and LAMP is performed on Botrytis cinerea. Detection, short detection cycle, high accuracy, high sensitivity, visual inspection results

Method used

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  • Detection primers, detection kits and application of tomato cinerea Lamp
  • Detection primers, detection kits and application of tomato cinerea Lamp
  • Detection primers, detection kits and application of tomato cinerea Lamp

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Design of specific primers for detection of Botrytis cinerea Loop-Mediated Isothermal Amplification (LAMP) and verification of primer specificity

[0038] 1. Extraction of genomic DNA of the tested strains

[0039] The genomic DNA of the tested strains (Table 1) was extracted by the CTAB method. The specific method is as follows: Take a small amount of mycelium powder in a 1.5mL 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 (25:24:1), shake gently to mix,...

Embodiment 2

[0055] Example 2: Detection Sensitivity of Botrytis cinerea Loop-Mediated Isothermal Amplification (LAMP) Detection

[0056] 1. Preparation of genomic DNA at different concentrations

[0057] Genomic DNA of Botrytis cinerea was diluted with sterile ultrapure water, and prepared into a series concentration of 10 times order of magnitude for subsequent use;

[0058] 2. Sensitivity determination and result observation of LAMP detection method

[0059] Genomic DNA of Botrytis cinerea at different concentrations was used as a template, and the outer primers F3 / B3 and inner primers FIP / BIP were used for LAMP amplification. The LAMP detection reaction system was 25 μL, including 1.0 μL of 5 μM outer primers F3 and B3, 1.0 μL each of 40 μM inner primers FIP and BIP, 12.5 μL of LAMP reaction mixture, 8 U Bst 1.0 μL of polymerase, 1.0 μL of DNA templates with different concentrations, made up to 25 μL with sterilized ultrapure water; LAMP reaction conditions: incubate at 63.5 °C for ...

Embodiment 3

[0062] Example 3: LAMP detection of Botrytis cinerea in diseased tissues

[0063] Sample collection: Collect tomato gray mold symptoms and healthy leaves from Zhouning, Sanming, and Liancheng in Fujian Province and bring them back to the laboratory for later use;

[0064] 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.

[0065] LAMP amplification detection and observation: Using the above-mentioned extracted DNA as a template, use the outer primer F3 / B3 and inner primer FIP / BIP for LAMP amplification. The LAMP detection reaction system is 25 μL, including 5 μM outer primer F3 and 1.0 μM each for B3...

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Abstract

The invention relates to a botrytis cinerea LAMP detection primer, a detection kit and application thereof. An outer side primer pair is F3 / B3; an inner side primer pair is FIP / BIP; the kit comprises an LAMP reaction mixed solution, one outer side primer pair F3 / B3, one outer side primer pair FIP / BIP, an 8U Bst polymerase and nucleic acid dye 1000*SYBR Green I. Through three steps of DNA (deoxyribonucleic acid) extraction of samples to be tested, loop-mediated isothermal amplification and color development detection, whether the botrytis cinerea is infected or not can be detected. The botrytis cinerea LAMP detection primer has the advantages that primers are designed according to the botrytis cinerea ITS sequence; the LAMP technology is used for detection; the specificity is high; the sensitivity is high; the speed is high; the accuracy is high; the sensitivity is high; the site application is convenient, and the like. The defects of long period, low sensitivity, high cost, difficult site application and the like in the prior art are overcome. The application potential is huge in the aspect of botrytis cinerea detection.

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 detection primer, a detection kit and an application thereof for Botrytis cinerea LAMP, which can be used for fast, sensitive and specific molecular detection of Botrytis cinerea, and can be used for Early diagnosis of Botrytis cinerea and monitoring and identification of the pathogen. Background technique [0002] tomato( Lycopersicon seculentum Mill) is a vegetable crop widely grown in the world, and it is also one of the main vegetable consumption varieties in my country. In recent years, tomato cultivation area has increased year by year. At the same time, tomato pest control has made great progress from theory to practice. Tomato is one of the vegetable crops with the most diseases, and the diseases seriously restrict the development of tomato industry. Among them, Botrytis cinerea ( Botrytis cinerea ) inf...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
Inventor 兰成忠姚锦爱阮宏椿吴玮
Owner INST OF PLANT PROTECTION FAAS
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