Real-time fluorescent PCR detection method of rhododendron bud wilt and its special set of reagents

A fusarium wilt and rhododendron technology, applied in the biological field, can solve the problems of difficulty in meeting the detection requirements of fast, accurate and sensitive identification, high professional quality requirements of detection personnel, time-consuming and labor-intensive problems, etc., to achieve significant application promotion value and avoid human factors Interference, sensitivity-enhancing effects

Active Publication Date: 2022-04-05
NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] So far, domestic and foreign methods for detecting rhododendron bud blight are limited to morphological detection methods, which are time-consuming and labor-intensive, taking more than 15 days, and are prone to missed detection, false negative results, and require relatively high professional quality for testing personnel. High, it is difficult to meet the detection requirements of rapid, accurate and sensitive identification

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  • Real-time fluorescent PCR detection method of rhododendron bud wilt and its special set of reagents
  • Real-time fluorescent PCR detection method of rhododendron bud wilt and its special set of reagents

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Embodiment 1, the preparation of the complete set of reagents that detects rhododendron bud wilt

[0062] The complete set of reagents for the detection of Rhododendron wilt bacterium provided by the present embodiment is made up of a primer pair named SA and a probe named SA-P. SA is composed of single-stranded DNA named SA-F and SA-R respectively, and the molar ratio of the two is 1:1. SA can be amplified from the genomic DNA of Rhododendron fusarium wilt to obtain the sequence shown in sequence 4 in the sequence table. DNA fragments. The sequences of each primer are as follows:

[0063] SA-F (forward primer, sequence 1 of the sequence listing): 5'-TGAGATAGCACCCTTTGTTTATGAGT-3';

[0064] SA-R (reverse primer, sequence 2 of the sequence listing): 5'-TGCTACTGCAAAGGGTTTTTCA-3';

[0065] The nucleotide sequence of the probe SAP is shown in Sequence 3 of the Sequence Listing, with a fluorescent reporter group FAM at the 5' end and a fluorescent quencher group MGB at the...

Embodiment 2

[0068] The specificity of the complete set of reagents of the detection rhododendron bud wilt bacterium of embodiment 2, embodiment 1

[0069] Samples to be tested: Rhododendron bud blight Pycnostysanus azaleae CBS113454, rhododendron wilt CBS 280.53, apple shell color monospore canker Botryosphaeria stevensii MYA 3697, apple canker Cytospora cincta ATCC 28972, soybean southern canker bacterium Diaporthe phaseolorum var.meridional4alis 6 , American-Australian stone fruit brown rot fungus Monilinia fructicola MF, grape stem blight fungus Phoma glomerata ATCC96757, pea foot rot fungus Phoma pinodella ATCC38814, oak sudden death fungus Phytophthora ramorumMYA 3238, soybean Phytophthora sojae ATCC200094, Pythium ATCC3 splendor 、苹果黑星病菌Venturia inaequalisATCC 11096、Verticillium albo-atrum CBS 102464、 Verticillium alfalfae VAA2、Verticillium dahliae VD1、Verticillium nubilum CBS 456.51、Verticilliumtricorpus CBS 447.54,菌除Verticillium alfalfae VAA2、 Verticillium dahliae VD1与美澳型核果褐腐病菌 Moni...

Embodiment 3

[0075] The sensitivity of the complete set of reagents of the detection rhododendron bud wilt bacterium of embodiment 3, embodiment 1

[0076] 1. Extract the genomic DNA of rhododendron bud blight CBS113454 bacterial strain (Netherlands Microorganism Culture Collection (CBS)), and use the genomic DNA as a template after gradient dilution.

[0077] 2. Using the genomic DNA obtained in step 1 as a template, perform real-time fluorescent PCR.

[0078] Reaction system (10μL): 5μL Universal PCR Master Mix, 0.5 μL SA-F (10 μmol / L), 0.5 μL SA-R (10 μmol / L), 0.5 μL SA-P (10 μmol / L), 1 μL genomic DNA, made up to 10 μL. In this reaction system, the concentration of SA-F is 0.5 μmol / L, the concentration of SA-R is 0.5 μmol / L, and the concentration of SA-P is 0.5 μmol / L. An equal volume of water was used instead of genomic DNA as a blank control.

[0079] There are 1-8 reaction system systems in total, and in reaction system 1, the DNA content of the template is 10ng.

[0080] In re...

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Abstract

The invention discloses a real-time fluorescent PCR detection method and a special kit of reagents for rhododendron bud wilt. The complete set of reagents disclosed by the present invention for detecting rhododendron bud blight is composed of a primer pair named SA and a probe named SA-P, and SA is composed of single-stranded DNAs whose names are respectively SA-F and SA-R; SA-F F is the single-stranded DNA molecule shown in sequence 1 of the sequence listing; SA-R is the single-stranded DNA molecule shown in sequence 2 of the sequence listing; the nucleotide sequence of SA-P is sequence 3 of the sequence listing. Experiments have proved that the complete set of reagents of the present invention is fast and simple to detect rhododendron bud blight, has strong specificity, high sensitivity, low cross-contamination, and can realize quantitative detection. It has great application and promotion value for the detection and prevention of rhododendron bud blight .

Description

technical field [0001] The invention relates to a real-time fluorescent PCR detection method for rhododendron fusarium wilt and a set of special reagents thereof in the field of biotechnology. Background technique [0002] Seifertia azaleae belongs to Fungi Kingdom (Fungi), Ascomycota, Eurotiomycetes, Chaetothyiales, and Herpotrichiellaceae. [0003] Rhododendron bud blight has been included in the list of imported plant quarantine pests in my country, and it is a quarantine plant pathogenic fungus. At present, the pathogen is distributed in Poland, Japan, and North America, and mainly damages flower buds, leaf buds, and twigs, and can cause the whole plant to die in severe cases. [0004] So far, domestic and foreign methods for detecting rhododendron bud blight are limited to morphological detection methods, which are time-consuming and labor-intensive, take a long time, take more than 15 days, and are easy to miss, false negative results occur, and the professional quali...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12Q1/04C12R1/645
CPCC12Q1/04C12Q1/686C12Q1/6895C12Q2561/113C12Q2563/107
Inventor 段维军郭立新段丽君李雪莲张慧丽陈先锋
Owner NINGBO ACAD OF SCI & TECH FOR INSPECTION & QUARANTINE
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