Real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting plantain mosaic virus in lily
A real-time fluorescence quantification, mosaic virus technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. Strong, sensitive and repeatable effects
Pending Publication Date: 2021-11-19
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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Problems solved by technology
[0005] In view of the above-mentioned shortcoming of prior art, the object of the present invention is to provide a kind of real-time fluorescent quantitative PCR method that detects plantain mosaic virus in lily, is used to solve the identification operation loaded down with trivial details to having P1AMV plant in the prior art, With defects such as low sensitivity and complex quantification, the present invention can provide technical support for accurate and efficient monitoring and control of PlAMV
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Embodiment 1
[0040] 1. Extraction and reverse transcription of lily total RNA
[0041] Take 50-100 mg of leaves of lily plants, use polysaccharide polyphenol plant tissue lysis method to extract total RNA from lily leaf samples, and then use reverse transcription kit II One-Step gDNA Removal and cDNASynthesis Super Mix to obtain cDNA.
[0042] 2. Design, synthesis and screening of primers
[0043] Select the PlAMV (GenBank: KX245539.1) sequenced in our laboratory as the template sequence, use DNASTAR software to analyze and compare the CP gene conservative sequence of PlAMV in the NCBI database, design and synthesize 4 pairs of PlAMV RT-qPCR detection primers CP-1F / R , CP-2F / R, CP-3F / R, CP-4F / R. Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.
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The invention provides a real-time fluorescent quantitative PCR (polymerase chain reaction) method for detecting plantain mosaic virus in lily. The method includes the following steps: taking leaves of a to-be-detected lily plant, and extracting total RNA; carrying out reverse transcription reaction by taking the extracted total RNA of the lily leaves as a template to obtain cDNA (complementary deoxyribonucleic acid); taking the obtained cDNA as a template, and carrying out real-time fluorescent quantitative PCR detection based on a fluorescent dye SYBRGreenI by utilizing a specific primer pair CP-2F / R; preparing a standard curve by taking plasmid PlAMV-CP2 standard substances containing detection gene segments with different concentrations as templates; judging the disease-carrying condition of the plant according to the detection result Cq value, and quantitatively calculating the virus of the disease-carrying plant according to the standard curve. The method has the advantages of rapidness, simplicity, convenience, high sensitivity and high repeatability in identification of lily plants infected with plantain mosaic viruses, the detection sensitivity of the method is 100 times that of conventional RT-PCR, and the method can be used for rapid diagnosis, quantitative detection, monitoring, prevention and control of plantain mosaic viruses in entry and exit ports and lily production.
Description
technical field [0001] The invention relates to the technical field of virus detection, in particular to a real-time fluorescent quantitative PCR method for detecting plantain mosaic virus in lilies. Background technique [0002] In production, lilies are propagated by asexual reproduction, and are susceptible to a variety of viruses, resulting in reduced bulb production, deformed flower buds, and litter, seriously affecting the yield and quality of lilies. The detection of PlAMV in lilies grown in my country has gradually become a common A major disease that endangers lily production. After lilies were infected with PlAMV, the veins of lilies were rusty, the flower buds withered and died, and the necrosis was severe in the later stage of the disease, resulting in the loss of commercial value of large-scale cut flowers. PlAMV belongs to the Alphaflexiviridae family (Alphaflexiviridae), a member of the genus Potexvirus). PlAMV is a single-stranded positive-sense RNA virus, a...
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IPC IPC(8): C12Q1/70C12Q1/6851C12R1/94
CPCC12Q1/701C12Q1/6851C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 徐雷锋明军杨盼盼宋蒙
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI