Method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing

Abstract

The invention discloses a method for real-time fluorescence isothermal quantitative detection of citrus huanglongbing. The method includes the steps: pretreating a detected sample, and rapidly extracting DNA; according to an HLB16SrDNA conserved sequence, designing two pairs of specific primers including an inner side primer HLB-F3, an inner side primer HLB-B3, an outer primer HLB-FIP and an outer primer HLB-BIP, and preparing the specific primers; carrying out a fluorescence nucleic acid isothermal amplification detection technology reaction; constructing a fluorescence nucleic acid isothermal amplification detection standard curve according to a relationship between plasmid concentrations of a template pMD18-T-HLB with different dilution and corresponding Tt values, wherein the X axis represents log values of starting template concentrations, and the Y axis represents the time taken for amplifying the different-concentration templates to reach thresholds; adopting quantitative detection result judgment, after ending an ESE-Quant TubeScanner reaction, displaying quantitative results by an instrument automatically according to the standard curve, after ending the reaction, instantaneously centrifuging to mix SYBRGreenI on an inner cover of a reaction tube into a reaction liquid, and judging results by adopting color development without opening the cover. The method has the advantages of high sensitivity, high specificity, low pollution, and stable reaction.

Description

technical field [0001] The invention belongs to the technical field of detection of citrus huanglongbing, in particular to a method for real-time fluorescence constant temperature quantitative detection of citrus huanglongbing. Background technique [0002] Citrus Huanglongbing (HLB) is one of the most devastating diseases in citrus production in the world, and it is an important target of plant quarantine at home and abroad. At present, HLB is mainly distributed in nearly 50 countries and regions in Asia, Africa, Oceania, South America and North America. Citrus huanglongbing pathogenic bacteria can infect a variety of citrus plants, and the symptoms include mottled yellowing, uniform yellowing, element-deficiency yellowing and "rubber" fruit, etc., shortening the economic life of plants, reducing yield and quality, and causing huge losses. Economic losses. Weihai, where most of the 19 citrus producing provinces (municipalities and autonomous regions) in China have suffere...

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Problems solved by technology

[0008] The purpose of the embodiments of the present invention is to provide a real-time fluorescence constant temperature quantitative detection method for citrus Huanglongbing, aiming to solve the problems of low sensitivity, low specificity, high pollution and unstable reaction existing in the existing citrus Huanglongbing detection method

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