Method and primer composition for detecting soybean fusarium oxysporum based on LAMP (loop-mediated isothermal amplification) technology

A primer composition and a technology for Fusarium oxysporum, which are applied in the biological field, can solve the problem of inability to clearly distinguish Fusarium fungi and the like, and achieve the effects of good practicability, low sensitivity, and overcoming a long period of time.

Active Publication Date: 2013-10-16
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Commonly used target genes in common PCR include the ribosomal gene transcription spacer (Internal transcribed space, ITS) [14], but many scholars believe that this target cannot clearly distinguish Fusarium fungi [10,11]

Method used

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  • Method and primer composition for detecting soybean fusarium oxysporum based on LAMP (loop-mediated isothermal amplification) technology
  • Method and primer composition for detecting soybean fusarium oxysporum based on LAMP (loop-mediated isothermal amplification) technology
  • Method and primer composition for detecting soybean fusarium oxysporum based on LAMP (loop-mediated isothermal amplification) technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Fusarium oxysporum detection kit, including 1mL detection solution + dye SYBR Green I25μL; 1mL detection solution includes: 32mM forward inner primer FIP, 32mM reverse inner primer BIP, 8mM forward outer primer F3, 8mM reverse outer primer B3 , 8mM Loop Primer LF, 8mM Loop Primer LB, 56mM dNTPs, 0.8M Tris-HCl (pH8.8), 0.4mM KCl, 0.4mM (NH4) 2 SO 4 , 0.24mM MgSO 4 , 4% Triton X-100, Bst DNA polymerase 320 units, add ultrapure water to prepare 1mL detection solution. Among them, four specific primers FIP, BIP, F3, B3 and two loop primers LF, LB for molecular detection of Fusarium oxysporum LAMP are as follows:

[0071]

[0072] The shelf life of this kit is 1 year.

Embodiment 2

[0073] Example 2 Detection of Fusarium oxysporum LAMP

[0074] In order to verify the feasibility of the LAMP method, the standard Fusarium oxysporum strain 6497 (purchased from the CBS database, Fusarium oxysporum f.sp.tracheiphilum (E.F.Smith) Snyder&Hansen-PP3; cond: OA, SNA 175.33 the same below) was selected. The DNA of the Fusarium oxysporum strain and other specialized strains of Fusarium isolated from the disease specimen was used as a template, and 4 μl of the DNA solution was taken, and 18 μl of the detection solution prepared in Example 1 and 3 μl of sterilized deionized water were added to carry out the LAMP reaction. The reaction procedure was: 62°C for 60 min, after amplification, 0.25 μL of dye SYBR Green I was added as a reaction indicator, observed with naked eyes, and irradiated with 245 nm wavelength ultraviolet light to observe fluorescence. The color change and fluorescence intensity of SYBR Green I were used as the result judgment standard. The results s...

Embodiment 3

[0075] The specificity test one of embodiment 3 Phytophthora soybean LAMP reaction

[0076] To verify the specificity of the LAMP method, standard Fusarium oxysporum strains and Fusarium strains different from Fusarium oxysporum (F. solani; F. graminearum; F. moniliforme; F. chrysogenum; Fusarium avenae; Fusarium nivale; Fusarium equiseti; Fusarium laminarum) DNA was used as a template, and 4 μl of DNA solution was taken, and the LAMP reaction and result observation were carried out according to the method in Example 2. The results showed that when LAMP primers were used to amplify the DNA template of Fusarium oxysporum, a yellow-green color change and green fluorescence were produced; while other species of Fusarium had no color change or strong green fluorescence ( image 3 ).

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Abstract

The invention discloses a method and a primer composition for detecting soybean fusarium oxysporum based on an LAMP (loop-mediated isothermal amplification) technology. The primer composition comprises four specific primers FIP, BIP, F3 and B3 for detecting the LAMP molecules of the soybean fusarium oxysporum, and two ring primers LF and LB used for increasing the reaction speed. The method is used for performing LAMP on DNAs to be detected by the primer composition provided by the invention; a detection result is observed by naked eyes or under the irradiation of ultraviolet light with the wavelength of 245 nm; the color change and the fluorescence intensity of SYBR Green I are taken as result judgment standards. The invention provides the new molecular detection method and the primer composition for detecting the soybean fusarium oxysporum, the soybean fusarium oxysporum is subjected to LAMP detection, the detection cycle is short, the accuracy is high, the sensitivity is high, and the detection result is observed by the naked eyes.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for detecting Fusarium oxysporum based on LAMP technology and a primer composition used. Background technique [0002] Soybean Fusarium wilt, also known as wilting disease, yellow leaf disease, root rot, is a worldwide soil-borne disease with wide distribution, serious damage and difficult control. Cromwell in the United States first reported soybean wilt caused by Fusarium in 1917 [2]. At present, the disease occurs to varying degrees in China[19,22,23], the United States[7,13], India, the Philippines, Japan and other countries every year, seriously affecting the yield of soybean and causing huge economic losses to agricultural production. . In my country, Fusarium wilt occurs more seriously in soybean producing areas in Northeast China. In 1997, Wang Changjia et al. conducted a disease investigation on soybeans with a planting area of ​​2314 hectares in some farms in Heilo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11G01N21/64
Inventor 郑小波陆辰晨戴婷婷王源超张海峰
Owner NANJING AGRICULTURAL UNIVERSITY
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