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Rapid and accurate detection method for Fusarium oxysporum based on RPA (recombinase polymerase amplification)-lateral flow chromatography test strip technology

A technology of Fusarium oxysporum and lateral flow chromatography, which is applied in the field of high-sensitivity and rapid detection of F. oxysporum, can solve problems such as no relevant application reports, and achieve good amplification effect, increased sensitivity, and good specificity. Effect

Inactive Publication Date: 2019-06-18
NANJING FORESTRY UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, RPA technology has been widely used in the detection of viruses on humans, animals or plants, but in the detection of plant pathogenic oomycetes, especially for the RPA detection of Fusarium oxysporum, there are no relevant application reports

Method used

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  • Rapid and accurate detection method for Fusarium oxysporum based on RPA (recombinase polymerase amplification)-lateral flow chromatography test strip technology
  • Rapid and accurate detection method for Fusarium oxysporum based on RPA (recombinase polymerase amplification)-lateral flow chromatography test strip technology
  • Rapid and accurate detection method for Fusarium oxysporum based on RPA (recombinase polymerase amplification)-lateral flow chromatography test strip technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] 1. According to the CYP51 sequence of Fusarium oxysporum (XM_018393091.1), Primer Premier 5.0 was used to design the forward primer sequence RPA-FoRPA-F, the reverse primer sequence RPA-FoRPA-R, and the probe sequence FoRPA-P. The specific sequence is as follows:

[0043] RPA-FoRPA-F:

[0044] RPA-FoRPA-R:

[0045] FoRPA-P:

[0046] 2. Extract the genomic DNA of the tested pathogenic bacteria strain. The specific extraction process is as follows:

[0047] Take a small amount of mycelium powder, add 900 μL 2% CTAB extract and 90 μL 10% SDS, vortex and mix well, put in a water bath at 60°C for 1 hour, and invert several times every 10 minutes. Centrifuge at 12000rpm for 10min, take the supernatant and add an equal volume of phenol / chloroform / isoamyl alcohol (25:24:1), mix by inverting, centrifuge at 12000rpm for 10min; transfer the supernatant to a new tube, add an equal volume of chloroform, Gently invert to mix and centrifuge at 12000rpm for 5min. Take the...

Embodiment 2

[0053] In order to verify the specificity of the RPA lateral flow chromatography test strip detection method, using Fusarium oxysporum strains and other Fusarium and pathogenic bacteria as test materials (Table 1), the results of the RPA lateral flow chromatography test strip detection method The test strip showing Fusarium oxysporum has two brown bands, one is in the quality control area and the other is in the detection area, the result is positive. For other Fusarium and pathogenic bacteria, only one brown band appears in the quality control area If there is no band in the detection area, the result is negative, indicating that the sample does not contain Fusarium oxysporum. Select different species of Fusarium oxysporum (Fusarium solani; Pythium; Pythium anthracnose; Phytophthora soybean; Verticillium dahliae; Rhizoctonia solani; Magnaporthe oryzae, etc.) DNA was used as a template for RPA lateral flow chromatography test strip detection.

[0054] Table 1 Fungal and oomyc...

Embodiment 3

[0062] Use Nanodro 2000 micro spectrophotometer to measure the concentration of DNA extracted from Example 2 to be 100ngμL -1 .

[0063] It was serially diluted to 100pg μL -1 , 10pg μL -1 , 1pg μL -1 and 100fg μL -1 According to the primers, reaction system and reaction conditions adopted in the embodiment of Example 1, RPA and PCR detection were performed on DNA with different concentrations.

[0064] The result is as Figure 4 As shown, the 25 μL reaction system contains 100 ng μL -1 , 10ngμL -1 , 1ngμL -1 , 100pgμL -1 There are two brown bands on the test strip of Fusarium oxysporum DNA, indicating a positive reaction. The 25 μL reaction system contains 10 pg μL respectively -1 , 1pgμL -1 , 100fgμL -1 A brown band appeared on the paper strip of Fusarium oxysporum DNA, indicating a negative reaction; the color development results showed that the sensitivity of the RPA-lateral flow chromatography test strip technology reached 100pgμL -1 ; The detection time of RP...

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Abstract

The invention discloses a rapid and accurate detection method for Fusarium oxysporum based on a RPA (recombinase polymerase amplification)-lateral flow chromatography test strip technology. The methodcomprises the steps as follows: 1) extracting DNA of a to-be-detected sample; 2) performing RPA with DNA as a template; 3) detecting an amplification product by a lateral flow chromatography test strip; when two brown bands appear on the test strip, one is located in the quality control area and the other is located in the detection area, the result is positive and indicates that the sample contains the Fusarium oxysporum; when only a brown band appears in the control area of the test strip and no band is located in the detection area, the result is negative and indicates that the sample doesnot contain the Fusarium oxysporum. The primer probe group has good RPA effect and strong band specificity and has no cross reaction with other bacteria, positive reaction occurs in the detection area, detection sensitivity is increased, a new technical platform is provided for detection of the Fusarium oxysporum, pathogens are detected at the early stage of disease infection, and the Fusarium oxysporum in nursery grounds or diseased plants can be detected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the detection of Fusarium oxysporum based on RPA-lateral flow chromatography technology, which is specially used for the high-sensitivity and rapid detection of Fusarium oxysporum, and can be used for early diagnosis and monitoring of pathogens in forests . It specifically relates to a method for rapid and accurate detection of Fusarium oxysporum based on RPA-lateral flow chromatography test strip technology. Background technique [0002] Pine seedling blight caused by Fusarium oxysporum infecting seedlings is an important disease that harms seedlings. In severe cases, it can cause 80% seedling death, seriously affecting the economic benefits of seedling growers. Fusarium oxysporum (F.oxysporum) is a filamentous fungus distributed worldwide. Its main morphological feature is that colonies grow radially on Potato Dextrose Agar (PDA) medium, and the growth rate is relatively fast. The ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11
Inventor 戴婷婷陆辰晨焦彬彬田雯
Owner NANJING FORESTRY UNIV
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