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Corn setosphaeria turcica LAMP detection primer as well as fast detection method and application thereof

A technology for the detection of Septoria maize and its detection method, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of cumbersome detection and identification procedures, high requirements for identification experience, and low accuracy, and achieve practical Good performance, simple and fast operation, high accuracy

Inactive Publication Date: 2018-09-18
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the problems in the prior art that the detection and identification procedures of Pseudomonas spp. are cumbersome, time-consuming, require high identification experience, low accuracy, and PCR detection needs to rely on amplification equipment and other equipment, the present invention provides a large-spot maize LAMP detection primers and simple, rapid, sensitive and specific visual detection method for spot fungus

Method used

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  • Corn setosphaeria turcica LAMP detection primer as well as fast detection method and application thereof
  • Corn setosphaeria turcica LAMP detection primer as well as fast detection method and application thereof
  • Corn setosphaeria turcica LAMP detection primer as well as fast detection method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Design of LAMP primer set for Pseudomonas maize

[0025] According to the β-tubulin gene sequence of S. maize, a LAMP detection primer set with specific amplification effect on S. maize was designed, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP). The nucleotide sequences are:

[0026] F3: 5'-GGAAGGGCACCATGTTGAC-3';

[0027] B3: 5'-GCTGGTGGAGAACTCTGAC-3';

[0028] FIP: 5'-ACCTCGTCTCCGCCGTCATGT-AGAGTTAAGCTGACCAGGGA-3';

[0029] BIP: 5'-GGTTCAGGTCGCCGTACGAG-CTGCATTGACAACGAGGCT-3'.

Embodiment 2

[0030] Example 2 Establishment of the detection method for LAMP of maize leaf spot

[0031] 1. Extraction of DNA from the sample to be tested:

[0032] ① When used to detect pure cultures of pathogenic bacteria, the CTAB method is used to extract the genomic DNA of the tested strains, and the specific steps are as follows:

[0033] (1) Take 0.1g of mycelium powder in a 1.5mL centrifuge tube, add 900μL of 2wt.% CTAB extract, shake and mix with an oscillator, bathe in water at 60°C for 60min, and centrifuge at 12000r / min for 15min at room temperature;

[0034] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (each volume ratio is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 min at room temperature;

[0035] (3) Take 500 μL of the supernatant, add an equal volume of chloroform and extract again, and centrifuge at 8000 r / min for 10 min at room temperature;

[0036](4) Take 350 μL of the supernatant, add 1 / 1...

Embodiment 3

[0044] Example 3 Determination of Specificity of Detection of Leptosphaeria maize LAMP

[0045] 1. Using the CTAB method to extract the genomic DNA of 3 strains from different sources of maize leaf spot, maize leaf spot, maize sheath blight, maize round spot, maize gray spot, and maize southern rust.

[0046] 2. Carry out LAMP amplification using the DNA extracted from the test bacteria as a template: LAMP reaction system 25 μL, the reaction system includes 0.2mmol / L F3, 0.2mmol / L B3, 1.6mmol / L FIP, 1.6mmol / L BIP, Bst DNA 8 U of polymerase, 50-100 ng of DNA template, 12.5 μL of LAMP reaction mixture (40 mM Tris-HCl, 20 mM (NH 4 ) 2 SO 4 , 20mM KCl, 16mM MgSO 4 , 1.6mol / L betaine, 2.0mM dNTPs, 0.2% Trion X-100), make up 25μL with sterile ultrapure water. The LAMP reaction conditions are incubation at 63-65°C for 45-60 minutes, and inactivation at 85°C for 5-10 minutes.

[0047] 3. Determination of LAMP reaction results: Visual observation with fluorescent dyes or agarose g...

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Abstract

The invention provides a corn setosphaeria turcica LAMP detection primer as well as a fast detection method and application thereof, is mainly applied to the specific detection of the corn setosphaeria turcica and belongs to the technical field of crop disease detection and biology. The corn setosphaeria turcica LAMP detection primer is designed, and comprises outer primers F3 and B3, and inner pripers FIP and BIP, shown in a sequence table. According to the corn setosphaeria turcica detection method built on the basis of the primer, the green fluorescence can be observed or trapezoidal stripswith LAMP features can occur through color development reaction after LAMP constant-temperature amplification or agarose gel electrophoresis detection. The LAMP detection primer and the detection method can realize the fast, sensitive and accurate detection of the corn setosphaeria turcica infected plants in the production practice; meanwhile, the primer and the detection method can be used for early diagnosis of the field diseases and the pathogenic bacterium monitoring and identification; the reliable technical and theoretical basis is provided for the early warning and prevention and control of the corn setosphaeria turcica.

Description

technical field [0001] The invention relates to a LAMP detection primer for the large spot of corn and its rapid detection method and application. Crop disease detection, identification, control and biotechnology fields. Background technique [0002] Corn is widely planted in many countries in the world due to its advantages of wide application, high yield and low requirements on planting conditions. In my country, corn is an important food and economic crop with the largest planting area and the second largest output. In addition to being used as food, the modern corn industry has also developed multiple uses such as energy, feed, fruits and vegetables. The healthy development of the corn industry plays a very positive and important role in improving my country's agricultural industrial structure, ensuring food security, and expanding the national economic aggregate. [0003] Northern corn leaf blight (NCLB) is a serious leaf blight fungal disease caused by the infection ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/45
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 杜宜新石妞妞阮宏椿陈福如甘林杨秀娟代玉立
Owner INST OF PLANT PROTECTION FAAS
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