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LAMP detection primer of bipolaris maydis and visualized detection method and application thereof

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

Inactive Publication Date: 2018-08-10
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems in the prior art that the detection and identification procedures for corn spot disease 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 small corn spot LAMP detection primers and simple, rapid, sensitive and specific visual detection method for spot fungus

Method used

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  • LAMP detection primer of bipolaris maydis and visualized detection method and application thereof
  • LAMP detection primer of bipolaris maydis and visualized detection method and application thereof
  • LAMP detection primer of bipolaris maydis and visualized detection method and application thereof

Examples

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Effect test

Embodiment 1

[0023] Embodiment 1 The design of the LAMP primer of corn spot bacterium

[0024] According to the highly conserved intraspecies Actin (ACT) gene sequence of P. maize, LAMP detection primers with specific amplification effect on P. maize were designed, including 2 outer primers (F3 and B3) and 2 inner primers (FIP and BIP), the nucleotide sequences of which are respectively:

[0025] F3: 5'-ACCACTGGTATCGTCCTCG-3';

[0026] B3: 5'-GCAGAGCTTCTCCTTGATGT-3';

[0027] FIP: 5'-CATGTCGACACGGGAGATGGC-TCTGGTGACGGTGTCACTC-3';

[0028] BIP: 5'-GGTCGTGACTTGACCGACTACC-CGATTTCTCGCTCGGCAG-3'.

Embodiment 2

[0029] Example 2 Establishment of LAMP Visual Detection Method for Corn Leaf Spot Bacteria

[0030] 1. Extraction of DNA from the sample to be tested:

[0031] 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:

[0032] (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 60°C water for 60min, and centrifuge at 12000r / min for 15min at room temperature;

[0033] (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;

[0034] (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;

[0035](4) Take 350 μL of the supernatant, ad...

Embodiment 3

[0043] Example 3 Determination of the specificity of detection of LAMP of corn leaf spot bacterium

[0044] 1. Using the CTAB method to extract the genomic DNA of 3 strains from different sources of corn leaf spot fungus, corn leaf spot fungus, corn leaf spot fungus, corn round spot fungus, maize southern rust fungus, and corn sheath blight fungus.

[0045] 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.

[0046] 3. Determination of LAMP reaction results: Visu...

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Abstract

The invention provides a LAMP detection primer of bipolaris maydis and a visualized detection method and application thereof, and belongs to the technical field of crop disease detection and biology.The LAMP detection primer and the visualized detection method are specially used for specified detection of the bipolaris maydis. The LAMP detection primer of the bipolaris maydis is designed, the primer comprises outer primers F3 and B3, and inner primers FIP and BIP, and the outer and inner primers are as shown in a sequence table. According to the visualized detection method established based on the primer, green fluorescence or ladder shape presented with LAMP characteristics can be observed through chromogenic reaction or agarose gel electrophoresis detection after LAMP isothermal amplification is conducted. By means of the LAMP detection primer and the detection method thereof, rapid, sensitive and accurate detection of the bipolaris maydis can be achieved in production practice, andthe LAMP detection primer and the detection method thereof can be used for the monitoring and identification of pathogenic bacteria and early diagnosis of field diseases to provide reliable technicaland theoretical basis for early warning, prevention and control of the bipolaris maydis.

Description

technical field [0001] The invention relates to a LAMP detection primer for corn spot disease and its visual detection method and application, which is specially used for the rapid visual detection of corn spot spot disease, and can realize the early diagnosis of corn spot spot disease and the monitoring and identification of the pathogen at the same time, belonging to Crop disease detection, identification, control and biotechnology fields. Background technique [0002] Corn is an important food crop and economic crop in my country. It also has multiple uses such as energy, feed, fruits and vegetables. It is the crop that ranks first in planting area and second in output, and is in great demand. The healthy development of the corn industry plays a very positive and important role in improving the structure of my country's agricultural industry and ensuring food security. Corn leaf spot is a fungal disease of leaves caused by the infection of Bipolaris maydis. Under the co...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119C12Q2563/107
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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