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Colletotrichum glycines Hori loop-mediated isothermal amplification detection primers and detection method thereof

A ring-mediated isothermal, anthracnose technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the cumbersome detection and identification procedures, high identification experience requirements, low accuracy, etc. problem, to achieve the effect of good practicability, simple and fast operation, and high accuracy

Inactive Publication Date: 2018-03-20
INST OF PLANT PROTECTION FAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the problems in the prior art that the detection and identification procedures for soybean anthracnose bacteria 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 soybean anthracnose bacteria Loop-mediated isothermal amplification (LAMP) detection primers and a simple, fast, sensitive and specific visual detection method

Method used

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  • Colletotrichum glycines Hori loop-mediated isothermal amplification detection primers and detection method thereof
  • Colletotrichum glycines Hori loop-mediated isothermal amplification detection primers and detection method thereof
  • Colletotrichum glycines Hori loop-mediated isothermal amplification detection primers and detection method thereof

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Effect test

Embodiment 1

[0029] The loop-mediated isothermal amplification (LAMP) primer design of embodiment 1 soybean anthracnose bacterium

[0030] According to the highly conserved intra-species ribosomal transcribed spacer (rDNA-ITS) sequence of Soybean anthracnose and the variability among families and species, the LAMP detection primer combination with specific amplification effect on Soybean anthracnose was designed, including 2 The nucleotide sequences of one outer primer (F3 and B3) and two inner primers (FIP and BIP) are:

[0031] F3: 5'-ATGCCTGTTCGAGCGTCA-3';

[0032] B3: 5'-TGGGGGTTTTACGGCTAGA-3';

[0033] FIP: 5'-CGCCACTACCTTTAAGGGCCT-TTTCAACCCTCAAGCTCTGC-3';

[0034] BIP: 5'-GACCCTCTCGGAGCCTCCTT-GTCCCTCCGAATCCCAATG-3'.

Embodiment 2

[0035] Example 2 Establishment of the Loop-Mediated Isothermal Amplification (LAMP) Detection Method of Soybean Anthracnose Bacteria

[0036] 1. Extraction of DNA from the sample to be tested:

[0037] 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:

[0038] (1) Take 0.1 g of mycelium powder in a 1.5 mL centrifuge tube, add 900 μL of 2wt.% CTAB extract, shake and mix with a shaker, put in a water bath at 60°C for 60 min, and centrifuge at 12,000 r / min for 15 min at room temperature;

[0039] (2) Take 700 μL of the supernatant, add an equal volume of phenol, chloroform, and isoamyl alcohol mixture (the volume ratio of each is 25:24:1), shake gently, and centrifuge at 8000 r / min for 10 min at room temperature;

[0040] (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 t...

Embodiment 3

[0049] Example 3 Loop-mediated isothermal amplification (LAMP) detection specificity determination of soybean anthracnose pathogen

[0050] 1. Using the CTAB method to extract the genomic DNA of 3 strains of soybean anthracnose, soybean head blight, soybean phytophthora, corn leaf spot, tomato cinerea, and mushroom brown rot.

[0051] 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 0.2 mmol / L F3 and B3, 1.6 mmol / L FIP and BIP, 8 U of Bst DNA polymerase, DNA template 50-100 ng, 12.5 uL 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, 2.0 mM dNTPs, 0.2% Trion X-100), make up 25 uL with sterile ultrapure water. The LAMP reaction conditions were incubation at 63-65°C for 45-60 min and inactivation at 85°C for 5-10 min.

[0052] 3. Determination of LAMP reaction results: Visual observation with fluorescent dyes or agar...

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Abstract

The invention provides Colletotrichum glycines Hori loop-mediated isothermal amplification (LAMP) detection primers and a detection method thereof, which are specially used for specific detection of Colletotrichum glycines Hori, and belongs to the field of crop disease detection and biotechnology. The Colletotrichum glycines Hori LAMP detection primers designed in the invention comprise outer primers F3 and B3 and inner primers FIP and BIP, sequences of which are as shown in SEQ ID NO.1-4. According to the Colletotrichum glycines Hori detection method established on the basis of the primers, green fluorescence can be observed or LAMP characteristic ladder bands appear through chromogenic reaction or agarose gel electrophoresis detection after LAMP. By the LAMP detection primers and the detection method thereof, rapid, sensitive and accurate detection of plants infected with soybean anthracnose can be realized in production practice. Meanwhile, the primers and the detection method can be used for early diagnosis of diseases in the field and monitoring and identification of germs, thus providing a reliable technology and theoretical basis for early warning and control of soybean anthracnose.

Description

technical field [0001] The invention relates to a soybean anthracnose bacterial ring-mediated isothermal amplification detection primer and a detection method thereof, which are specially used for the rapid molecular detection of the soybean anthracnose pathogen, and at the same time can realize the early diagnosis of the soybean anthracnose in the field and the monitoring and identification of the pathogen, belonging to crops Disease detection, identification, control and biotechnology fields. Background technique [0002] China is the origin of soybean. Soybean is not only an important food crop, but also an important oil crop, and an important feed crop. It occupies a very important position in my country's agricultural production and social and economic life. It is the main source of plant protein for people to eat. one. In the 1930s, my country was the largest soybean producer in the world, with an average annual output of more than 10 million tons, accounting for 90% o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6848C12Q1/04C12N15/11
CPCC12Q1/6848C12Q1/6895C12Q2531/119C12Q2563/107
Inventor 杜宜新石妞妞阮宏椿陈文乐甘林陈福如杨秀娟代玉立
Owner INST OF PLANT PROTECTION FAAS