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LAMP (Loop-Mediated Isothermal Amplification) detection primer, kit and LAMP rapid detection method for pathogenic bacteria of leaf spot bacteria

A technology for detection kits and detection methods, which can be applied in microorganism-based methods, biochemical equipment and methods, DNA/RNA fragments, etc., can solve problems such as unfavorable plant protection, low efficiency, etc. less demanding effects

Pending Publication Date: 2022-05-13
HUNAN AGRICULTURAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, PCR technology is commonly used in the detection of pathogenic bacteria, but the detection efficiency of this technology is low, which is not conducive to the protection of plants

Method used

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  • LAMP (Loop-Mediated Isothermal Amplification) detection primer, kit and LAMP rapid detection method for pathogenic bacteria of leaf spot bacteria
  • LAMP (Loop-Mediated Isothermal Amplification) detection primer, kit and LAMP rapid detection method for pathogenic bacteria of leaf spot bacteria
  • LAMP (Loop-Mediated Isothermal Amplification) detection primer, kit and LAMP rapid detection method for pathogenic bacteria of leaf spot bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0045] The invention provides a LAMP detection primer for the leaf spot pathogen:

[0046] Based on the conserved region of the leaf spot pathogen EF-1α sequence gene, primers were designed online using Primer Explore V5, including 2 outer primers F3 and B3 and 2 inner primers FIP and BIP. The primers used were completed by Sangon Bioengineering (Shanghai) Co., Ltd., and the primer sequences are as follows:

[0047] F3: 5'-ACCAGTGCGGTGGTATCG-3';

[0048] B3: 5'-CAAGAGCGCCTGTCACTC-3';

[0049] FIP:

[0050] 5'-GGATCGATGGGAAGAAGGGCGCAAGCGAACCATCGAGAAGT-3';

[0051] BIP: 5'-CATACGACGACTCGACAAGCGCCCACCCACCCAAAAAAACGAC-3'.

[0052]Construct the LAMP detection reaction system, the LAMP detection reaction system includes 10uM FIP and BIP primers 4μL each, 10uM F3 and B3 primers 0.5μL each, 10mM dNTPs 2.5μL, 8U / μL Bst DNA polymerase 1μL, 50mM MgSO 4 4 μL, 2.5 μL of 10×isothermal amplification buffer, 1 μL of template DNA, and make up to 25 μL with sterilized ultrapure water. Dur...

experiment example -L

[0056] Experimental example - LAMP detection primer specificity detection;

[0057] LAMP detects primer specificity detection: day lily leaf spot pathogen, wild rice stem rot pathogen (Fusarium andiyazi), citrus anthracnose pathogen (Colletotrichum gloeosporioides), Rhizoctonia solani (Rhizoctonia solani), sorghum epicoccus (Epicoccum nigrum) according to the embodiment The LAMP detection method is used for detection, and the LAMP primer specificity is judged by adding SYBR Green I dye staining and 1% agarose gel electrophoresis to observe the results. The detection results are as follows: figure 1 As shown in the figure, a shown in the figure is: SYBR Green I detection of LAMP products, b is gel electrophoresis detection of LAMP products, M is DL2000 marker; other numbers 1 to 3 are day lily leaf spot pathogen; 4 is Zizania sheath rot fungus ; 5 is glyospora anthracnose; 6 is Rhizoctonia solani; 7 is Eperococcus sorghum; 8 is negative control of sterile water.

[0058] The t...

experiment example

[0062] Experimental example - detection of pathogenic bacteria in actual samples

[0063] The operation method is as follows:

[0064] The total DNA was extracted from day lily leaves artificially inoculated with the pathogen of leaf spot disease 4 days after inoculation, and used as a template for LAMP amplification. Pathogenic bacteria, and pathogenic bacteria were detected on the inoculated plants, such as image 3 As described in a, 3 in the figure is the artificial inoculation of day lily leaf LAMP detection; M is DL 2000marker; another 1 is positive day lily leaf spot bacterial strain; 2 and 3 are leaves inoculated with day lily leaf spot pathogen; 4 and 5 are healthy plants not inoculated with pathogenic bacteria; 6 is the negative control of sterile water.

[0065] In addition, 2 leaf samples collected in Jinyun County, Lishui City, Zhejiang Province, showing the symptoms of daylily leaf spot disease were isolated from the tissues. Through the identification of strai...

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Abstract

The invention provides an LAMP (loop-mediated isothermal amplification) detection primer for leaf spot disease pathogenic bacteria, and relates to the technical field of plant pathogenic bacteria detection, the primer comprises a forward outer primer F3, a reverse outer primer B3, a forward inner primer FIP and a reverse inner primer BIP, and the primer series are as follows: F3: 5 '-ACCAGTGCGGGGTATCG-3', B3: 5 '-CAAGAGCGCCTGTCAC-3', FIP: 5 '-GGATCGGGAAGGCGCAAAGCGCCACCACCAAAAAAAAGCGCCACCACCAAAAAAACGAC-3', and BIP: 5 '-

Description

technical field [0001] The invention relates to the technical field of detection of plant pathogenic bacteria, in particular to a LAMP detection primer, a kit and a rapid detection method for LAMP of leaf spot pathogen. Background technique [0002] Day lily (Hemerocallis citrina Baroni), also known as day lily, calming vegetable, forget worry grass, scientific name Hemerocallis citrina Baroni, is a perennial herbaceous plant of Liliaceae. Its edible part is the flower bud at the end of the bud, which is rich in nutrition and contains a variety of sugars, proteins, vitamins, inorganic salts and a variety of amino acids necessary for human body. It is one of the traditional characteristic vegetables in my country. The plant is widely grown in China, North Korea, and Japan, and has ornamental and vegetable value. In addition, the synthetic metabolites of flower buds of day lily are rich in rutin, hesperidin, colchicine, etc., which are effective in treating anxiety, edema, de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11C12R1/77
CPCC12Q1/6895C12Q1/6844C12Q2531/119C12Q2565/125
Inventor 朱俊子钟杰李晓刚马雅敏
Owner HUNAN AGRICULTURAL UNIV
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