Real-time fluorescence RPA detection primer of ceratocystis fimbriata and application of real-time RPA detection primer

A black spot bacteria and real-time fluorescence technology, which is applied in the measurement/testing of microorganisms, DNA/RNA fragments, microorganisms, etc., can solve the problems that are difficult to meet the needs of real-time and on-site detection of plant pathogens, and achieve real-time rapid detection and accurate results , the effect of easy operation

Active Publication Date: 2020-09-25
INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the defect that the existing PCR technology and LAMP constant temperature amplification technology are difficult to meet the real-time and on-site detection needs of plant pathogens, the purpose of the present invention is to provide real-time and rapid detection of sweet potato black spot fungus by using the RPA method

Method used

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  • Real-time fluorescence RPA detection primer of ceratocystis fimbriata and application of real-time RPA detection primer
  • Real-time fluorescence RPA detection primer of ceratocystis fimbriata and application of real-time RPA detection primer
  • Real-time fluorescence RPA detection primer of ceratocystis fimbriata and application of real-time RPA detection primer

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Effect test

Embodiment 1

[0034] Embodiment 1, sweet potato black spot fungus RPA primer design and screening

[0035] (1) RPA primer design: Based on the conserved sequence of the β-tubulin gene of the sweet potato black spot bacterium, 5 upstream primers and 5 downstream primers, and a probe were designed according to the design principles of RPA primers and probes (see Table 1), primers and probes were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0036] Table 1. Candidate RPA primers and probes for sweet potato black spot fungus

[0037]

[0038]

[0039] Among them: FAM-dT is a thymine nucleotide carrying a fluorescein group, THF is tetrahydrofuran, BHQ1-dT is a thymine nucleotide carrying a fluorescence quenching group BHQ1, and C3-Spacer is a nucleotide at the 3′ end A spacer arm is introduced to prevent chain extension.

[0040] (2) Extraction of fungal genomic DNA: using the Fungal gDNA Kit (Biomiga) kit to extract and operate according to the instructions.

[0041] (3) RP...

Embodiment 2

[0048] Embodiment 2, RPA primer of the present invention is to the specific detection test of sweet potato black spot bacterium

[0049] Proceed as follows:

[0050] (1) Fungi to be tested: Monilochaetes infuscans, Fusarium semimitectum, Fusarium solani, sweet potato parasite unidentified JXX2, sweet potato soft rot fungus (Rhizopus stolonifer), sweet potato parasite Unidentified JXX3, F. solani, Cladosporium anthropophilum, F. oxysporum, F. fujikuroi, Ceratocystis fimbriata A total of 11 species are preserved in the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences.

[0051] (2) Extraction of fungal genomic DNA: using the Fungal gDNA Kit (Biomiga) kit to extract and operate according to the instructions.

[0052] (3) RPA reaction system: according to According to the instructions of the exo kit, add the following components in sequence to the reaction tube containing RPA lyophilized powder: 29.5 μL of rehydration buffer, 2.1 μL of upstream ...

Embodiment 3

[0055] Embodiment 3, the present invention is used to detect the sensitivity test of the RPA primer of sweet potato black spot bacterium

[0056] Proceed as follows:

[0057] (1) DNA preparation: a) to the detection sensitivity of sweet potato black spot fungus genomic DNA: extract the genomic DNA of sweet potato black spot fungus with the method described in embodiment 2 steps (2), dilute with 10 times of gradient dilution method, respectively Diluted to original concentration: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 . b) Detection sensitivity to the amount of spores of the sweet potato black spot fungus: the number of extracted sweet potato black spot fungus spores is: 3.9×10 6 For each, the extracted genomic DNA was diluted in a 10-fold gradient, and diluted to the original concentration: 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 .

[0058] (2) RPA reaction system: refer to step (3) of Example 2, and 2 μL of genomic DNA of each gradient conc...

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Abstract

The invention discloses a real-time fluorescence RPA detection primer of ceratocystis fimbriata. The primer consists of nucleotide sequences as shown in SEQID No:1 and SEQID No:2. The invention further discloses a combiantion of the RPA detection primer and a probe. The probe consists of a nucleotide sequence as shown in SEQID No:3. The invention further discloses an application of the real-time fluorescence RPA detection primer to detection of the ceratocystis fimbriata. The real-time fluorescence RPA detection primer is high in specificity, and particularly better in specificity after the probe is used. Secondly, the RPA primer is high in sensitivity, and can perform detection only needing 10 or below spores. The detection speed is high, and detection can be completed within 20 minutes.In addition, the detection method is low in reaction temperature, simple to operate and low in detection cost, and is suitable for port quarantine inspection and field site real-time detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a real-time fluorescent RPA detection primer for sweet potato black spot fungus, and also relates to the application of the RPA detection primer. Background technique [0002] Sweet potato black spot (also known as black scar disease, commonly known as black plaster, black furuncle, black wound, etc.) is a fungal disease caused by sweet potato long beak shell fungus (Ceratocystis fimbriata Ellis&Halsted), which can cause rotten cellar during storage of sweet potato, Rotten beds and dead seedlings during the seedling raising period, the annual yield loss of sweet potato production in my country due to this disease is 5% to 10%, and in severe cases it can reach 20% to 50%, or even higher. It has become one of the three major sweet potato diseases in my country . In addition, sweet potatoes infected by the fungus Ipomoea can also produce furan terpenes such as ipomeamarone, w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2561/113C12Q2563/107
Inventor 高波马娟李秀花李焦生王容燕陈书龙
Owner INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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