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A loop-mediated isothermal amplification primer composition for detecting Fusarium fusarium and its application

A technology of Fusarium flavus and primer composition, which is applied in the field of warm amplification primer composition, can solve the problem of inability to distinguish Fusarium fungi and the like, and achieves the effects of increasing the time required for detection, simple operation and strong practicability

Active Publication Date: 2019-01-25
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Target genes commonly used in common PCR, such as ribosomal gene transcription spacer (Internal transcribed space, ITS) [25] Inability to accurately differentiate Fusarium fungi at the species level

Method used

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  • A loop-mediated isothermal amplification primer composition for detecting Fusarium fusarium and its application
  • A loop-mediated isothermal amplification primer composition for detecting Fusarium fusarium and its application
  • A loop-mediated isothermal amplification primer composition for detecting Fusarium fusarium and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 A kind of LAMP detection kit for detecting Fusarium flavum

[0060] Kit reaction system: detection solution.

[0061] The detection solution preparation method is as follows:

[0062] 2.5μL 10×ThermoPol Buffer, 8mM MgSO 4 , 1.2mM dNTPs, 1.6μM forward inner primer CYP51C-FIP, 1.6μM reverse inner primer CYP51C-BIP, 0.4μM forward outer primer CYP51C-F3, 0.4μM reverse outer primer CYP51C-B3, 0.8μM loop primer CYP51C -LF, 0.8μM Loop Primer CYP51C-LB, 0.8M Betaine, 20mM Tris-HCl(PH 8.8), 10mM KCl, 10mM(NH4) 2 SO 4 , 0.1% Trion-X, 8U·μL -1 Bst DNA polymerase, 180mM HNB, shelf life is 1 year.

Embodiment 2

[0063] Embodiment 2 The universality investigation of the kit of the present invention

[0064] Select the standard strain of Fusarium xanthus shown in Table 2 and the DNA of Fusarium xanthus from different regions as templates, take 2 μL DNA solution, add 24 μL detection solution to carry out LAMP reaction, the reaction program is: 62 ° C, 85 min; After observing the color change of the reaction tube, the samples of Fusarium xanthus from different regions all turned into sky blue, and the negative control remained purple. It shows that this technique is applicable to the detection of Fusarium chrysogenum from different regions. figure 2 Seven representative strains were selected and listed.

Embodiment 3

[0065] Embodiment 3 The kit specificity investigation of the present invention

[0066] Select Fusarium chrysogenum standard bacterial strain, and Fusarium such as its similar species Fusarium graminearum shown in table 2, and other genus pathogenic bacteria (panthrax anthracnose, gloeospora anthracnose, multi-main corynesporium, soybean purple spot bacterium , Soybean charcoal rot fungus, Rhizoctonia solani, Rhizoctonia solani, Rhizoctonia holly, Helminthias maize, Aspergillus oryzae, Magnaporthe grisea, Southern stem canker of soybean, Northern stem canker of soybean, Brown rot of soybean stem , Phytophthora sojae, Phytophthora soybean) DNA as a template, take 2 μL DNA solution, add 24 μL detection solution to carry out LAMP reaction, the reaction program is: 62 ° C, 85 min; after the reaction, observe the color change of the reaction tube, containing Samples with Fusarium xanthus should turn azure, while samples containing other species and negative controls are purple. Th...

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Abstract

The invention discloses a loop-mediated isothermal amplification primer combination for detecting yellow fusarium solani and application of the loop-mediated isothermal amplification primer combination. The primer combination is composed of a forward-direction outer primer CYP51C-F3 as indicated by SEQ ID No. 2, a reverse-direction outer primer CYP51C-B3 as indicated by SEQ ID No. 3, a forward-direction inner primer CYP51C-FIP as indicated by SEQ ID No. 4, a reverse-direction inner primer CYP51C-BIP as indicated by SEQ ID No. 5, a loop primer CYP51C-LF as indicated by SEQ ID No. 6 and a loop primer CYP51C-LB as indicated by SEQ ID No. 7. By means of the loop-mediated isothermal amplification primer combination, the problems that existing yellow fusarium solani detection methods are long in required cycle, complex, troublesome and low in specificity, and time and labor are consumed are solved, the primer combination is short in required detection time (only 85 minutes are needed), great in specificity and high in sensitivity, and a detection result can be observed visually.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a loop-mediated isothermal amplification primer composition for detecting Fusarium chrysogenum and its application. Background technique [0002] Soybean root rot is a general term for diseases caused by a variety of pathogenic bacteria that cause soybean roots and stem bases to rot. It is one of the most serious diseases in soybean production because of its wide distribution, serious damage, more than a dozen types of pathogens, and the symptoms of diseases caused by it are difficult to distinguish and difficult to control. Fusarium soybean root rot is a soybean root rot caused by fungi of the genus Fusarium, first reported by Crommell in the United States in 1917 [1-3] , currently in China [4-6] ,United States [7] , India, the Philippines, Japan and other countries have reported that it is an important disease in the soybean producing areas of Northeast my country and the H...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 郑小波曾丹丹田擎张海峰王源超
Owner NANJING AGRICULTURAL UNIVERSITY
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