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LAMP (Loop-mediated isothermal amplification) detection primer for ceratocystis fimbriata and application thereof

A technology of black spot bacteria and detection primers, which is applied to the determination/inspection of microorganisms, microorganisms, recombinant DNA technology, etc., can solve the problems of long cycle, poor specificity and low sensitivity of detection methods, and achieves reliable results, strong specificity, Sensitive effect

Pending Publication Date: 2019-08-06
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide a kind of LAMP detection primer and application of sweet potato black spot fungus for the biological detection method of sweet potato black spot fungus in the prior art.

Method used

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  • LAMP (Loop-mediated isothermal amplification) detection primer for ceratocystis fimbriata and application thereof
  • LAMP (Loop-mediated isothermal amplification) detection primer for ceratocystis fimbriata and application thereof
  • LAMP (Loop-mediated isothermal amplification) detection primer for ceratocystis fimbriata and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The design of embodiment 1 sweet potato black spot fungus LAMP detection primer

[0044] 1. Sweet Potato Rhizorrhizae ( Ceratocystis fimbriata Ellis et Halsted) Tsr1 Gene acquisition

[0045] (1) Extraction of Genomic DNA of Sweet Potato Black Spot Fungus:

[0046] Genomic DNA of sweet potato black spot fungus was extracted by CTAB method, the specific process was as follows: take 50 mg freeze-dried mycelia powder in a 1.5 ml centrifuge tube, add 900 μl 2% CTAB (cetyltrimethylammonium bromide) Extract (2% CTAB; 100mmol / L Tris-HCl (trishydroxymethylaminomethane hydrochloride), pH 8.0; 20 mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4 mol / L NaCl) and 90 μl 10% SDS (sodium dodecylbenzene sulfonate), mix well, put in a water bath at 55-60°C for 1.5 h, shake and mix once every 10 min, centrifuge (12,000 rpm) for 15 min after 1.5 h in water bath , take the supernatant and add the same volume of phenol / chloroform / isoamyl alcohol as the supernatant (t...

Embodiment 2

[0057] Embodiment 2: Visual detection of LAMP primers to sweet potato black spot bacteria

[0058] (1) Extraction of Genomic DNA of Sweet Potato Black Spot Fungus:

[0059] Genomic DNA of sweet potato black spot fungus was extracted by CTAB method, the specific process was as follows: take 50 mg freeze-dried mycelia powder in a 1.5 ml centrifuge tube, add 900 μl 2% CTAB (cetyltrimethylammonium bromide) Extract (2% CTAB; 100mmol / L Tris-HCl (trishydroxymethylaminomethane hydrochloride), pH 8.0; 20 mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4 mol / L NaCl) and 90 μl 10% SDS (sodium dodecylbenzenesulfonate) and mix well, put in a water bath at 55-60°C for 1.5 h, shake and mix once every 10 min, centrifuge (12,000 rpm) for 15 min after 1.5 h in water bath , take the supernatant and add the same volume of phenol / chloroform / isoamyl alcohol as the supernatant (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1), centrifuge (12,000rpm) for 5 min...

Embodiment 3

[0064] Embodiment 3: LAMP primer is to the specific amplification of sweet potato black spot bacterium

[0065] Using 2 strains of sweet potato black spot fungus and 24 other pathogenic fungi and oomycetes from Fujian and Sichuan in my country as the test materials, the specificity of the detection primers was verified by LAMP.

[0066] (1) Tested strains:

[0067]

[0068] (2) Extraction of genomic DNA of the tested strains:

[0069] The genomic DNA of the tested strain was extracted by the CTAB method, and the specific process was 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) to extract solution (2% CTAB; 100mmol / L Tris-HCl (trishydroxymethylaminomethane hydrochloride), pH 8.0; 20 mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4 mol / L NaCl ) and 90 μl 10% SDS (sodium dodecylbenzene sulfonate) and mix well, put in a water bath at 55-60°C for 1.5 h, shake and mi...

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Abstract

The invention discloses an LAMP (Loop-mediated isothermal amplification) primer for ceratocystis fimbriata and application thereof, and belongs to the technical field of detection, identification andprevention and control of crop diseases. The LAMP detection primer for the ceratocystis fimbriata comprises two external primers F3 and B3, and two internal primers FIP and BIP. After LAMP on the LAMPdetection primer is carried out, color development reaction or agarose gel electrophoresis is performed, and the phenomenon that reaction liquid shows green or a ladder-shaped tape which shows the LAMP characterization can be observed. The LAMP primer for ceratocystis fimbriata and a detection method disclosed by the invention can be applied to quick, sensitive and accurate detection of plants infected with the ceratocystis fimbriata in production practice and the ceratocystis fimbriata in soil, and also can be applied to early diagnosis of diseases in the fields as well as monitoring and identification of pathogenic bacteria; and therefore, a reliable technological and theoretical basis is provided for preventing and controlling the diseases caused by the ceratocystis fimbriata.

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 LAMP detection primer for sweet potato black spot fungus and an application thereof. [0002] A LAMP detection primer for sweet potato black spot fungus and a rapid detection method thereof are specially used for high-sensitivity rapid molecular detection of sweet potato black spot fungus, and can be used for early diagnosis of sweet potato black spot disease and monitoring and identification of the pathogen. Background technique [0003] Sweet potato is one of the main food crops in my country, and it is also an important light industrial raw material and feed crop. In addition to processed starch and alcohol, the consumption of sweet potatoes in my country has increased the proportion of fresh food and leafy vegetables. The National Development and Reform Commission has selected sweet potatoes as the preferred non-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2531/119
Inventor 王荣波陈庆河刘裴清李本金翁启勇
Owner INST OF PLANT PROTECTION FAAS
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