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A combination of primers and probes for the detection of Phytophthora syringae based on rpa-lateral flow chromatography and its application

A technology of Phytophthora syringae and lateral flow chromatography, which is applied in the biological field, can solve problems such as no relevant application reports, and achieve good amplification effect, increased sensitivity, and good specificity

Active Publication Date: 2019-10-22
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 quarantine Phytophthora syringae, there are no relevant application reports

Method used

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  • A combination of primers and probes for the detection of Phytophthora syringae based on rpa-lateral flow chromatography and its application
  • A combination of primers and probes for the detection of Phytophthora syringae based on rpa-lateral flow chromatography and its application
  • A combination of primers and probes for the detection of Phytophthora syringae based on rpa-lateral flow chromatography and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The genomic DNA of the tested pathogenic bacteria strain was extracted, and the specific extraction process was as follows:

[0039] 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 12000 rpm for 10 min, pour off the supernatant, wash the precipitate twice with 70% ethanol, and dry at room temperature. Add appropriate amount of sterilized ultrapu...

Embodiment 2

[0041] The length of RPA primers is generally 30 to 35 nucleotides. Too short primers will seriously affect the activity of recombinases. Long primers do not necessarily improve the amplification performance, but instead increase the possibility of forming secondary structures, increasing the noise. In addition, when designing primers, try to avoid sequences that are easy to form secondary structures, primer-primer interactions, and hairpin structures, so as to reduce the formation of primer-dimers, and the size of the amplified product should not exceed 500bp. According to the above-mentioned primer design principle, the present invention uses Primer Premier 5.0 to design forward primer sequence RPA-PsyYPT-F, reverse primer sequence RPA-PsyYPT-R, probe sequence according to the Ypt1 sequence (KJ755159.1) of Phytophthora syringae PsyYPT-P.

[0042] The specific sequence is as follows: the forward primer is RPA-PsyYPT-F: 5'-TCTGCTAGCAGACTGCTGACTGTTCTATCT-3'; the reverse primer...

Embodiment 3

[0046] In order to verify the specificity of the RPA lateral flow chromatography test strip detection method, with 1 strain of Phytophthora syringae and other oomycetes and pathogenic fungi as test materials (Table 1), the RPA lateral flow chromatography test strip detection method The results showed that there were two brown bands on the test strip of Phytophthora syringae, one in the quality control area and one in the detection area, the result was positive, and the test strips of other oomycetes and pathogenic fungi only appeared in the quality control area A brown band with no band in the test area is negative, indicating that Phytophthora syringae is not present in the sample. Select different species of Phytophthora syringae (Phytophthora winter; Phytophthora parasitic; Phytophthora infestans; Phytophthora woody; Phytophthora strawberry; Phytophthora ramie, etc.) Magnaporthe oryzae; Rhizoctonia solani; Verticillium dahliae; Pythium ultima) DNA was used as a template for...

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Abstract

The invention discloses a combination of primers and probes for detecting Phytophthora syringae by RPA-lateral flow chromatography and an application thereof. The primer probe set used in the present invention has good RPA amplification effect, strong band specificity, no cross-reaction with other germs, and a positive reaction in the detection area, which increases the detection sensitivity. The invention adopts RPA chromatography technology for the first time to establish a method for rapid detection of Phytophthora syringae, and through the evaluation of specificity and sensitivity, it can be used for actual sample detection, and at the same time provides a new technical platform for the detection of Phytophthora quarantine. The pathogen was identified in the early stage of infection, and it was able to detect Phytophthora syringae in entry-exit fruits. The invention also has great significance for the blight caused by Phytophthora syringae and for reducing blind use of pesticides, reducing production costs and reducing environmental pollution of pesticides.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the detection of Phytophthora syringae based on RPA-lateral flow chromatography technology, which is specially used for the high-sensitivity and rapid detection of Phytophthora syringae carried by customs entry and exit soybeans, and can be used for the early stage of field soybean blight Diagnosis and monitoring of pathogens. Background technique [0002] Phytophthorasyringae (Phytophthorasyringae) is an important plant quarantine pathogen in my country. Its main hosts include citrus, apple, peach, pear, clove, oak and other 14 families and 29 genera. kind of disease. The pathogen can infect citrus and cause fruit brown rot, sticky gum flow, root neck foot rot and root rot, affecting fruit yield and quality, causing major economic losses, and seriously threatening the world's citrus production. Its distribution covers all major citrus producing areas in the world, but it has not been...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
Inventor 戴婷婷胡涛徐月焦彬彬陆辰晨沈浩田雯
Owner NANJING FORESTRY UNIV
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