A combination of primers and probes for the detection of Fusarium solani rot based on rpa-lateral flow chromatography and its application

A technology for Fusarium solani and lateral flow chromatography, which is applied in the biological field, can solve problems such as no relevant application reports, and achieves good amplification effect, increased sensitivity and good specificity.

Active Publication Date: 2019-10-18
NANJING FORESTRY UNIV
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  • Summary
  • 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 solani solani, there are no relevant application reports

Method used

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  • A combination of primers and probes for the detection of Fusarium solani rot based on rpa-lateral flow chromatography and its application
  • A combination of primers and probes for the detection of Fusarium solani rot based on rpa-lateral flow chromatography and its application
  • A combination of primers and probes for the detection of Fusarium solani rot based on rpa-lateral flow chromatography and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The genomic DNA of the tested pathogenic bacteria strain was extracted, and the specific extraction process was as follows:

[0033] 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 inversion, and 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 supernatant and transfer it to a new tube, add 2 times the volume of absolute ethanol and 1 / 10 volume of 3M NaAc (pH5.2), and precipitate at -20°C (>1h). Centrifuge at 12000rpm for 10min, pour off the supernatant, wash the precipitate twice with 70% ethanol, and dry at room temperature. Add an appropriate amount of sterilized ultrap...

Embodiment 2

[0035] Using the extracted DNA as a template and using the designed RPA primers, perform the RPA reaction in the following reaction system:

[0036] Sample detection: Add 29.5 μL of buffer buffer, 2.1 μL of 10 μM upstream primer, 2.1 μL of 10 μM downstream primer, and 0.6 μL of probe to a 0.2 mL TwistAmp reaction unit tube (TwistAmp Basickits, Twist) containing lyophilized enzyme powder , DNA 2.0μL, MgAc 2.5μL were added to the inside of the PCR tube cap, and deionized water was added to 50μL; the RPA amplification system was thoroughly mixed, centrifuged at 5,000×g for 10s, placed on a metal bath at 39°C for 30min, and incubated for 4 Minutes, the reaction tube was mixed again, centrifuged for 3-5 s, and placed in a water bath at 39°C to continue the reaction for 30 min.

[0037] Wherein, the length of the RPA primer is generally 30 to 35 nucleotides. Too short primers will seriously affect the activity of the recombinase. Long primers do not necessarily improve the amplific...

Embodiment 3

[0042] In order to verify the specificity of the RPA lateral flow chromatography test strip detection method, Fusarium solani solani and other Fusarium and pathogenic bacteria were used as test materials (Table 1), and the RPA lateral flow chromatography test strip detection method The results show that there are two brown bands on the test strip of Fusarium solani rot, one is in the quality control area and the other is in the detection area, the result is positive. The test strips of other Fusarium solani and pathogenic bacteria only appear in the quality control area A brown band with no band in the test area is negative, indicating that Fusarium solani solani is not present in the sample.

[0043] Select different species of Fusarium solani rot (Fusarium solani rot; Fusarium oxysporum; Fusarium graminearum; Fusarium moniliforme; Fusarium equiseti; Pythium ultima; Anthracnose flathead; Phytophthora sojae; Verticillium dahliae; Rhizoctonia solani; Magnaporthe oryzae, etc.) w...

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Abstract

The invention discloses a primer and probe composition for detecting fusarium solani based on an RPA-lateral flow chromatographic technology and application of the composition. The application methodcomprises the specific steps that 1, a to-be-detected sample DNA is extracted; 2, with the DNA as a template, RPA amplification is conducted; 3, a lateral flow chromatographic test strip is applied for amplification product detection; when two brown strips exist on the test strip, one strip is located in a quality control area, and the other strip is located in a detection area, a result is positive, and it is expressed that a sample contains the fusarium solani; when a brown strip only exists in the quality control area of the test strip and a strip does not exist in the detection area of thetest strip, a result is negative, and it is expressed that the sample does not contain the fusarium solani. The primer and probe composition has a good RPA amplification effect and high strip specificity and does not conduct a cross reaction with other germs, positive control is achieved in the quality control area, the detection sensitivity is improved, a novel technology platform is provided for detection of the fusarium solani, pathogens are identified in the early period of disease infection, and the fusarium solani in a plant nursery field or disease plants can be detected.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a method for detecting Fusarium solani rot based on RPA-lateral flow chromatography technology, and special primers and probe combinations. Background technique [0002] Fusarium solani ( Fusarium solani ) infection of seedlings caused by pine blight is an important disease that harms seedlings. In severe cases, it can cause 80% of seedlings to die, seriously affecting the economic benefits of seedling growers. Fusarium solani ( F. solani ) were cultured on PDA medium at 25°C for 4 days, the colony diameter was 25-46 mm, and for 7 days, the colony diameter was 65-70 mm. Aerial hyphae are cashmere-like, white to light gray. Most strains do not produce pigment but are cream-colored, and a few strains can produce purple pigment on the back of the colony. A large number of pseudoprimitive microconidia are produced, elliptic, ovate or reniform, relatively wide, 0-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
Inventor 戴婷婷陆辰晨焦彬彬田雯胡涛廖婷婷许新
Owner NANJING FORESTRY UNIV
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