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Primer and probe combination for detecting peronophythora litchii based on RPA-lateral flow chromatography technology and detection method of peronophythora litchii

A litchi downy mildew and lateral flow chromatography technology, applied in the biological field, can solve the problems of few plant pathogenic oomycetes and no related application reports, and achieve reliable results, simple and fast operation, and good practicability

Pending Publication Date: 2021-01-29
INST OF PLANT PROTECTION FAAS +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the detection of RPA-lateral flow chromatography technology is mainly used in the detection of human and animal pathogens, food safety and environmental sanitation, but there are few reports on the detection of plant pathogenic oomycetes, especially the detection of Lychee peronospora at home and abroad. Related Application Reports

Method used

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  • Primer and probe combination for detecting peronophythora litchii based on RPA-lateral flow chromatography technology and detection method of peronophythora litchii
  • Primer and probe combination for detecting peronophythora litchii based on RPA-lateral flow chromatography technology and detection method of peronophythora litchii
  • Primer and probe combination for detecting peronophythora litchii based on RPA-lateral flow chromatography technology and detection method of peronophythora litchii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Design of primers and probes for the detection of Phytophthora lychee RPA-lateral flow chromatography

[0044] (1) Acquisition of Rh gene from Peronophythora litchi

[0045] The genome data of Pe.litchi was downloaded from GenBank (ID: PRJNA290406), and the hidden Markor of the ammonium transporter domain was downloaded from the Pfam database (http: / / pfam.sanger.ac.uk / ). Hidden Markov Model (HMM), model accession number is PF00909. The HMMER 3.2.1 program (http: / / hmmer.org / ) was used to search and compare the proteins in the whole genome of Phytophthora litchie, and the E-value threshold of the target functional domain was set to 1e-10. Subsequently, the compared candidate genes were submitted to NCBI for comparison and verification with the identified sequences, and the nucleotide sequence of the Rh gene was obtained, as shown in SEQ ID NO.1.

[0046] 5’-ATGTGTCTCATCTTCTTCGCCCTGAAATTCGACATGCCCAGTCCGAAAAACAACGACGAAGACACTGTCAGCACTCTAAACTACTACCCCATGTACATGGATGT...

Embodiment 2

[0054] Example 2 RPA-Lateral Flow Chromatography-based Detection Primer and Probe Combination for Visual Detection of Lychee Peronospora

[0055] (1) Extraction of Phytophthora lychee Genomic DNA:

[0056] Adopt CTAB method to extract Phytophthora lychee Genomic DNA, the specific process is as follows: get 50mg freeze-dried mycelia powder in a 1.5ml centrifuge tube, add 900 μ l 2% CTAB (cetyltrimethylammonium bromide) extract (2% CTAB; 100mmol / L Tris-HCl (trishydroxymethylaminomethane hydrochloride), pH 8.0; 20mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4mol / L NaCl) and 90μl 10% SDS (sodium dodecylbenzenesulfonate) and mix well, put in water bath at 55~60℃ for 1.5h, oscillate and mix once every 10min, centrifuge (12,000rpm) for 15min after 1.5h in water bath, take the supernatant and add Phenol / chloroform / isoamyl alcohol (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1) equal to that of the supernatant, centrifuged (12,000rpm) for 5...

Embodiment 3

[0061] The reaction time that embodiment 3 litchi downy mildew RPA detects

[0062] (1) Genomic DNA extraction of P. litchi Pyrosophthora: The CTAB method in Example 2 was used to extract the genomic DNA of P. litchi Pyrosophthora.

[0063] (2) RPA reaction system: 50 μl reaction system, including 29.5 μl reaction buffer (Rehydration buffer), 2.1 μl each of 10 μM PlRPA-F and biotin-labeled PlRPA-R, 0.6 μl probe 10 μM PlRPALF-P, template DNA to be tested 2.0 μl, 11.2 μl of sterile double distilled water, mix the above components and add to RPA lyophilized enzyme powder, then add 2.5 μl 280mM magnesium acetate (MgAc) and mix by inversion; replace the template DNA with an equal amount of wxya 2 O was used as a negative control.

[0064] (3) RPA reaction: Incubate the reaction system in step (2) at 40°C for 5min, mix the reaction tube again, and continue to incubate at 40°C. The total reaction time is 5min, 10min, 15min, 20min, 25min, 30min, 35min. Among them, 5min, 10min, 15m...

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Abstract

The invention discloses a primer and probe combination for detecting peronophythora litchii based on an RPA lateral flow chromatography technology and a detection method of peronophythora litchii andbelongs to the technical field of biology. The invention discloses a primer and probe combination for detecting peronophythora litchii based on an RPA lateral flow chromatography technology. The primer and probe combination comprises two primers PlRPA-F, a biotin-labeled PlRPA-R and a probe PlRPALFD-P, wherein the RPA detection primer and probe combination is subjected to constant-temperature amplification at 40 DEG C and then is detected by a lateral flow chromatography test strip, and the test strip can be observed to present a positive strip in a detection area. The RPA lateral flow chromatography technology detection primer and probe combination and the detection method thereof disclosed by the invention can be used for rapidly, sensitively and accurately detecting peronophythora litchii infected plants and fruits in production practice, and can also be used for early diagnosis of field diseases and monitoring and identification of germs; reliable technical and theoretical bases are provided for prevention and treatment of diseases caused by peronophythora litchii.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a combination of primers and probes based on RPA-lateral flow chromatography technology for detecting P. litchi and its detection method. Background technique [0002] Litchi (Litchi chinensis) is a famous and high-quality fruit in the south of my country. The cultivated area and annual output account for 84.5% and 70.5% of the world's respectively, and the annual output value reaches 6.288 billion yuan. It is the most competitive fruit in the world in my country. A subtropical and tropical fruit. Litchi frost blight is currently the most important disease in litchi production, which is caused by the infection of Peronophythora litchi. It mainly harms the near-ripe fruit, and also harms flower spikes, young fruits, fruit stalks, fruiting branchlets and leaves, thereby causing a large number of flower drop, fruit drop, cracked fruit and rotten fruit. It is also an impo...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11C12Q1/04C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 王荣波陈庆河赵玉梅刘裴清李本金翁启勇
Owner INST OF PLANT PROTECTION FAAS
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